Chapter 5 Membrane-bound enzymes

CHAPTER 5

Membrane-bound enzymes R.B. FREEDMAN Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ, U.K

1. The role and significance of membrane-bound enzymes (a) Introduction

A very high proportion of intracellular enzymes are membrane-bound according to a simple functional criterion, namely that they appear bound to, or associated with, a membranous fraction when a cell homogenate is subjected to a standard scheme of sub-cellular fractionation. The usefulness and limitations of this criterion will be discussed below (Section 2a) but the significance of the basic finding is plain. A cell is not a bag of enzymes and intermediary metabolites, but a highly organised and structured entity, and the specific disposition of enzymes within sub-cellular membranes is a central feature of this organisation. Tabulations of the sub-cellular locations of enzymes (e.g. [1D show that large numbers of enzymes in typical, eukaryotic cells are associated with the plasma membrane, the endoplasmic reticulum, the membranes of mitochondria and chloroplasts, and the membranes of other organelles. Many of these activities are of course used as markers for the specific organelles in analytical and preparative applications of sub-cellular fractionation. Despite the fact that they form the majority of intracellular enzymes, and that they catalyse many of the most central and significant reactions of metabolism, membrane-bound enzymes have been less well characterised than "soluble" enzymes because of difficulties in isolating them and in studying their kinetic and structural properties (see Sections 2b, 4a, 4b). Only in the last decade, with the appearance of some consensus on the broad structural aspects of membranes and with the development of techniques for solubilizingmembrane-bound enzymes with retention of activity, has real progress been made. Several membrane-bound enzymes have now been purified to homogeneity, subjected to the standard techniques of protein structure determination, and reconstituted with other well-defined components in lamellar or vesicular model systems to reproduce the physiological catalytic activity. Examples of such enzymes are considered in Section5. Although most membranebound enzymes have been studied in much less depth, it is now possible to detect themes and patterns and make useful generalisations. Several reviews on membrane-bound enzymes have appeared in recent years; those dealing with specific enzymes or specific organelles will be cited below, as FineanjMichell (eds.) Membrane structure © Elsevier/ North-Holland Biomedical Press, 1981

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appropriate, but more general surveys should be mentioned at this point. A significant review which brought together much of the early work on membranebound enzymes was that of Coleman [2]. Bacterial membrane-bound enzymes have been reviewed by Salton [3]. More recently, Sandermann [4] and Gennis and Jonas [5] have discussed the influence of interactions with lipids on the properties of membrane-bound enzymes, and DePierre and Ernster [6] have reviewed the enzyme topology of intracellular membranes in terms of the lateral and transverse dispositions of enzymes in specific membranes. A series of volumes edited by Martonosi [7] entitled "The Enzymes of Biological Membranes" gives a broad coverage of the subject with volumes on techniques, on enzymes involved in biosynthesis of cell components, on enzymes involved in transport phenomena and on enzymesinvolved in energy transduction and receptor systems. (b) Functional classification of membrane-bound enzymes

Membrane-bound enzymes catalyse an extraordinary variety of activities and it is helpful to think of these in terms of functional categories. Such a classification is inevitably somewhat arbitrary and the system proposed below is neither exhaustive nor exclusive, but it does provide some framework for considering the functions of membrane-bound enzymes. In brief, we can consider membrane-bound enzymes which are involved in the transfer of molecules between distinct regions, enzymes acting on substrates which are located in or on a membrane, enzymes producing products which are components of membranes, and enzymes which are part of multi-enzyme sequences for which the membrane acts as an organising matrix. Some membrane-bound enzymes, particularly those involved in energy transduction, can be considered as belonging in more than one of these categories. (i) Enzymes involved in translocation phenomena

Many aspects of metabolism are vectorial. Membrane proteins which facilitate the movement of specific solutes across membranes are not in themselves regarded as enzymes, although many aspects of their function-their specificity and high turnover rate, their kinetics and responses to inhibitors- make it inevitable that their combination with their substrates is regarded in the same terms as enzymesubstrate interactions. Nevertheless, such transport systems as the adenine nucleotide translocator of the inner mitochondrial membrane and the general anion (bicarbonate) transporter of the erythrocyte membrane do not catalyse a net chemical reaction and so are not. considered as enzymes. But many translocation systems do involve chemical reaction, especially where the translocation, considered in isolation, is endergonic and is coupled to an exergonic metabolic reaction. Such "active" transport systems include cases where the translocated substrates are themselves chemically modified in the metabolic reaction; these are known as group translocation processes. The best known example is the bacterial translocation

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system for sugars where the overall reaction is (phosphoenolpyruvate)intemal + (sugar)extemal ~ (pyruvate)intemal + (sugar phosphate)intemal This system is considered in detail in Section Sa. In other active transport systems the translocated substrates are unchanged chemically, but their movement is linked to a chemical reaction. The commonest examples are the cation-translocating ATPases, particularly the Na+ ,K +-translocating system which is widely distributed in the plasma membranes of most eukaryotic cells [8-10] and the Ca2+-translocating system of the muscle sarcoplasmic reticulum [11,12]. These systems are, of course, intrinsically vectorial and in broken membranes or solubilised preparations this aspect of their physiological function is obscured; they appear simply as ATPases which are stimulated by particular cations. In order to characterise such systems fully, techniques have been developed for reconstituting the solubilised enzymes in artificial membranes where vectorial functions can be expressed (see Section 4c). Chapter6 deals with membrane transport proteins in more detail. There may be other membrane-bound enzymes whose vectorial character is not yet appreciated. The cases referred to above were first investigated as the enzymic machinery of transport systems which were already defined in physiological terms. But in a recent example, a well-known membrane-bound enzyme, glucose-6phosphatase, was found to consist of two distinct functional components, a highly specific transporter which conveys glucose-6-phosphate from the cytoplasm (or the exterior of a microsomal vesicle)into the lumen of the endoplasmic reticulum (or the interior of microsomal vesicles) and a non-specific phosphohydrolase which accepts substrates only from the luminal (interior) phase [13,14]. Thus, at the last enzymic step in gluconeogenesis, glucose is liberated within the lumen of the endoplasmic reticulum. This discovery of an intrinsic transport role for the enzyme emerged from detailed kinetic studies, in particular from comparisons of the properties of the intact enzyme in microsomal vesicles, where a permeability barrier is maintained, with those of the enzyme in detergent-treated and permeable preparations. This finding explained many previous observations on the activity of the system towards other sugar substrates and of the effects of endocrine, nutritional and pharmacological status on glucose-6-phosphatase activity. It confirmed earlier suggestive evidence that the sites for initial interaction with glucose-6-phosphate and for release of glucose were on opposite sides of the membrane [15]. This recent development in our understanding of an enzyme which had been studied for many years, emphasises the fact that the vectorial character of membrane-bound enzymes can easily be overlooked if there is no obvious connection between the reaction catalysed and physiological transport phenomena. (ii) Enzymes associated with information transfer

Neurotransmitter and polypeptide hormones act on their target cells without (neces-

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sarily) entering them; their effects are mediated by receptor systems located in the plasma membranes of the target cells. These receptor systems can be envisaged as consisting of three functional components (this does not necessarily correspond to the number of macromolecules involved)- a receptor component which participates in the initial interaction with hormone or neurotransmitter, an effector component which carries' out the primary functional response, and a transducer component which links receptor to effector. In many cases the effector component is a system controlling the permeability of the cell membrane to a specific ion such as Na+ or Ca2+. In others the effector is an enzyme; many hormones bring about their effects via a "second messenger" system in which the hormone activates the membranebound enzyme adenylate cyclase, and the intracellular effects of the hormone are brought about by the product, adenosine-3',5'-monophosphate (cyclic AMP). sugar acceptor

reaction product

enzyme UDP

-

Me"

•

•

sugar donor

UDP free nuc leotide

Glycosyl transferase reoction

Initial adhesive recognition as a result of transferase-

~

Reoction completion

and

substrate complex UDP - XJ UDP

consequent cell

modification

Cell

< .

Seporation

Fig. 1. Model for generalised cell-cell recognition. Each cell is represented with a cell surface trisaccharide sugar acceptor and surface glycosyltransferase specific for that sugar acceptor. Enzyme-substrate interaction occurs only between transferase and acceptors on adjacent cells. Adhesion is stable so long as the requisite sugar nucleotide is not made available. In the presence of this sugar nucleotide, catalysis occurs, forcing the cells to separate, producing cell surfaces with the added monosaccharide, X. From [16,17).

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Adenylate cyclase is the classic example of a membrane-bound enzyme involved in an information transfer process and is considered in detail in Section 5b. But it is unlikely to be unique. Analogous "second messenger" systems involving other nucleotides and possibly other classes of compound, such as prostaglandins, have been suggested, but little is known yet of their enzymology. Membrane-bound enzymes may also be important in the direct communication between neighbouring cells which is important for the control of growth and cell division. The cell surface glycosyl transferases are a group of enzymes which have been suggested to play such a role [16,17]. Some glycosyl transferases, which transfer sugar residues from sugar nucleotides to glycoprotein acceptors, are associated with the plasma membrane fraction and can be detected on whole cells. They could be enzyme mediators of cell-cell interactions, transferring monosaccharides to acceptors on neighbouring cells (Fig. 1). Thus, (i) platelet sialyl transferases are thought to be important in platelet-platelet aggregation during haemostasis; (ii) there is evidence that comparable enzymes are involved in the specific adhesion between cells in embryonic tissues; and (iii) there is a stimulation of glycosyl transferase activities on mixing of algal gametes of opposite mating type, which may be important in ensuring specific gamete attachment. More significantly, there is a considerable body of evidence suggesting that in culture cells these activities may be important in the phenomenon of contact inhibition of cell division. In several normal cell types, low density cultures show high glycosyl transferase activities and activity declines as culture density increases, whereas corresponding transformed cells show high glycosyl transferase activities that are independent of culture density (transformed cells in general do not show contact inhibition of growth and division). Shur and Roth [l7] have given a critical review of the work of these enzymes. (iii) Enzymes acting on locally concentrated substrates

Enzymes of the two previous categories are necessarily membrane-bound because their functions involve communication between two phases which are separated by a membrane. In other cases, the membrane is significant not as a barrier between aqueous phases but as a non-polar phase or a rather specialised surface. Thus, many cellular metabolites are concentrated either in membranes, because of their preferential solubility in non-polar media, or at the surface of membranes, because of the presence of specific binding sites. Enzymes acting on such membrane-located substrates are often themselves membrane-bound. This generalisation applies in particular to the enzymes of phospholipid and steroid metabolism, and to enzymes acting on very non-polar polyisoprenoid substrates such as dolichol and bactoprenol; an enzyme of this type is discussed in Section 5c. However, many quite polar metabolites are found concentrated on membranes or in their vicinity, and enzymes acting on these molecules are commonly membranebound. In this group are the enzymes which break down neurotransmitters or polypeptide hormones close to their sites of action. Post-synaptic cell membranes at cholinergic junctions contain a very active acetyl-cholinesterase, fat cell plasma membranes contain an active system for degrading insulin, etc.

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Another group of enzymes acting on "local" substrates are those responsible for the post-translational modifications of secretory proteins in animal cells [18]. Proteins to be secreted are synthesised on ribosomes bound to the endoplasmic reticulum and passed across this membrane into the lumen during translation; thereafter they move through membrane-limited regions-the lumen of the endoplasmic reticulum, the cisternae of the Golgi body and secretory vesicles. During the course of this movement, most such proteins are subject to covalent modification involving some combination of disulphide bond formation, glycosylation, and partial proteolysis. In addition, many important secreted proteins undergo more specific modifications, such as hydroxylation of lysyl and prolyl residues in connective tissue proteins, and carboxylation of glutamyl residues in proteins involved in blood coagulation. All these enzymes acting on secretory proteins are located in the membranes of the endoplasmic reticulum or Golgi body and hence line the route followed by the proteins between synthesis and secretion. (iv) Enzymes involved in the biosynthesis of membrane components

Several lipids, polysaccharides and other complex molecules are found exclusivelyin membranes and many are highly insoluble in aqueous media or (in the case of bacterial membranes) are extensively cross-linked. Enzymes involved in the synthesis of these components are generally membrane-bound, and a well-characterised example, the enzyme(s) involved in incorporation of carbohydrate residues into the complex lipopolysaccharide of the bacterial envelope, is considered in Section 5d. (v) Enzyme components of organized multi-enzyme sequences

Many examples are known of several enzymes coexisting in a single isolatable functional entity; such entities are known as multi-enzyme complexes [19] or multifunctional enzymes [20], depending on whether the enzyme active sites are located on different polypeptides or on a single multifunctional polypeptide. Structures catalysing several reactions in the decarboxylation of e-ketoacids [21] and in the biosynthesis of aromatic amino acids [22] are well-known examples. The association of several enzymes within such organised structures provides a number of possible functional advantages. The fact that products of one enzyme can pass directly to become substrates at the active site of an adjoining enzyme, without equilibrating with the bulk medium, can enhance catalytic efficiency and may also permit subtle regulation, since the sequestered metabolites are effectively within a distinct metabolic compartment [23,24]. It is quite possible that associations between enzymes to produce multi-enzyme complexes or aggregates are more common in vivo than is generally appreciated, but are destroyed by conventional techniques for tissue disruption and fractionation [25]. The best known of such multi-enzyme complexes and aggregates are self-organising structures which are generally regarded as free in the cell (but see Section 2a). However, it is now becoming clear that many membrane-bound enzymes should be considered as members of analogous membrane-associated multi-enzyme sequences in which all the enzymes of a pathway are associated with the same membrane. The

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membrane would, in such a case, play an organisational role which would make possible catalytic and regulatory advantages through the "channelling" of substrates or the direct interaction of membrane-associated enzymes. The obvious examples are membrane-associated electron-transfer chains in which many membrane-bound enzymes function as intermediate electron carriers between reduced substrates and O2 or other oxidants. The mitochondrial, bacterial and chloroplast electron-transfer chains associated with energy transduction are the obvious examples. The component enzymes of these chains are associated in "complexes" which could, individually, be classified in category (i), namely enzymes involved in translocation phenomena. Each complex catalyses a redox reaction which is coupled, by a mechanism which is still undefined, to a translocation of protons (e.g. [26]). The energy-coupling ATPases could likewise be regarded as complex vectorial enzymes (Section Sf). But when considered together these enzymes form an extensive membrane-associated multi-enzyme sequence. The extent to which the efficient functioning of this sequence depends on direct interactions between the component enzymes and their organisation in the energy-coupling membrane constitute one of the most controversial areas in membrane biology (Section 3d). Another example in which the role of the membrane is still controversial is the electron-transfer system in liver endoplasmic reticulum membranes that is responsible for the oxidation and detoxification of many drugs and other foreign compounds. The electron-transfer chain in this system consists of a haemoprotein, cytochrome P-450, which binds O2 and the substrate, and a flavoprotein, cytochrome P-450 reductase, which transfers electrons to the cytochrome from NADPH. The overall reaction is a mixed-function oxidation: H+

+ NADPH + O2 + Substrate-H ~ NADP + + H 20 + Substrate-OH

Both cytochrome P-450 and its reductase are extremely hydrophobic proteins which form a significant fraction of the total protein in liver microsomal membranes. The substrates oxidised or hydroxylated by this system are usually non-polar and it could HEXOSE-6-PHOSPHATE DEHYDROGENASE

CONJUGATING ENZYMES

MONOOXYGENASE

Reductase!

P-450

I

I I

I I 2 [H] I I

I

ROH

7

?

RH

CeHg0 - PhOSPhat e 5

U9P RO-glucuronide

H 20

R-dihydrodiol

Fig. 2. Multi-enzyme sequence of xenobiotic oxidation and conjugation in endoplasmic reticulum membranes.

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R.B. Freedman

be argued that these enzymes are examples of category (iii), enzymes acting on substrates located in membranes. However, there is now evidence that the electrontransfer chain is only a part of a more extensive multi-enzyme sequence of detoxification. Thus the NADPH involved in the mixed-function oxidation does not appear to be drawn from the bulk cytoplasmic pool, but may be generated by a membranebound hexose-phosphate dehydrogenase [27,28]. Likewise the products of the mixed function oxidation are further transformed, and the enzymes involved in these later conjugation steps, such as epoxide hydratase, UDP-glucuronyl transferase and glutathione-S-transferase, are also located in endoplasmic reticulum. The kinetic evidence for "channelling" of intermediates is not yet conclusive, but this system should certainly be regarded as a membrane-associated multi-enzyme sequence (Fig. 2). Another microsomal electron transfer chain involving the components cytochrome bs and cytochrome bs reductase has been characterised in some detail; this system is considered in Section 5e. (c) Membrane-bound enzymes and concepts of membrane structure

It is obvious that the way in which membrane-bound enzymes operate cannot be pictured with any success in the absence of a plausible view of membrane organisation in general. In the last decade, views about membrane-bound enzymes have been significantly influenced by three major developments in our understanding of membranes as a whole. The first such development arose from spectroscopic and other physical studies on membrane protein, from chemical studies on the extraction of membrane proteins (including membrane-bound enzymes) and from thermodynamic considerations of how lipids and proteins might interact in a membrane. These lines of work led to the recognition of the functional distinction between extrinsic (peripheral) and intrinsic (integral) proteins and to detailed characterisation of the different modes of interaction between specific membrane proteins and the other components of a membrane. The second development was the increasing emphasis given to the fact that membranes are intrinsically asymmetric structures which separate distinct aqueous regions, so that enzymes and other membrane components have defined transverse orientations. Finally the realisation that the bulk lipids of most membranes are in a fluid state and that lipid and some protein components show extensive lateral mobility has obviously had enormous impact on thinking about how membrane-bound enzymes operate. These developments, summarised initially by Singer [29], are dealt with in detail in Chapters 1- 3 of this volume but their influence is felt in all the subsequent discussion of membrane-bound enzymes in this chapter.

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2. Problems in the study of membrane-bound enzymes (a) Problems of definition

At the outset we defined membrane-bound enzymes as those which appear in a membranous fraction when a homogenate is subjected to a standard scheme of sub-cellular fractionation. This definition is of course inferior to the one we would wish to use, namely that a membrane-bound enzyme is one which functions in or on a membrane in a living cell, but we do not have reliable and general techniques for detecting and establishing the locations of enzymes within intact living cells. In a few cases cytochemistry with the electron microscope is possible; the enzyme acts on an artificial substrate to generate an electron-opaque product easily detected in the microscope. Several requirements must be satisfied for this approach to be reliable [30]. Firstly the enzyme must survive "fixation" of the tissue, which usually involves reaction with glutaraldehyde. Secondly the enzyme must have a broad enough substrate specificity to act on the required non-physiological substrates. Thirdly, the primary reaction product must be precipitable rapidly, so that it does not diffuse away from the immediate vicinity of the enzyme. Thus phosphatases can often be detected easily since the phosphate or analogous product can be precipitated as the lead salt. Similarly, oxidoreductases can be located if the natural oxidant can be replaced by ferricyanide; in this case the product can be precipitated as electronopaque cupric ferrocyanide. Given the limited applicability of this technique, most membrane-bound enzymes have to be identified by their presence in a membranous fraction from a homogenate. Such an approach can obviously give rise to two kinds of artefacts, namely false positives- enzymes which are free in intact cells but bind to sub-cellular membranes during the course of homogenisation and fractionation-and false negatives- enzymes which are membrane-bound in the cell but are displaced during preparation. The former class can be exemplified by the "honorary" enzyme, haemoglobin. Anyone who has tried to prepare cell membranes from human red blood cells will know that it is difficult to prepare these membranes free from haemoglobin. It is present in the cell interior at such high concentration, and it can bind to biological membranes in so many conditions, that stringent control of temperature, ionic strength and pH in the lysing medium are required to produce haemoglobin-free "ghosts" [31]. False negatives can be illustrated by the case of cytochrome C, an essential component of the mitochondrial electron transfer chain that is located, like the other components of the chain, in the mitochondrial inner membrane. Cytochrome C is a basic protein and its interaction with other components of the inner mitochondrial membrane is almost entirely electrostatic; it is a classic extrinsic protein. As a consequence, its interaction with the membrane is highly sensitive to ionic strength and it is easily lost if mitochondria are exposed to 5 mM aluminium sulphate or even to 100-150 mM KCl. These examples demonstrate that the presence or absence of an enzyme in a membrane fraction can be influenced by the conditions and procedures used in lysis

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and fractionation. Temperature, pH, ionic strength, the concentrations of specific ions, chelating agents or enzyme substrates can all affect the attachment of enzymes to membranes. The problem is that it is extremely difficult to determine whether the conditions favouring the attachment of a particular enzyme to a particular membrane are stabilising an attachment which exists in the intact cell, or promoting an artefactual attachment during isolation. In some cases extensive careful work has allowed this distinction to be made, but the conclusions are usually controversial. The best example is that of the enzymes of glycolysis in the human red blood cell. Enzymes of glycolysis are commonly found associated with isolated red blood cell membranes. Recoveries of these enzymes in the membrane fraction are highly dependent on conditions of lysis and washing, and for most of the enzymes only a small fraction of the total activity is found in the membrane fraction. However, glyceraldehyde-3-phosphate dehydrogenase (G-3-PDH) and, to a lesser extent, aldolase, phosphoglycerate kinase, and pyruvate kinase are quite tightly bound and appear preferentially in the membrane fraction [32,33]. The case of G-3-PDH is particularly striking; red cell membranes prepared by the standard technique which minimises contamination with haemoglobin contain anything up to 90% of the total G-3-PDH activity of a haemolysate. The enzyme comprises 7% of the total protein in the isolated membranes [34] and is one of the prominent protein bands in the pattern obtained when red cell membrane proteins are resolved by SDS-PAGE (band 6). The interaction between this enzyme and the remainder of the red cell membrane has been characterised in some detail and the evidence suggests that the binding is at specific sites on the cytoplasmic surface of the membrane, is significant in physiological conditions and is affected by relevant concentrations of metabolites such as NAD + and NADH [34- 37]. The enzyme is displaced from the membrane by EDTA, by detergents, and by trypsin and also by 0.5M NaCl. Enzyme displaced by 0.5M NaCl can rebind to the depleted membranes and this binding has been characterised in terms of affinity and the number and location of sites [34,35]. There are 3.10 5 high-affinity binding sites per red cell and 50% of these can be occupied at physiological ionic strength (0.15M NaCl). Furthermore, the binding sites are located on the cytoplasmic face of the membrane; binding is seen with membrane fragments and "inside-out" vesicles, but resealed membranes with the normal orientation do not bind added enzyme [35]. If membrane fragments containing bound enzyme are resealed, the enzyme activity becomes latent (see Section 3c), confirming the location of the binding sites on the "cytoplasmic" surface; the native enzyme is also "latent". The number of binding sites is similar to the number of copies of the enzyme in whole red cells, which also supports this view. The extensive characterisation of the molecular organisation of the red cell membrane by impermeant reagents, cross-linking reagents and proteolytic dissection has confirmed that the enzyme occurs at the cytoplasmic surface of the membrane, bound to the intrinsic membrane protein, known as band 3, which is responsible for the translocation of bicarbonate and other anions [38,39] (Fig. 3). Further study of the binding of G-3-PDH to red cell membranes in vitro has

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PAS1( 2) PAS 3

2

~ 5

4.2 7

~6

?

?

Fig. 3. A schematic representation of the major polypeptides of the human red cell membrane, numbered according to mobility in SDS-PAGE. Band3 is the anion-transport protein; Band6 is glyceraldehyde-3phosphate dehydrogenase. From [39].

shown that the binding is affected by specific substrates as well as by more general conditions such as ionic strength [37,40]. NAD + promotes binding while NADH favours dissociation. In several other enzymes similar influences of substrates on the interaction between enzyme and sub-cellular membranes have been noted [25,41]. In many cases the kinetic and regulatory properties of such enzymes are altered on binding to membranes and this has led to the proposal that such reversible binding is a genuine cellular phenomenon permitting sensitive regulation of enzyme activity. This proposal implies that the lability of enzyme-membrane interactions is not just a nuisance in the isolation and identification of membrane-bound enzymes but is a functionally significant property. Enzymes capable of reversible and metabolitedependent interconversion between membrane-bound and free forms have been termed "ambiquitous" enzymes [41]. G-3-PDH is not the only membrane-bound or "ambiquitous" glycolytic enzyme in red cells. Phosphoglycerate kinase is also commonly found bound to isolated red cell membranes, and this enzyme can also be removed by high ionic strength and rebound subsequently; the binding is promoted by NAD + and ADP and opposed by NADH and ATP [42]. This property may be relevant to the generation and utilisation of ATP in red cells since these cells derive their ATP from glycolysis (the steps catalysed by phosphoglycerate kinase and pyruvate kinase) and use the bulk of their ATP in driving ion-translocation across the membrane. Some time ago, physiological evidence led to the proposal that the enzymes

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G-3-PDH, phosphoglycerate kinase and Na+ ,K + -ATPase formed a multi-enzyme sequence in the red cell membrane, with 1,3-diphosphoglycerate and ATP being channelled directly from one enzyme to the next [43,44]. This proposal has recently been revived by evidence that Na+ and K + ions, and ouabain, a specific inhibitor of the ATPase, influence the binding of glycolytic enzymes (G-3-PDH, phosphoglycerate kinase and phosphoglycerate mutase) to red cell membranes and influence the binding of substrates to these membrane-bound enzymes [45,46]. The binding of ouabain to the external face of red cell membranes appears to inhibit the binding of a complex of glycolytic enzymes to the cytoplasmic face of the membrane, implying some linkage between the ouabain-sensitive ATPase and the glycolytic enzymes. All the data summarised in this section emphasise that the binding of many enzymes to membranes in vitro is sensitive to a variety of environmental factors and that not all binding observed in vitro is necessarily of physiological significance. This limitation ought constantly to be kept in mind. Nevertheless, it is possible to characterise such in vitro interactions sufficiently to be reasonably confident about whether or not they are artefactual. The glycolytic enzymes in red cells, especially G-3-PDH, seem to have an interesting and functionally significant interaction with the cell membrane, and G-3-PDH can be regarded as a major extrinsic protein of the membrane. (b) Problems in assay and kinetic characterisation

For the characterisation of an enzyme it is essential to have good methods for assaying it and interpreting its kinetics. Membrane-bound enzymes present a number of specific problems in this respect. At the most basic level membrane-bound enzymes can be technically difficult to assay in that membrane preparations are turbid, may sediment under gravity and (where intact vesicles are present) may swell or contract in response to changes in osmotic pressure; all of these properties cause difficulties in spectrophotometric assays. In addition, membrane preparations sometimes contain several similar enzyme activities (e.g. phosphatases) which may interfere with each other's assays. A more fundamental difficulty arises from the fact that membrane suspensions are multicompartmental systems in which membranes may form permeability barriers between distinct aqueous regions. In such systems the full catalytic activities of some membrane-bound enzymes become detectable only when the membranes are fragmented by sonication or made permeable by detergents. Such enzymes are said to be latent. This phenomenon is valuable in establishing the transverse distribution of enzymes in membranes but it is a problem in the quantitative assessment of the extent of purification of a membrane enzyme as it is isolated from its crude source and also in kinetic studies where it becomes necessary to work with solubilised enzymes or at least with enzymes which have lost their vectorial properties. In kinetic studies also the location of the enzyme at an interface between a non-polar phase and an aqueous phase leads to problems arising from the distribution of substrate between the two phases. Thus, lipophilic substrates such as the xenobiotics

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2-acetylamino-fluorene or 4-dimethylamino-azobenzene become solubilised in hepatocyte membranes to an extent of about 99%, and even endogenous compounds such as oestrone are 89% membrane-bound [47]. Such preferential distribution of lipophilic substrates into the membrane phase will have a profound effect on the rates of reaction catalysed by membrane-bound enzymes but this is rarely considered in kinetic analyses. An explicit treatment of this case is given by Parry et al. [48]. They assume that the system can be treated as consisting of an aqueous and a membrane phase, that the substrate simply partitions between the two phases according to a partition coefficient K p , that a membrane-bound enzyme can either interact with substrate in the aqueous phase or with substrate dissolved in the membrane phase (but not with both), and that the enzyme-substrate interaction can intrinsically be described by the simple conventional formal scheme E+S

k +I

k

+2

~ ES ~ L_ 1

E + products.

The initial rate equations which they derive can be expressed in the following form:

for an enzyme interacting with substrates in the aqueous phase, and

for an enzyme interacting with substrates in the membrane phase, where eo is the total concentration of enzyme active sites, s is the total substrate concentration in the whole system, K m is (k_ t +k+ 2)/k+ 1, K p is the partition coefficient for the substrate between the aqueous and membrane phases and A is the fraction of the total volume of the system occupied by the membrane phase. Since eo is linearly related to A (the enzyme is a component of the membrane), v is a complex function of eo and plots of initial reaction rate against enzyme concentration are not linear. Furthermore, although these equations show a hyperbolic relationship between v and s, and enzymes conforming to this model will therefore appear to follow Michaelis-Menten kinetics, the apparent K m is a function of A and hence varies with the concentration of enzyme (membrane) present. Such variation of apparent K m has been noted in some kinetic analyses of membrane-bound enzymes (see refs. in [48]), and has probably been overlooked in many others. The other important conclusion from this treatment is that, no matter whether the enzyme interacts with substrate in the aqueous or the membrane phase, the apparent K m is a function both of the "true" K m and of K p and of A. As a result,apparent K m values derived from conventional studies using the total substrate concentration as

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the variable will be significantly different from true K m values, except in the limiting case of an enzyme acting on a substrate in the aqueous phase where either the substrate is very hydrophilic (K p ~ 0) or the membrane volume is very small

(A ~ 0).

The central assumption of the above treatment- that the interaction of substrate with membrane can be described as a simple partition-may not be appropriate in all cases. Sometimes the interaction is better described as a reversible interaction with a finite number of binding sites in the membrane which can be characterised by a dissociation constant; in this case the substrate is distributed between the aqueous phase, the active site of the enzyme and other saturable binding sites on the membrane. Such a case has been analysed by Gatt and Bartfai [49]; the equation derived has the form

v=

K 1s+K2s 2 K 3+K4s+S 2

where the constants are functions of the intrinsic rate-constants k + I' k -I' k +2 etc., the total enzyme concentration eo and of parameters describing the association of the substrate with the non-enzymic sites on the membrane. In this case, as before, initial rate is not a linear function of eo, but this expression also includes second power terms of s in both numerator and denominator, so that the relationship between v and s is not hyperbolic but sigmoidal. Gatt and Bartfai [49] point out that if the complexities of interaction between a lipophilic substrate and a membranebound enzyme are overlooked these kinetic characteristics may be incorrectly interpreted as demonstrating co-operative interactions in the enzyme, inhibition by excess substrate and other phenomena. Further kinetic complexities may result if the lipophilic substrate undergoes self-association (e.g. to form micelles) as a function of concentration. Rate equations for this situation have been derived [50]. (c) Problems of strategy

The practical and interpretative difficulties in studying the catalytic properties of membrane-bound enzymes suggest that these are best studied after extraction of the enzyme from the membrane, and purification. But a membrane-bound enzyme is naturally associated with other protein and lipid components of the membrane in which the enzyme functions; removing these "contaminants" often alters the properties of the enzyme. This phenomenon, which has been termed "allotopy", emphasizes the importance of the membrane environment in the functioning of membrane-bound enzymes. Solubilisation may produce changes in stability, in affinity and specificity for substrates and effectors, in pH optimum and other kinetic properties (see Section 3a). Where a membrane-bound enzyme interacts with a membrane component which is essential for regulation of activity, these properties will be lost on solubilisation. In addition, of course, vectorial properties of membrane-bound enzymes are completely lost on solubilisation.

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So there is a problem of strategy. How useful is it to study solubilised membranebound enzymes? Should one use the physiological substrates to assay them or an artificial substrate which gives higher activity? Should one assay in the presence of lipids or detergents which activate the enzyme? Can one be sure that no important functional components of the membrane-bound enzyme (e.g. effectors or effectorbinding sites) have been lost on solubilisation? These questions only emphasise the fact that a solubilised membrane enzyme is an artificial system lacking many features of the cellular situation. Work with highly purified materials will allow clear molecular conclusions to be drawn, but at the cost of physiological relevance. The most productive approach is to combine the fullest possible characterisation of the enzyme in its membrane environment (Section3) with studies on the solubilised purified enzyme (Sections 4a,b) and then to attempt to reconstitute the "native" properties using fully defined components (Section 4c).

3. The significance of the membrane environment (a) Lipid dependence

It is a common observation that the activity of membrane-bound enzymes can be abolished or diminished by treatment of the membranes with organic solvents, detergents or phospholipases. These treatments respectively extract membrane lipids, disrupt the interactions between membrane lipids and proteins, and chemically degrade membrane lipids, so it has naturally been concluded that the activity of many membrane-bound enzymes is "lipid-dependent". For the most part this is probably a reasonable conclusion, but trivial explanations such as direct inhibition of the enzyme by solvents or detergents or by the products (e.g, lysophospholipids, fatty acids) of phospholipase action cannot be ignored. Steps can be taken to reduce the possibility of direct inhibition but the establishment of genuine lipid dependence requires stricter criteria. Fleischer and coworkers, in a study of the mitochondrial enzyme ,8-hydroxybutyrate dehydrogenase (see Section 4c), insisted that not only should the enzyme inactivation be proportional to the lipid removed but also that the enzyme should be reactivatable on readdition of lipid and that the reactivation should be proportional to the extent of incorporation of added lipid. Conclusions from reactivation studies are, however, also limited by technical considerations in that the extent of reincorporation of added lipid is critically dependent on the physical condition of both enzyme and lipid components in the reconstitution experiment. It may be necessary to include detergent in order to maintain the membrane enzyme in aqueous solution and the physical state of the added lipid component will be dependent on its composition, the conditions of temperature, pH and ionic strength, and also on the methods used for its dispersion. Under these circumstances, failure to reactivate a membrane enzyme cannot be considered as evidence for the absence of lipid dependence. In early work on lipid-dependent, membrane-bound enzymes there was great

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concern to establish whether reactivation was specifically dependent on the presence of a particular lipid class or of particular fatty-acyl groups; it was hoped that such findings might allow the identification of the lipids which actually interact with the enzyme in its natural membrane environment. Accumulated experience has caused these simple expectations to be revised. For example, the extrinsic bacterial membrane protein pyruvate oxidase is activated by the addition of phospholipids to the homogeneous, lipid-free enzyme. When lipids are added simply in suspension, lysophosphatidylethanolamine is the most effective activator; when the lipids are added as micellar dispersions all are more effective than as suspensions and the differences between lipids are minimal [52]. So these experiments do not show that the enzyme has a specific requirement for lyso-PE nor that in the bacterial membrane it is specifically associated with this lipid; the results presumably reflect some optimal physical properties of suspensions of this lipid. Another example is the Na+ ,K +-ATPase of the plasma membrane whose lipid dependence has been studied in much detail [8,53,54]. The fully delipidated enzyme is inactive and can be reactivated by many amphiphiles including alkylphosphate detergents, and several phospholipids. However, of these effective activators, only phosphatidylserine (PS) and phosphatidylinositol (PI) are actually present in the native membranes from which the enzyme is derived and in partially purified, active, lipid-containing preparations. It is tempting to interpret this as showing that the enzyme interacts with PS or PI in the intact cell membrane, but this should probably be resisted. Preparations in which both PS and PI have been completely converted to other phospholipids by specific enzymes still retain considerable activity [54]. Many of the early claims for specificity in reactivation of lipid-dependent enzymes have been modified by later work [4]. Claims for specificity in lipid dependence of selected enzymes are considered in Sections 5a-d. The concept of lipid dependence can be clarified and a large number of experimental observations rationalised by asking how the lipid interacts with the enzyme and what actual functions are performed by the lipids in lipid-dependent enzymes. A number of cases could be considered. (1) The lipid is effectively an allosteric activator, binding to the enzyme at a specific site or sites. (2) The lipid provides a specific solvation shell or "annulus" of tightly associated lipid. (3) The lipid provides an extensive non-polar region such as a micelle, in which the enzyme is effectively dissolved and in which environment its interaction with substrates and catalytic activity are most efficient. (4) The lipid forms vesicles, providing an oriented, curved bilayer in which the enzyme can take up an asymmetric disposition and demonstrate its characteristic vectorial activity. The lipid interacting with the enzymes in one of these ways may be required to facilitate interaction between enzyme and substrate to stabilise an active conformation of the enzyme, to disperse aggregates of lipid-free enzyme or to permit the components of a multi-component enzyme system to interact productively. These possibilities have not often been distinguished. The importance of lipids in the binding of non-polar substrates is shown by the fact that in some cases, the observation of "lipid dependence" is conditional on the use of certain substrates and not others. For example, with the NADH and succinate

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dehydrogenases of the mitochondrial electron transfer chain, the solubilised lipid-free enzymes are active with artificial electron acceptors such as ferricyanide, but are active with the physiological oxidant ubiquinone only in the presence of lipids. With complex, vectorial membrane-bound enzymes, different aspects of the enzyme's activity may show different lipid requirements. Thus the activity of the sarcoplasmic reticulum Ca2+-ATPase can be maintained in a detergent micelle [55] but the demonstration of Ca2+ ion translocation requires the incorporation of the enzyme into a lipid bilayer. In the case of hormone-sensitive adenylate cyclases, hormone-binding, basal adenylate cyclase activity and hormone-stimulated adenylate cyclase activity can all show distinct lipid-dependencies (see Section 5b). (b) Effects of the physical state of membrane lipids

Many membrane-bound enzymes, as well as other membrane-bound functional systems such as transporters, show a complex temperature dependence. In particular, when the activity of such systems is expressed as an Arrhenius plot (log activity vs. I IT) the result is often not a single straight line but two or more lines with different gradients, or with discontinuities [56-60]. These experiments are usually conducted in the temperature range 1O-40°C. The interpretation of such phenomena is complex, but fundamentally they indicate that the activation energy of the process is not constant over the temperature range studied. For some membrane-bound enzymes this unusual temperature dependence arises because the enzymes are influenced by their membrane environment and respond to changes in the physical state of this membrane environment. In lipid-water systems, a pure lipid component may undergo a thermotropic order ~ disorder (gel ~ liquid crystal) transition at a characteristic temperature dependent on the nature of the lipid, ionic strength etc. The transition reflects the appearance of a free rotation around C-C bonds in the lipid alkyl chains, so that the non-polar region of such systems above the transition temperature is effectively fluid. Such transitions can be observed by differential scanning calorimetry, by X-ray diffraction and by numerous spectroscopic techniques [58,61]. Similar phenomena occur in lipid mixtures, but instead of a simple phase transition occurring at a sharp transition temperature there is more complex phase behaviour; the transition extends over a range of temperatures. At intermediate temperatures, regions of "ordered" and "fluid" lipid, differing in lipid composition, coexist. In bilayers comprising mixed lipids these coexisting regions of "fluid" and "ordered" lipid will form patches along the bilayer surface- this is known as a lateral phase separation [62]. The presence of cholesterol in such lipid systems reduces the enthalpy change in such phase transitions and extends the temperature range over which they occur, but it does not abolish the transition. Thus these temperature-dependent lateral phaseseparation effects might be expected to occur in biological membranes. In natural membranes there may be an asymmetric distribution of lipids between the two faces of the membrane and so the two halves of the lipid bilayer could undergo independent phase transitions and lateral phase separations.

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Physical techniques indicate that membranes can indeed undergo temperaturedependent structure transitions analogous to those in lipid systems; membrane lipids appear to be responsible for these transitions. Firstly, in many cases the transitions are comparable in membranes and in total lipid extracts from those membranes. Secondly, the transitions can be modified or abolished by treatment of membranes with phospholipases. Thirdly, and most convincingly, changes in the lipid composition of membranes lead to characteristic changes in the phase transition temperatures observed both in intact membranes and in extracted lipids. In general, increases in the proportion of unsaturated fatty acids lower the temperature at which the gel-liquid crystal transition occurs and increase the mobility of fatty acyl chains above the transition temperature. The temperatures at which discontinuities occur in Arrhenius plots of membrane-bound enzymes often correspond to temperatures at which changes in the physical state of the membrane can be detected [56-58,60]. These findings of corresponding changes in the physical state of membrane lipids and functional properties of membrane-bound enzymes are strong evidence for an effect of the lipid environment on such enzymes, but the actual mechanism of such effects is not really clear. Early work in this field tended to simplify the nature of the phase change occurring in natural membranes and to suggest that membrane-bound enzymes were simply coupled to some generalised membrane physical state. Growing appreciation of the heterogeneity and asymmetry of membranes has modified this view. Lipids and enzymes are not uniformly distributed in the transverse direction and there is also heterogeneity in the lateral direction as shown by the phenomenon of lateral phase separation and also by evidence from electron microscopy, chemical cross-linking, and the isolation of distinct functional regions from single continuous membranes (see below). Thus the "lipid environment" in a membrane is not uniform. So it is not surprising that (i) the breaks in activation energy for some enzymes and transporters in E. coli membranes do not occur at the same temperature as the apparent transition temperature detected by X-ray diffraction [63], (ii) one enzyme in a membrane sometimes shows a break or non-linearity in its Arrhenius plot at a specific temperature whereas another shows no discontinuity at all [64-66] or shows a discontinuity at a different temperature [67,68]. In some cases a single enzyme may show several discontinuities [69,70] and these have been interpreted as corresponding to the lower and upper extremes of the complex phase transitions occurring in each half of the membrane lipid bilayer (see below for a further example of this). All of these findings- and those quoted are only a fraction of those which have been observed-show that there is not a single "membrane physical state" which influences all enzymes in a particular membrane. Different enzymes occupy different membrane environments; some may extend through the bilayer, others are located in one half of the bilayer; some may lrave a tightly bound shell of specific lipid which exchanges only slowly with the bulk lipid of the membrane, others may have no direct interaction with lipids but may be extrinsic proteins bound to the membrane by interaction with intrinsic proteins, and others again may be anchored to the non-polar region of the membrane by a hydrophobic "tail" but may have their

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functionally active region out of contact with membrane lipids. All these factors could influence the extent to which the enzymes are affected by the physical state of membrane lipids and by which fraction of the total membrane lipid they are affected. Phase transitions and lateral phase separations in membranes are interesting in providing insights into the responsiveness of membrane-bound enzymes to their environment, but it is uncertain whether any enzymes are physiologically regulated by such transitions. The evidence suggests that enzymes in general require lipid fluidity in their immediate environment [60]. However, this does not mean that the phenomenon is merely a laboratory curiosity. Microorganisms, plants and poikilothermic higher organisms may be exposed to widely varying temperatures which could alter the physical state of their membranes. Such organisms appear to be adapted to respond to this problem by altering their membrane lipid content to maintain the membranes in the liquid crystal state; this phenomenon has been referred to as "viscotropic regulation" [71]. Thus in many wild-type bacteria, the temperature of growth affects lipid composition, the proportion of unsaturated and branched fatty acids usually being inversely proportional to growth temperature [72]. Similar effects are observed even in higher animals such as the goldfish; fish maintained at 5°C contain higher proportions of mono- and poly-unsaturated fatty acids in the phospholipids of synaptosomal membranes than do fish maintained at 25°C [73]; these changes in composition do not abolish but significantly reduce the changes in "fluidity" which would otherwise be observed in these membranes as a function of temperature. The metabolic control mechanisms underlying these changes in composition ate not yet clear. Some evidence in bacteria and protozoa implicates temperature-dependent effects on the level of fatty acyl-CoA desaturase activity [74]. Even more remarkable phenomena are observed in studies on membrane-bound enzymes in animals which hibernate. Arrhenius plots of the respiratory chain activity in liver mitochondria from active ground squirrels give two straight lines; below a certain temperature the activation energy of the overall reaction becomes very high and respiration is very slow [75]. This kind of behaviour is typical of that seen in studies of the temperature dependence of respiratory chain activity in homeothermic animals and most plants [76,77] (Fig.4). However, in liver mitochondria from hibernating ground squirrels, whose body temperature falls below lOoC, the Arrhenius plot is linear down to 5°C and moderately active respiration can be maintained down to this temperature. This behaviour is typical of that seen in poikilothermic animals and in plants which are resistant to chill damage. These differences in functional response to temperature are correlated with differences in physical behaviour. In mitochondria from homeotherms, spin-labeled fatty acids can reveal phase transitions in the membrane at the temperature corresponding to the break in the Arrhenius plot of respiratory activity, but no such transition is observed in mitochondria from poikilotherms [78]. Thus the mitochondria of organisms adapted to withstand low temperatures show changes in the physical properties of the mitochondrial membrane which permit reasonable rates of respiration to be maintained at low temperatures.

180

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A more recent extensive study of the effects of hibernation on enzymes of the plasma membrane of hamster liver exemplifiesmany of the phenomena discussed in this section. Houslay and Palmer [79] studied eight enzyme activities in plasma membranes isolated from livers of control and hibernating hamsters; the animals' body temperatures were 38° and 4-5°C, respectively. All the enzymes showed sharp changes in gradient in their Arrhenius plots (temperature range 1°-42°C) and some common patterns emerged. In control hamster membranes four enzymes showed two break points in their Arrhenius plots at 25° and l3°C; these were glucagon-stimulated ouabain-sensitive ATPase (Na+,K + -A'TPase) adenylate cyclase, 5 and ouabain-insensitive ATPase (Mg2+-ATPase). In membranes from hibernating hamsters, these four enzymes again showed two break points in Arrhenius plots, but these occurred at 25° and 4°C, respectively. The fact that four enzymes show the same temperature effects, and the same change in these effects on hibernation, makes it most unlikely that the temperature-dependencies represent individual properties of the enzymes and likely that all the enzymes are responsive to temperature-dependent changes in the membrane as a whole. Three other enzyme activities - basal adenylate cyclase, F - -stimulated adenylate cyclase and cyclic AMP phosphodiesterase-all showed single breaks in their Arrhenius plot at 25-26°C both in control and in hibernating hamster plasma membranes. A final enzyme, alkaline phosphodiesterase showed a single break point in its Arrhenius plot which occurred at l3°C in membranes from control hamsters and at 4°C in membranes from hibernating hamsters. Two further pieces of information are required to interpret these findings. Membrane phase transitions can be indirectly detected by the fluorescent probe 4-anilinonaphthalene-sulphonate; they are observed at 25° and at l3°C in membranes from control hamsters and at 25° and 4°C in membranes from hibernating animals. Furthermore the enzymesin question have known transverse dispositions in f-nucleotidase,

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181

Fig. 5. Disposition of membrane proteins in hamster liver plasma membranes. A schematic representation of the localization of some of the proteins of hamster liver plasma membranes. The temperature at which lipid phase separations occur are given (T, "C) and the active site of enzymes or binding site on receptors is indicated by (1WWIIoIIIW). Abbreviations: GR, glucagon receptor; H, glucagon, hormone; A, catalytic unit of adenylate cyclase; SP, (Na + + K + )-stimulated ATPase; M, Mg 2+ -dependent ATPase; PDI, phosphodiesterase I; ePD, cyclic AMP phosphodiesterase; N, 5'-nucleotidase. From [79].

the membranes established by techniques discussed in the next section and in Chapter 3. The catalytic unit of adenylate cyclase, responsible for both basal and fluoride-stimulated adenylate cyclase activity is located on the cytoplasmic surface of plasma membranes (see Section 5b); these activities show a single break at 25°C. The glucagon receptor is located on the external surface and glucagon stimulation of adenylate cyclase activity requires coupling of these two components to form a unit spanning the membrane. The Na + ,K + -ATPase is likewise known to span the membrane. Both these enzymes show two breaks, at 25° and at a lower temperature which is l3°C in controls and 4°C in hibernating animals. In fact all the data on these enzymes can be rationalised by the proposal that the break temperatures reflect lipid phase separations occurring separately in the inner (cytoplasmic) and outer (external) halves of the lipid bilayer in these membranes-at 25°C in the inner half and at l3°C in the outer half in membranes from control animals, but at 25°C in the inner half and 4°C in the outer half of membranes from hibernating animals. Then the first four enzyme activities discussed above detect changes in both halves, the next three detect changes in the inner half only, and alkaline phosphodiesterase, which is known to be exposed on the outer surface, detects changes in the outer half only (Fig. 5). According to this interpretation, the important change in physical properties of the membrane accompanying hibernation is apparently confined to the outer half of the bilayer. (c) Transverse disposition

Effects on the properties of membrane-bound enzymes dependent on their location relative to the plane of the lipid bilayer have been mentioned in the previous section and in discussions of "latency" of membrane-bound enzyme activity (Section 2b). All membrane-bound enzymes that have been studied in this respect are intrinsically asymmetric, but this may not be immediately obvious if the enzyme acts on freely permeant substrates or on substrates dissolved in the non-polar membrane phase

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(Section 2b). This transverse asymmetry can be maintained only because proteins flip from one side of the membrane to the other, or reorient themselves relative to the bilayer, at very slow rates, if at all. Methods for establishing the transverse disposition of membrane-bound enzymes include all those used to study asymmetry of other membrane proteins (see Chapter 3). They include accessibility of the enzyme to labeled impermeant chemical reagents (monitored by the appearance of label in the appropriate band after SDS-PAGE), accessibility to proteases (monitored by loss of enzymic activity or disappearance of the appropriate band after SDS-PAGE) and accessibility to specific antibodies (monitored by inhibition of enzyme activity or by electron microscopy if ferritincoupled antibodies are used). In addition to these methods, the transverse disposition of enzymes can be established by exploiting latency. As examples we can consider contrasting findings on mitochondrial and bacterial glycerol-3-phosphate dehydrogenase assayed using the impermeant oxidant, ferricyanide. The dehydrogenase activity is fully detectable in intact mitochondria, using this oxidant, implying that the enzyme-active site is exposed on the outer face of the inner mitochondrial membrane [80]. But no enzyme activity is observed with intact bacteria using this oxidant. Furthermore, E. coli mutants that are deficient in transport of glycerol-3-phosphate cannot use it as sole carbon source. These findings suggest that the enzyme in bacteria faces the interior of the cell [81]. Of course all of these methods require that the enzyme is present in a membrane which can be obtained as a closed and impermeable structure with a defined orientation. Thus plasma membrane enzymes can be studied using whole cells (external surface only accessible) and using membrane fragments (both surfaces accessible); enzymes of the inner mitochondrial membrane can be studied using intact mitochondria where the outer surface (C-face) of the inner membrane is accessible (since the outer membrane is freely permeable to small molecules) and using sub-mitochondrial particles in which the matrix face (M-face) of the inner membrane is accessible. In the last few years, efforts have been made to generate vesicles with opposite orientations from those usually available and there have been some successes. Experiments on the interaction of erythrocyte enzymes with "insideout" vesiclesderived from red blood cell membranes were described in Section 2a. In all work of this kind it must be recognised that isolated preparations of membrane fragments and vesicles are unlikely to be all of one orientation. The transverse topology of the energy-coupling enzymes of the inner mitochondrial membrane have been studied particularly intensively, not least because full information on the transverse disposition of all the components might resolve the outstanding controversies in the mechanism of energy coupling. From this vast body of work, some assignments are well-established but the locations of other functional components are still obscure (see [6,82,83] for reviews). In brief, the well-established transverse features of the respiratory chain are as follows (Fig.6). (l) The initial sites of interaction of NADH and succinate with their dehydrogenases are at the matrix surface (M-face) of the inner mitochondrial membrane; these substrates are normally generated within the matrix in intact

183

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mitochondria, and added NADH or succinate are not readily oxidised by intact mitochondria but are oxidised by sub-mitochondrial particles. Furthermore the dehydrogenases are sensitive to digestion by trypsin in inside-out sub-mitochondrial particles. Although the dehydrogenases may span the membrane, there is no evidence for functional interactions at the C-face. (2) Cytochrome e is located at the C-face of the membrane; it is easily extractable from mitochondria but not from sub-mitochondrial particles. Anti-cytochrome e antibodies specifically label the C-face. Cytochrome e interacts at the C-face with its reductant, cytochrome e l , and with its oxidant, cytochrome a. (3) Cytochrome oxidase (complex IV) spans the membrane, with cytochrome a and the site of interaction with cytochrome e at the C-face, and cytochrome a 3 and the site of interaction with O2 at the M-face. These conclusions are based on work with antibodies and on inhibitors of the individual electron transfers. (4) The locations of other electron transfer centres, such as iron-sulphur centres, ubiquinone, the cytochromes b and the Cu centres of cytochrome oxidase, have not been clearly established by the standard techniques; they are probably embedded within the membrane but little more can be said with confidence. (5) These findings, taken together, are consistent with a "single loop" model for the transverse organization of the mitochondrial electron transfer chain (M-face ~ C-face ~ M-face). Work on the topology of the mitochondrial electron transfer chain is now concentrating on establishing the positions not only of the functional centres in these enzymes but of the individual polypeptides. Each of the electron transfer complexes contains several polypeptides and their locations are being probed by labeling techniques (see Fig. 6b). The transverse topology of mitochondrial energy-coupling ATPase is discussed in Section 5f. The transverse dispositions of membrane-bound enzymes are not arbitrary. For enzymes involved in translocation or communication it is obvious that the transverse disposition is determined by the function. But in other cases, too, the disposition is related to the role of the enzyme. Examples already considered include the glycolytic enzymes which are located on the cytoplasmic surface of the red cell membrane acting on intracellular metabolites; enzymes catalysing post-translational modifications of proteins which are generally located on the luminal face of the endoplasmic reticulum, facing the compartment through which the newly synthesised proteins move; acetylcholinesterase and other membrane enzymes degrading neurotransmitters which are located on the outer face of innervated cells. (d) Lateral disposition

The distribution of membrane-bound enzymes in the plane of the membrane is a subject on which there has been extensive speculation, but very little clear evidence. On the one hand, the physical data' on protein mobilities accumulated during the last decade imply that many membrane proteins should be regarded as solutes in a fluid lipid phase, capable of quite rapid (and unlimited?) lateral diffusion (see Chapter2). On the other hand, in some membranes there is evident lateral specialisation into

Membrane-bound enzymes

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Fig. 7. Schematic diagram of mature enterocyte, emphasising the distinct regions of plasma membrane, differing in morphology and function. From [172].

regions clearly different in structure and physiological function. This is true of the plasma membranes of most cells, especially epithelial cells which have distinct surfaces specialised for absorption or secretion. These specialised regions have distinct protein and enzyme contents, but they have not yet been extensively characterised [84,85] (Fig.7). Even the liver cell plasma membrane is structurally and functionally differentiated into regions in contact, respectively, with sinusoids, bile canaliculi and other liver cells [86]. Lateral specialisation is also observed in prokaryotes, a popular example being Halobacterium halobium, which, when illuminated in conditions of oxygen limitation, produces specialised purple membrane patches which contain the light-energy transducing protein, bacteriorhodopsin. All these lateral specialisations of membranes must be maintained either by strong direct interactions between the components of the specialised membrane, or by interactions between membrane components and some extramembranous framework, such as cytoskeletal elements. Very little is known about the precise mechanisms involved. Apart from this large-scale lateral specialisation there is also the question of whether there is specialisation on a smaller scale; do the component enzymes of membrane-bound multi-enzyme sequences diffuse freely through the whole area of the membrane interacting through random collisions, or do they have more or less permanent functional interactions leading to formation of defined complexes? There are few reliable techniques for detecting and identifying protein-protein interactions

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in membranes (or for that matter, lipid-lipid and lipid-protein interactions). Cross-linking techniques, which have been successful in establishing the quaternary structure of other systems, such as oligomeric enzymes, multi-enzyme complexes and the bacterial ribosome, have provided some useful information but their interpretation is complex [87,88]. It is difficult to distinguish cross-links formed between species which interact transiently and those between components of stable complexes. Thus, although cross-linking can establish that some membrane enzymes are oligomeric (such as the Na" ,K + -ATPase, the sarcoplasmic reticulum CaH -ATPase and the "complexes" of the mitochondrial electron transfer chain), it has not yet succeeded in establishing whether such complicated enzymes exist in the membrane as independent entities, as aggregates, or as organised multi-enzyme sequences [87]. In the case of the mitochondrial electron transfer chain, the high concentration of protein in the inner mitochondrial membrane and the stoichiometric relationship which exists between some of the components (Complex III: cytochrome c: complex IV = 1 :2: 2 [6]) suggests the presence of an organised multi-enzyme system rather than independently diffusing components. Perhaps new electron microscopic techniques will resolve this question [89]. The same arguments can be presented with respect to lipid components. The physical data imply that lipids are in general freely mobile in the lateral plane and hence would be expected to give a homogeneous lateral distribution. On the other hand regions of distinct lipid composition can occur either as a result of lateral phase separations (see Section 3b) or through interactions between specific lipids and integral membrane proteins. The existence of such specific strong interactions within membranes is a matter for some controversy at present [90,91]. The evidence for the existence of a defined lipid "annulus" around some membrane proteins is: (i) using certain probes it appears that not all membrane lipid participates in the bulk lipid phase transition, so the remainder may be firmly associated with proteins; (ii) certain isolated integral membrane enzymes require a defined minimum lipid: protein ratio to maintain activity, the minimum amount of lipid being close to that needed to form a monomolecular "shell" around the integral protein; and (iii) in intact membranes and in reconstituted systems comprising integral proteins and defined lipids, spin labels commonly give two signals implying the existence of two different lipid environments, a fluid bulk phase and a limited region of immobile lipid. Although this is not universally accepted (see e.g. [91]), these findings are generally interpreted as showing that some integral proteins are surrounded by an annulus or boundary layer of firmly bound, "immobile" lipid which exchanges only slowly with the bulk lipid. However, there is no evidence yet as to the composition of such layers in native membranes, so that it is not yet possible to give definite examples of specific lateral heterogeneity of lipid distribution. The question of the extent to which the several functional components of a multi-enzyme sequence can be associated in a membrane as a reasonably stable entity is particularly interesting in the case of the endoplasmic reticulum membrane. This membrane in liver cells is rich in enzyme activities and, in particular, contains several multi-enzyme sequences; the enzymes catalysing post-translational modifica-

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tions of proteins (see Section lb (iii», the NADH-dependent electron transfer chain responsible for fatty acid desaturation (see Section 5e), the NADPH-dependent electron transfer chain responsible for oxidation and hydroxylation of steroids and numerous foreign compounds, and the conjugating enzymes which act on the hydroxylated products of this mixed-function oxidase. Most of the interest has concentrated on the organisation of the individual electron transfer chains and possible interactions between them. The NADH-dependent electron transfer chain consists of cytochrome b., a flavo-protein known as NADH-cytochrome bs reductase, and an ill-characterised cyanide-sensitive factor. A large body of data implies that the cytochrome and its reductase are highly mobile both in microsomal membranes and in reconstituted systems (see Section 5e). The NADPH-dependent electron transfer chain comprises cytochrome P-450 and NADPH-cytochrome P-450 reductase. Several lines of evidence point to some form of separation of these two electron transfer chains in microsomes, rather than their distribution at random. Various methods of subfractionation of microsomes partially resolve the two chains from each other without separating them into their individual components [92,93]. Although it is unlikely that distinct regions wholly specialised for one function occur, these results suggest that the membrane contains functional electron transfer complexes analogous to those found in mitochondrial membranes [94]. Fractionation of solubilised microsomes can also lead to isolation of such putative complexes [95]. The evidence for organisation within each electron transfer chain is weaker. Recent rapid reaction studies have detected two rates for the reduction of cytochrome P-450 by its reductase; this has been interpreted as implying that each molecule of cytochrome P-450 reductase is surrounded by a cluster of molecules of the cytochrome, with further molecules of cytochrome P-450 slowlyexchanging with those in the cluster [96]. On the other hand, other kinetic evidence has been interpreted in terms of random interactions between fully mobile species [97]. Evidence has been sought for structural interactions between this electron-transfer chain and the membrane-bound microsomal enzymes which act on its immediate products, such as epoxide hydratase, UDP-glucuronyl transferase and a recently detected microsomal glutathione-S-transferase, but the evidence to date is fairly weak [98,99] (see Section lb). In general, the evidence on the endoplasmic reticulum indicates that there are no distinct regions in the lateral plane permanently specialised for distinct functions. Any specialisation must be on a very small scale. There may be long-term associations of components of multi-enzyme sequences, but there is also good evidence that many of these components are mobile. These opposite conclusions emphasise a conceptual problem. The model of a multi-enzyme complex based on analogy with, say, the pyruvate dehydrogenase complex, and implying fixed protein-protein interactions and structural organisation, may not be appropriate for multi-enzyme systems in membranes. The evidence that microsomal proteins involved in such systems are "mobile" is based on the fact that they move freely in reconstituted membrane systems (see Section 5e) and that they are not tied functionally to a single

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partner in the native microsomal membrane. However, this does not necessarily imply that they diffuse freely throughout the entire endoplasmic reticulum. Our lack of techniques for detecting preferential interactions between protein components of membranes and for mapping lateral dispositions makes this a major area of uncertainty in membrane enzymology at present.

4. Purification and reconstitution (a) Solubilisation

For most detailed characterisation it is essential to obtain membrane-bound enzymes as homogeneous preparations in solution, as it is for other enzymes. This approach has some theoretical problems (see Section 2c) and even greater practical ones. These problems are minimal in the case of "extrinsic" membrane-bound enzymes, which, by definition, can be detached from membranes and brought into aqueous solution using mechanical disruption or particular conditions of pH, ionic strength, EDTA etc. The subsequent purification and characterisation of such enzymes presents no special problems, although it is possible that aspects of their function affected by association with the membrane may be overlooked. Examples of such enzymes are the erythrocyte G-3-PDH (Section 2a) and the soluble "F."-ATPase from inner mitochondrial membranes. The latter is the extrinsic portion of the complete energy-transducing mitochondrial ATPase and can be released from the membrane by a variety of treatments; its properties are distinctly different from those of the complete enzyme (see Section 5f). The real difficulties in solubilisation are presented by the intrinsic membranebound enzymes which, by definition, can only be solubilised in conditions which disrupt the integrity of the membrane as a whole. In such conditions, the strong and specific interactions between a membrane-bound enzyme and the other protein and lipid components of the membrane may be ruptured, and the structure and activity of the membrane-bound enzyme itself are likely to be affected. Some useful characterisation can be carried out on inactivated enzymes (primary structure, structural domains, raising of antibodies etc.) but without their activity, enzymes are difficult to identify. The strange story of mitochondrial structural protein [100,101] -a preparation consisting mainly of denatured mitochondrial ATPase-is a warning against working with inactivated membrane-bound enzymes. So the priority in much work on solubilisation has been to find treatments which will render the enzymes both soluble and active. In addition, it is important that the solubilised enzymes be separable from each other by reasonably convenient techniques. So the full criteria for a successful solubilising agent or solubilisation procedure for membrane-bound enzymes are that in the resultant preparation the enzymes should be soluble, separable and active. These criteria are hard to meet and the problem is not simply methodological. Intrinsic membrane-bound enzymes are adapted to function within a membrane

Membrane-bound enzymes

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environment; their tertiary and quaternary structures and the pattern of arrangement of polar and non-polar surface regions allow them to interact with other membrane components in a defined way, and the tertiary and quaternary structures are reciprocally stabilised by these interactions. Free in aqueous solution, such proteins might be expected to aggregate, precipitate or unfold from their native state. The aim in solubilisation is to provide an environment for the solubilised enzyme which itself can contribute the essential interactions necessary to maintain the enzyme in its native state without the formation of a macroscopic sedimentable particle. The main agents which have been used to solubilise intrinsic membranebound enzymes are organic solvents, phospholipases and detergents; non-ionic detergents and bile salts are, by some way, the most successful at solubilising with retention of activity. Organic solvents were extensively used in early studies on membrane proteins, as they are the conventional reagents for solubilising membrane lipids. In general, treatment of a membrane with alcohols, chloroform, hydrocarbons etc. tends to leave the membrane proteins as an insoluble aggregated residue with little surviving enzyme activity. Some enzymes may be extractable from such a residue into aqueous solution (as in the classical purification methods involving preparation of an "acetone powder") but these are often enzymes which could be solubilised by other techniques. Some enzymes are soluble in an aqueous solution saturated with nbutanol, and others are actually soluble in organic solvents; these "proteolipids" include a low M, component of the membrane portion of the mitochondrial A'TPase which has been suggested to form the "H + -channel" essential for coupling ATP synthesis to the H + -translocation steps in respiration (see Section Sf). The isoprenoid alcohol kinase of S. aureus membranes (Section Sc) is also soluble in organic solvents, and can be purified in such media without irreversible loss of enzymic activity. Phospholipases have been used to solubilise membrane-bound enzymes but their frequent contamination with proteases makes them suspect as general agents for this purpose (see e.g. the case of cytochrome bs discussed in Section Se). Part of the solubilising action of phospholipases comes not simply from the destruction of membrane phospholipids but from the formation of lyso-phospholipids and fatty acids which are themselves quite effective surfactants. The most successful agents for solubilisation of membrane enzymes are detergents. The mechanism by which detergents bind to lipids and proteins and disrupt membrane structure are discussed in a detailed review by Helenius and Simons [102]. The properties of proteins solubilised by detergents have been reviewed by Tanford and Reynolds [103]. The large body of empirical experience and physicochemical theory dealing with the action of detergents on membrane proteins can only be treated briefly here. The detergents used in solubilising membrane enzymes are all soluble amphiphilic compounds and can be considered to fall into three main groups: (1) ionic detergents such as long chain alkyl sulphates and alkyl ammonium salts which form large micelles (approx. 100 molecules/micelle) at concentrations above approx. 1mM, but are present as monomers in solution at lower concentra-

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R.B. Freedman

tions, (2) salts of the naturally occurring bile acids (cholate, deoxycholate etc.), which are soluble as monomers at low concentrations but form small aggregates (less than 10 molecules) at about 10 mM and above, (3) non-ionic detergents which are poorly soluble as monomers and form large micelles at concentrations above about 0.1 mM. At low concentrations all these detergents bind to biological membranes, perturbing their structure and activity to a greater or lesser extent. At higher concentrations the detergents convert the membranes into a mixture of mixed lipid-detergent micelles and lipid-protein-detergent complexes. In the presence of excess detergent the products are small lipid-detergent mixed micelles with a high ratio of detergent: lipid, and proteins solubilised as protein-detergent complexes. The properties of the detergent-protein complexes are determined by the mode of detergent binding. Detergents of type (1), such as sodium dodecyl sulphate, bind co-operatively to most membrane proteins inducing denaturation, massive detergent binding and the formation of rod-shaped protein-SDS complexes. The bile salts and non-ionic detergents bind much less extensively and usually without inducing denaturation. Various factors contribute to this difference in behaviour; SDS and related ionic detergents have flexible non-polar groups whereas those of the bile salts and non-ionic detergents (see below) are either more rigid or more bulky, limiting the possible interactions between detergents and proteins. In addition, in the case of the non-ionic detergents the effective concentration of free detergent monomers in solution is kept low because of the low critical micellar concentration. Although the theory of detergent-protein interactions is not thoroughly understood, especially for the bile salts, the empirical experience is clear cut. SDS and analogous ionic detergents are very effective solubilising agents which delipidate most membrane proteins and dissociate most protein-protein interactions within membranes. While these detergents are excellent for dissolving, separating and enumerating membrane components they generally inactivate membrane-bound enzymes, and though reactivation has been reported in some cases [104], inactivation is usually irreversible. Bile salts are also effective solubilisers, but their effects are less vigorous than those of SDS: the protein-protein and protein-lipid interactions characteristic of the membrane are frequently retained after solubilisation (presumably because these interactions are stronger than the potential interactions between the proteins and bile salts) and membrane enzymes are often not denatured. Non-ionic detergents are generally, though not always, even gentler than the bile salts. On these grounds, non-ionic detergents are now recognised as the most suitable agents for solubilisation of membrane enzymes with retention of activity, but there are no reliable rules for their use. Partly this is because of our ignorance of the molecular organisation of most membrane enzymes and of the nature of their interactions with detergents. But it is also partly because non-ionic detergents are rather heterogeneous preparations which discourage the user from attempting to think about their action in chemical structural terms. The most widely used non-ionic detergents are polyoxyethylene derivatives of general formula R-(OCH2CH2)n-OH where R can be a straight-chain alkyl group (Brij, Lubrol), a branched-chain alkyl

Membrane-bound enzymes

191

group (Emulgen, Renex), a phenyl group with a straight-chain alkyl substituent (Triton N), or a phenyl group with a branched-chain alkyl substituent (Triton X, Nonidet). The value of n is not fixed in a given preparation but an average value is usually defined. Different preparations in a series differ in their average value of n. The overall character of these detergents is often defined in terms of the hydrophiliclipophilic balance (HLB); the most successful detergents for solubilising membrane enzymes generally have an HLB of 12.5-14.5 [102]. Factors to be considered in the choice of a detergent for solubilisation are discussed by Tanford and Reynolds [103]. (b) Fractionation and purification of intrinsic membrane enzymes

Extrinsic membrane enzymes soluble in aqueous media can be purified like conventional enzymes. Proteolipids, soluble in organic solvents, can be purified by chromatographic techniques appropriate to these solvents. But the starting point for most purifications of membrane-bound enzymes is the membrane solubilised by excess non-ionic detergent. As noted above, in such a system the enzymes are present in complexes with the detergent and these complexes often retain some of the lipids and other proteins that were strongly bound to the enzyme in the membrane. A major advantage of the use of non-ionic detergents for solubilisation is that such complexes can be fractionated by methods comparable to those used for enzymes in free solution so long as detergent is present throughout. Thus crude fractionation can be achieved quite simply by selective precipitation with ammonium sulphate or with polyethylene glycol. This' often is the first step in purification schemes for membrane-bound enzymes. The partially purified preparation can then be purified further by ion-exchange chromatography or hydrophobic interaction chromatography, a technique based on the affinities of membrane proteins for hydrocarbonsubstituted agarose beads. Affinity chromatography can also be extremely useful in resolution of detergent-solubilised membrane-bound enzymes and is the preferred method for isolation of membrane-bound receptors whose specific affinity for a certain class of ligand is their definitive biological characteristic (e.g. [105]). Lipiddetergent mixed micelles are major contaminants of solubilised membrane enzymes; they can generally be separated from protein-detergent complexes on the basis of differences in size or buoyant density using gel-filtration or density-gradient centrifugation. Enzymes solubilised by bile salts can be partially purified by selective precipitation (this is the method used in the classic preparation of complexes of the mitochondrial respiratory chain), but, for further purification, preparations dissolved in cholate or deoxycholate are often transferred into non-ionic detergents. This is made easier by the small size of bile salt aggregates and the fact that bile salts are present as monomers at quite high concentrations so that these materials are easily removed by dialysis after displacement from proteins by an excess of a non-ionic detergent. A typical purification scheme for a membrane-bound enzyme is shown in Fig. 8 [106,107]. Protein-SDS complexes can be fractionated on the basis of particle size either by

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R.B. Freedman

PROCEDURE

COMMENTS

Pre-treat rabbits with the inducer ,,-naphthoflavone

Maximises concentration of this form of cytochrome P-450 in the liver

~

Isolate liver microsomes and wash extensively with pyrophosphate buffer ~

Solubilise in cholate (cholate-protein 3 : 1 by weight) ~

Fractionate by polyethyleneglycol precipitation; 8-10% and 10-12% cuts retained

Minimises contamination with adsorbed proteins and removes some extrinsic membrane proteins Bile salts are effective solubilising agents. From this point buffers contain 20% glycerol and ;;>1 mM EDTA to stabilise cytochrome P-450

~

Resuspended pellets, dialyse and redissolve in Renex 690 (0.5%) ~

Apply to DEAE-cellulose column and elute with buffer containing 0.5% Renex ~

Apply to hydroxyapatite-silica gel column, elute successively with 0.01 M phosphate containing 0.3% Renex, 0.1 M phosphate containing 0.3% Renex and 0.3 M phosphate containing 0.1% Renex

Dialysis removes the cholate and the material is transferred to solution in non-ionic detergent; Renex 690 = polyoxyethylenet l Ojnonylphenyl ether Ion-exchange chromatography is possible in presence of non-ionic detergent; various cytochrome P-450 species partly resolved Final purification in individual cytochrome P-450 species by ion-exchange and adsorption chromatography

~

Treat with Amberlite XAD-2 and then with calcium phosphate gel, elute from gel with 0.8 M phosphate, dialyse and store.

Initial treatment reduces phosphate ion concentration; adsorption on calcium phosphate gel permits removal of the non-ionic detergent.

Fig. 8. Purification protocol for rabbit liver cytochrome P-450 (LM-4) (based on [106,107]).

electrophoresis in polyacrylamide gels (the standard technique for enumerating and estimating the M, of membrane polypeptides) or by gel-filtration in the presence of detergent. Some extremely insoluble membrane proteins can be purified by a technique which has come to be known as "reverse solubilisation"; the membrane is subjected to successive gentle extractions which solubilise most membrane proteins but selectively leave the enzyme of interest in suspension as part of the depleted membrane. This technique has been successful both with the plasma membrane Na+ ,K +ATPase and with the sarcoplasmic reticulum Ca2+-ATPase (see next chapter). Purified membrane enzymes can be studied as the protein-detergent complexes [103], but many kinds of characterisation require removal of the detergent. This is often difficult to achieve since .the free proteins generally aggregate in aqueous solution. It is easier, in fact, to exchange detergent for detergent or lipid for detergent than to isolate the protein completely. This introduces the topic of reconstitution.

Membrane-bound enzymes

193

(c) Reconstitution

All "reconstituted" membrane enzymes, whatever their composition and however they are prepared, are models of the native enzyme in its membrane environment. In general, the term reconstitution is used to refer to procedures for combining solubilised membrane enzymes with natural lipids to yield an active product. Some types of reconstitution experiment are intended to probe the apparent lipid requirements for membrane enzymes (see Section 3a); others are intended to produce simple model systems in which the properties of the enzyme can be studied in a system comprising only well-defined components and in the absence of interference from other functions of the same membrane. Most forms of reconstitution involve combining the purified enzyme protein with lipids- either a total membrane lipid extract, or a single lipid class or defined lipid mixture, or even with synthetic and totally defined lipids. The variables in these procedures are then the physical state of the added lipid and the techniques used for removing detergent. In some cases, useful information can be derived by studies in which the purified enzyme is not combined with defined lipids but with the natural membrane from which it was derived. This type of reconstitution includes the experiments discussed above on the binding of extrinsic G-3-PDH to variously treated preparations of red cell membranes and studies on the functional interaction of added exogenous cytochrome bs with microsomal membranes (Section 5e). Much of the effort in reconstitution studies of membrane enzymes has been directed to reconstituting vectorial functions of enzymes involved in translocation processes, and reconstitution of integrated multi-enzyme sequences. Clearly these are the most demanding functions of membrane enzymes to simulate in a model system; they will be considered below. But a considerable body of work has also been carried out on recombining solubilised enzymes with natural lipids to regenerate simple catalytic activities alone. (Examples are considered in Sections Sa, b, c, and d.) For many membrane enzymes this probably only requires conversion of the solubilised enzyme to an appropriate enzyme-lipid complex. In most cases the formation of such a complex requires the displacement and removal of the detergent to which the enzyme was previously bound, but a handful of intrinsic membrane enzymes are soluble in aqueous solutions in the absence of detergents: this simplifies the process of reconstitution considerably. The classic example of this simplest form of reconstitution is the mitochondrial enzyme ,8-hydroxybutyrate dehydrogenase studied by Fleischer and colleagues [108112]. The enzyme can be solubilised in the absence of any lipids or detergents by treatment of mitochondria with phospholipase A and then extracting the membranes with solutions of high ionic strength. The purified soluble enzyme exists as monomer and dimer in dilute solution but aggregates at low ionic strength. It is only active after addition of phospholipids; phosphatidylcholine is the only individual lipid to reactivate, but more efficient reactivation is produced by mixtures of lipids including phosphatidylcholine and especially by total mitochondrial lipid which comprises 40% phosphatidylcholine. Activation requires a brief (5-10 min) preincubation of

194

R.B. Freedman

the soluble enzyme with the lipid which is added as an aqueous microdispersion. The membrane-bound enzyme and lipid-activated soluble enzyme are very similar in kinetic parameters and mechanism; differences in the composition of added lipid are reflected in differences in dissociation constants for the leading substrates NAD + and NADH. The apoenzyme, in the absence of any added lipid, is unable to bind these substrates. The conclusions from this body of work are that the purified enzyme binds phospholipids to form a lipoprotein complex which can bind the nicotinamide coenzymes and is enzymatically active. Reactivation is marked at quite low ratios of lipid to enzyme (2-5 mol phosphatidylcholinejmol enzyme subunit) and there is no evidence for the formation of a lipid bilayer. The enzyme thus reconstituted as a lipoprotein complex closely resembles the native enzyme in kinetic properties. In the majority of cases reconstitution involves the displacement of the detergent which maintains the solubilised enzyme in solution. This is usually achieved by addition of an excess of the required lipid followed by some treatment to separate the enzyme-lipid complex from detergent molecules and micelles; the common techniques exploit the larger size of the enzyme-lipid complex and use dialysis, gel-filtration or centrifugation. The main practical problem in such work is the slow rate of exchange found with the major non-ionic detergents, which is related to their limited solubility as free monomers. For this reason, solubilised enzymes are often transferred into solution in the more rapidly displaceable bile salts before reconstitution; alternatively bile salts may be added to the reconstitution mixture as "catalysts" of exchange. In a large number of cases the initial solubilisation in bile salts or non-ionic detergents does not completely dilipidate the membrane-bound enzyme; in these cases extensive dilution to reduce the detergent contentration often leads to reformation of membranous structures. Such procedures are simple and convenient and were most important in studies on the reconstitution of the mitochondrial electron transfer chain, but they do not allow control of the lipid content of the reconstituted enzyme. The central problem in many reconstitution studies is to combine enzyme and lipid in circumstances where a bilayer-type membrane can form, and (in cases where vectorial function is important) to obtain preparations in which the enzyme has a defined orientation with respect to the bilayer. It is not possible to design procedures from first principles to achieve these results, since the principles themselves are not clearly understood, but there is now a good deal of accumulated experience and a number of quite successful methods are available for reconstitution of functional membrane processes in artificial lipid vesicles [113-115]. No method is universally successful and each has its drawbacks. The detergent-dialysis procedure involves addition of the solubilised enzyme to a preparation of lipids which has been sonicated in the presence of a detergent (usually cholate), followed by dialysis to remove detergent. The method is timeconsuming and involves prolonged exposure of the protein to the detergent but it has been widely used. The dialysis step can in some cases be replaced by gel-filtration, or simply by dilution; although they reduce the exposure to detergent these tech-

Membrane-bound enzymes

195

niques are successful in fewer cases. The sonication procedure involves simply sonicating the enzyme preparation with the added lipids; it is rapid and does not involve detergents but reproducibility is poor and it is often difficult to establish and maintain optimal conditions. The incorporation procedure involves adding the enzyme to preformed liposomes; incorporation of the protein occurs in the presence of low concentrations of cholate and lysolecithin, or in their absence if the liposomes themselves contain a significant proportion of acidic phospholipids. In none of these methods is it possible to control the orientation in which vectorial enzymes are inserted into the vesicles. With some enzymes and some procedures, a clearly preferential orientation is obtained and the reconstituted system can be treated as a vectorial enzyme preparation. The Na+ ,K + -ATPase reproducibly inserts "wrong-way-round" into lipid vesicles, so that it hydrolyses external ATP, pumps Na+ ions into the interior of the vesicle and is only inhibited by ouabain which is trapped inside the vesicles. But in some cases where significant amounts of both orientations are produced it is important to be able to establish the proportions of the two orientations and, if possible, to remove or inhibit the vesicles containing the enzyme in the undesired orientation. Inhibition by antibodies directed against external facing components is a useful procedure in such cases. The point to emphasise in discussing these methods of reconstitution is that the rates of lipid binding and exchange are strongly dependent on the physical state of the lipid, the degree of aggregation of the protein, the amount of endogenous bound lipid and the nature and concentration of detergents present. The protein and lipids combine but do not come to equilibrium. The products are therefore meta-stable states whose nature is determined by the details of the experimental procedure. Most of the emphasis in reconstitution studies has been on forming homogeneous systems-lipid/protein complexes or vesicle dispersions. But interesting work has also been done using other model systems, principally monolayers at the air-water interface (see the bacterial UDP-sugar transferases, Section 5d), and planar bilayer lipid membranes (BLMs). Planar BLMs have for a long time been the preferred model lipid bilayer system for biophysical and electrical studies, and methods are now available for forming such membranes from mixtures of lipids and proteins [116].

5. Examples (a) Bacterial phosphoenolpyruoate.sugar phosphotransferase systems

In many obligate and facultative anaerobic bacteria, a group-translocation system catalysing a coupled chemical transformation and translocation is the main mode of uptake of sugars. This process is energetically attractive for organisms which obtain the bulk of their energy from glycolysis. One molecule of the phosphate donor phosphoenolpyruvate (PEP) is required to bring about both transport and phosphorylation of one molecule of sugar substrate, whereas conventional active transport and metabolic phosphorylation would consume two molecules of ATP (or their

196

R.B. Freedman out

in

pyruvate

e

Ip -HPr[---------..

n

~ !

m

e

PE P - - - - ' - - - - -

sugar phosphate

]]

B

sugar

Fig. 9. Outline of the PEP:sugar phosphotransferase system of group translocation.

equivalent). The sugars transported by this mechanism differ from organism to organism and the detailed composition and function of the system also differs from organism to organism (and to some extent from sugar to sugar). There have been a number of recent excellent reviews of phospho transferase systems which include accounts of the physiological and genetic aspects [117- 119]. Here the structural aspects will be summarized briefly. All phosphotransferase systems involve a set of soluble proteins and a set of membrane-associated proteins (Fig.9). The soluble proteins are involved in a series of phosphate-transfer steps; they comprise a heat-stable small protein known as HPr and an enzyme known as enzyme I which catalyses phosphate transfer from PEP to N 1 of a histidyl residue of HPr. These proteins are usually constitutive and they have no sugar specificity; in fact they are not directly involved in sugar phosphorylation or translocation. The remaining components of the system are generally known as enzyme II, although in the well-characterised cases it is clear that more than one enzyme is involved; they are membrane-associated and responsible for phosphorylation and translocation of the sugar. The enzyme II components are often inducible and sugar-specific so that a single organism may contain several different enzyme II systems. The best characterised of these are those in E. coli responsible for translocation of glucose, fructose and mannose. Washed membranes from E. coli catalyse phosphate transfer from phospho-HPr to any of these sugars without addition of any other factor. Evidence from work with mutants and variously induced organisms shows that this phosphotransferase reaction is brought about by the system which catalyses vectorial phosphorylation (i.e. phosphate transfer and translocation) in whole cells. The molecular characterisation of this system has given some information on the components, their interactions and the phosphotransferase reaction, but not, as yet, on the translocation process. The systems have been reconstituted to give effective phosphate transfer in a non-vectorial reaction, but the coupled group-translocation has not yet been reconstituted.

Membrane-bound enzymes

197

The composition of these membrane-bound "enzyme II" systems has been established by selective extraction of the E. coli membranes. Components known as enzyme IIA are solubilised with butanol and urea but a component known as enzyme lIB can only be solubilised with detergents. Both fractions have been purified to some extent. There appear to be distinct forms of enzyme IIA, each specific for glucose, mannose or fructose, but there is probably only a single form of enzyme lIB. Enzyme IIA proteins are reasonably soluble in aqueous media, whereas enzyme lIB aggregates in the absence of detergents. The phosphotransferase activity can be recovered by mixing the resolved components in the presence of lipid and divalent metal ions. The conditions of reconstitution are critical. When various phospholipids were added as lipid-Triton X-lOO mixed micelles (molar ratio 5: 1) only phosphatidylglycerol was effective in regenerating enzyme activity. This lipid must be preincubated with enzyme lIB in the presence of divalent ions (Ca2+ or Mg2+) before addition of enzyme IIA, phosphoHPr and the sugar. The preincubation leads to formation of sedimentable enzymelipid complexes which are amorphous in structure rather than lamellar; the optimal enzyme-lipid molar ratio is 1: 50. The conditions for reconstitution are interesting. Divalent metal ions are essential for reconstitution of many membranous systems [120], but their role is not clear. The specific requirement for phosphatidylglycerol is interesting in that PG is only a minor component of the E. coli membrane lipids; the major lipid, phosphatidylethanolamine, was ineffective. This has been claimed to indicate a specific PG-requirement for the enzyme in the native membrane [118]; however, the arguments mentioned in Section 3a, 4c make it possible that it only reflects more rapid and complete complex formation with the acidic PG. The properties of the individual components suggest that there is initial phosphate transfer from phospho-HPr to enzyme IIA and that enzyme lIB catalyses the final transfer of phosphate to the sugar. The fact that the complete vectorial translocation system has not yet been reconstituted makes it impossible, at this stage, to speculate on the mechanism of the coupled chemical reaction and translocation catalysed by enzyme lIB; however, the fact that the ultimate phosphate donor is a membrane-associated protein is clearly significant. (b) Hormone-sensitive adenylate cyclases

Adenylate cyclase is .widely distributed in the plasma membranes of cells of higher organisms, where it is activated by any of a large number of hormones, depending on the cell in question. It is the classic example of a membrane-bound enzyme with a function in communication; the hormones, including polypeptides and catecholamines, can activate without entering the target cell, whereas the enzyme's substrates and products are also impermeant but are intracellular. So the hormonally controlled enzyme functionally spans the membrane and determines the intracellular concentration of cyclic AMP in response to the extracellular concentrations of hormones. The molecular analysis of this transmembrane communication system has drawn

198

R.B. Freedman

on a number of experimental systems including, in particular, the glucagon-responsive enzyme in liver plasma membranes, the adrenaline-responsive enzyme in heart muscle, the fat cell enzyme which responds to numerous hormones and the adrenaline-responsive enzyme in avian erythrocytes. Early work on the hormone-sensitive adenylate cyclases has been reviewed [105,121,122]. This work led to the following picture. (l) The hormone-sensitive cyclase is the molecular machinery of primary hormone action in these tissues. In most cases concentrations for half-maximal activation of cyclase in broken cell preparations correspond to concentrations giving half-maximal physiological response in the intact tissue; there is good correlation between the potency of hormone analogues in intact tissues and their ability to activate adenylate cyclase, and the kinetics of physiological effect and enzyme activation also correlate. (2) The hormone-binding site is extracellular and clearly distinct from the enzyme active site, so that the system operates, in effect, as a membrane-spanning allosteric enzyme (but see below). Evidence for this picture is provided by the fact that the enzyme is very easily "desensitised" by solubilisation, or by various treatments of the membrane. For example, when fat cells are briefly incubated with trypsin they lose the glucagon stimulation of their adenylate cyclase activity, but not the basal cyclase activity or the stimulation of cyclase by F - ions. However, when fat cell ghosts (plasma-membrane fragments) are treated in the same way, all adenylate cyclase activity is lost. This experiment confirms that the glucagon effector site is extracellular, that the active site is intracellular and that glucagon and F - influence activity via different routes. (3)Hormone binding and enzyme activity are functions of distinct protein components. After solubilisation, hormone-binding and enzymically active components can be resolved from each other, implying that different proteins are responsible for these distinct functions. Furthermore, in cells responsive to many hormones, the same active enzyme components are involved in the response to each hormone; thus although several different hormones alone can activate adenylate cyclase in fat cells and they operate through distinct receptors (binding is not competitive) their effects are not additive when they are all present together. (4)Lipids are clearly important in the transduction process linking hormone binding to cyclase activation. Early work with lipases and detergents showed that hormone binding and hormone activation of cyclase were easily destroyed in conditions which did not affect basal cyclase activity or stimulation by F - . More specific work with purified lipases [123] suggests a specific role for acidic phospholipids; phospholipase C from B. cereus, which is specific for acidic phospholipids, destroys glucagon response of liver plasma-membrane adenylate cyclase without affecting basal activity while the corresponding enzyme from C. perfringens, which is relatively more specific towards zwitterionic phospholipids, has no such effect. These findings on the separateness of hormone-binding and active sites and on the influence of lipids can be rationalised in terms of the fluidity of plasma membranes. Rather than being pictured as "regulatory" and "catalytic" components of a multi-component enzyme with a permanent static organisation, the hormone-

Membrane-bound enzymes

199

binding and catalytic components of the system are viewed as independent mobile elements located respectively in the outer and inner halves of the membrane lipid bilayer. This "mobile receptor" hypothesis [124] has recently received support from two kinds of experiment. (i) Cell fusion. Cells containing adrenaline receptors but no adenylate cyclase (turkey erythrocytes in which the cyclase had been selectively and irreversibly inactivated) were fused with cells containing adenylate cyclase which was not responsive to adrenaline (Friend erythroleukaernia cells); within a short time adrenaline-stimulated adenylate cyclase activity was detectable in the fused cell population [125]. This experiment has now been repeated with a number of cell pairings and the finding is quite general (for review, see [126]); the receptors and active enzyme component are clearly independent mobile species. (ii) Radiation inactivation. The M, of the receptor and catalytic components of the system have been estimated in the intact membrane using the technique of radiation inactivation which indicates the size of target presented by the system under study to an electron beam generated by a linear accelerator [127]. When rat liver cell membranes were irradiated in the uncoupled state (no glucagon present) the apparent Mrs were 217000 for the glucagon-binding component, 160000 for the adenylate cyclase component and 389000 for glucagon-stimulated adenylate cyclase. Irradiation in the presence of glucagon gave Mrs for all the systems in the range 300000-400000. These findings suggest that the glucagon-receptor and cyclase-catalytic component are separate entities which combine in the presence of glucagon to form a 1: 1 complex. More recent data [128] suggest that the situation is probably more complicated than this, but they are still consistent with the "mobile receptor" model. It has been known for some time that intracellular GTP is essential for hormone activation of adenylate cyclase, and this has generally been pictured as a standard allosteric effect, but until recently no clear molecular picture emerged. Now, however, a model of this effect has been presented [129] based on the discovery of hormone-stimulated GTPase activities, and the effects of non-hydrolysable GTP analogues. In summary, the model is as follows. Adenylate cyclase is activated when GTP, or an analogue, but not GDP, is bound to a specific protein (Mr 42000) activator of the cyclase. The hormone and GTP bind independently to distinct protein subunits of the complete hormone-sensitive adenylate cyclase. The binding of hormone also stimulates a GTPase activity which is probably catalysed by the GTP-binding component of the cyclase system. When hormone and GTP are simultaneously present bound to their effector sites, the cyclase becomes activated, but so does the GTPase, which converts GTP to GDP and hence returns the cyclase to the inactive state. In the presence of GTP, therefore, the level of cyclase activation depends on the relative rates of activation and GTP hydrolysis. But in the presence of non-hydrolysable analogues (GppNHp or GTPyS) the cyclase is converted into a permanently activated state. The specific role of the hormone is to enhance the rate of displacement of GDP from the guanine nucleotide effector site. This model has been vindicated from an unexpected source. The well-known action of cholera toxin, which permanently activates adenylate cyclases from practically any source, has been shown to derive from its ability to inhibit the GTPase component, so that

200

R.B. Freedman

L TTTTT~TT __ TTTTTTTTT[3jTT ----L. TTUT. TTTTTTT 1111111 0]1 ( \ l1111Ull 11[]]llllllil ) 11111111[;1 TT~TTTTTTTT Inactive

h

GTP

h

GOP

Active

h

Active

h

I

rnocnve

h"'" hormone

r = receptor component c = catalytic component

Fig. 10. Summary scheme of properties of hormone-dependent adenylate cyclase complex.

activation by GTP becomes irreversible [130]. All these aspects are summarised in Fig. 10. A final question concerns the precise mode of coupling. Given that the hormonebinding unit and the catalytic unit are independently mobile in the absence of hormone, what events follow hormone binding? Do these units then combine to form a coupled unit which remains intact for as long as hormone is present? This was implied, though not stated, in the earliest formulations of the "mobile receptor" model, but very detailed kinetic and binding studies [129,131] suggest an alternative model in which collision between the hormone-receptor complex and the catalytic unit is essential for activation, but permanent coupling between the components is not required. The complete formulation of the model is then: E + GTP

+=%

E.GTP

H + R +=% H.R.

~ (HR.E.GTP +=% HR.E* .GTP) -> HR + E*GTP -> E + GDP + Pi

where E is the enzyme catalytic component, H the hormone, R the receptor component and E* is the active form of the catalytic component. Sophisticated models of this kind make it clear that the lipid dependence observed in early work on this enzyme cannot have a simple interpretation. Much of the lipid dependence may reflect the requirement for fluidity in the membrane to facilitate interaction between hormone-binding and catalytic components. For example, it has been shown [132] that the "collision" between hormone-receptor complex and catalytic unit is diffusion-controlled and increases linearly with membrane fluidity. This kind of model also emphasises that the functional aspects of the system are intimately dependent on the membrane environment and will be difficult to simulate in solubilised systems. (c) C55-isoprenoid alcohol phosphokinase

The phosphate ester of the highly lipophilic isoprenoid alcohol, bactoprenol, functions as a lipid carrier in bacterial cell wall biosynthesis. The enzyme which catalyses

R.B. Freedman

201

formation of this phosphate ester is a highly hydrophobic intrinsic membrane protein, which is soluble in organic solvents such as n-butanol (see [133] for review). This property has been exploited to purify the enzyme to homogeneity from S. aureus membranes; the enzyme is extracted from the membranes with acidic butanol, together with most of the membrane lipid and is then purified and freed from lipids by selective precipitation, gel-filtration and ion-exchange chromatography-all carried out with the enzyme dissolved in n-butanol or methanol. The homogeneous product, which has a M, 17000 subunit, has an unusually high proportion of non-polar amino acids (approx. 60%), and is very stable, which has simplified its characterisation. Although the enzyme is purified in non-polar solvents, its activity cannot be demonstrated in such solvents. For assay, the enzyme is added to the Css-isoprenoid alcohol substrate and to some amphiphile (phospholipid or detergent) and the organic solvent is evaporated off. The residue is then dispersed in aqueous medium and ATP is added to initiate reaction. No activity is observed in the absence of a lipid activator or analogue. This provides an interesting system for examining systematically the requirements of the enzyme for activation and the precise role of the activator [134]. The nature of the complex formed in these assay conditions has not been precisely defined, but in the presence of Triton X-lOO the enzyme can be obtained dispersed in Triton-phosphatidylcholine-enzyme mixed micelles which have a similar activity [135]. Many natural phospholipids activate the enzyme when present at 1mM. Egg phosphatidylcholine is more effective than any purified bacterial lipids, but lysophospholipids are even more effective. However, the effectiveness of the various lipids is influenced by the temperature at which the preincubation of the enzyme, lipid and isoprenoid alcohol is carried out, and many ineffective lipids become effective in the presence of low concentrations of deoxycholate or Triton X-lOO, which are not effective alone. This suggests that fluidity of the lipid activator is important for reactivation. This is confirmed by the fact that in any fixed conditions pure phosphatidylcholines containing short fatty acyl groups are more effective than those with longer chains, and the fact that dioleoylphosphatidylcholine is an effective activator at 25°C, while its saturated analogue distearoylphosphatidylcholine is not. The effectiveness of long-chain, saturated phosphatidylcholines to activate is a function of the assay temperature; activity is only seen above the temperature at which the isoprenoid alcohol-activator lipid mixture begins to undergo the gel-liquid crystal phase transition. In these reconstituted systems, the activating lipid is in great molar excess (> 1000) and there is no evidence for the formation of a defined complex. Activation appears to depend on two factors, namely fluidity of the lipid and the extent of hydration of the lipid headgroups. The role of fluidity is confirmed by spin label studies on the Triton-lipid-enzyme mixed micelles described above. The rate of rotational motion of the spin probe varies with the nature of the lipid as does the extent of kinase activation; there is a good correlation between these two parameters. Likewise, when the fatty acyl group is held constant through the use of a series

202

Membrane-bound enzymes

of oleoyl derivatives, but the headgroup is varied, there is a good correlation between kinase activation and the water binding ability of the activating lipid [4]. The fact that both the mobility properties of the non-polar region and specific features of the "headgroup" are important in activation is also shown by a study of the use of synthetic non-ionic detergents as activators [136]. The acyl esters of sorbitan (Span series) are the most effective, but again activation depends on the presence of short or unsaturated fatty acyl groups, except at the highest assay temperatures. In general, the best activators are rather hydrophobic detergents; work with mixed detergents indicates a hydrophile-lipophile balance of 7 to be optimal. Polyoxyethylene detergents such as Tritons and Brijs are poor activators in this system. (d) Glycosyltransferases of bacterial cell-wall lipopolysaccharide synthesis

The characteristic lipopolysaccharide of Gram-negative bacteria (LPS), responsible for O-antigen specificity, consists of a non-polar lipid core to which a branched chain of carbohydrate groups is attached (Fig. 11). The enzymes which catalyse incorporation of these carbohydrate groups are located in the bacterial inner membrane and use intracellular UDP-sugars as substrates. Mutants defective in the synthesis of the carbohydrate portions of LPS are known, so that incomplete LPS can be isolated for use as acceptor in studies on individual sugar transferase enzymes. Rothfield and colleagues have studied two such enzymes in particular, the UDP-galactose:LPS galactosyltransferase and the UDP-glucose:LPS glucosyltransferase (see [137] for review). Incomplete LPS lacking galactose residues is not in itself a substrate for the galactosyltransferase; phospholipid is required to form a complex with the acceptor

I-Core oligosaccharide-e--i P

I

Hep Gal GlcNAc I

I

I

(KDOh - Hep-Hep -Glc-Gal-Glc-O-sidechain Itt L P I KDO, 2-keto-3-deoxyoctonic acid P Hep, L-glycero-D-mannoheptose I Glc, glucose D GlcNAc, N-acetylglucosamine Gal, galactose A Arrows denote the bonds formed in reactions catalysed by the enzymes described in the text Fig. II. Outline structure of the cell surface lipopolysaccharide (LPS) of Gram-negative bacteria.

Membrane-bound enzymes

203

LPS. The most effective natural phospholipid is phosphatidylethanolamine (PE). A complex of PE and LPS can be formed and isolated by density gradient centrifugation, and can interact with purified galactosyltransferase to form a ternary complex which can also be isolated. This ternary complex is enzymically active and will incorporate labeled galactose from UDP-gal. Similar complexes can be formed with glucose-deficient LPS, glucosyltransferase and PE [138] and a quaternary complex containing both enzymes and capable of successive incorporation of glucose and galactose can also be isolated [139]. The enzymes in this system are extrinsic membrane enzymes and the primary role of the activating lipid appears to be to modify the conformation or other properties of the LPS acceptor. Similar conclusions can be drawn from the reconstitution of this system in monolayers at air-water interfaces. Formation of an enzymically active monolayer requires the sequential addition of the components in the following order: (i) PE to form the initial monolayer, (ii) LPS injected into the aqueous phase, (iii) enzyme injected into the aqueous phase. PE is the only component which alone can form a monolayer, but if the enzyme is added next, LPS will not subsequently penetrate the monolayer. Divalent cations, preferably Mg2+ , are required for interaction between the enzymes and the binary PE/LPS monolayer. In this work, the incorporation of each of the components into the monolayer can be recorded as a change in surface pressure, indicating that some molecular rearrangement of the components of the monolayer accompanies incorporation of the new component. By this means enzymically active ternary monolayers (containing galactosyltransferase) and quaternary monolayers (containing both glucosyl- and galactosyltransferases) can be obtained. Labeled sugars are incorporated into the monolayer following injection of labeled UDP-sugars into the aqueous phase. The monolayer in these studies is an interesting model of the natural membrane, and the kinetic parameters of the enzymic activity are similar in the intact membrane, the reconstituted complex in the dispersed state, and the monolayer. More recent work has shown that the effect of the lipid activator in these systems is not simply on the LPS. Fluorescence energy transfer studies on galactosyltransferase labeled with pyridoxal phosphate indicate that the enzyme protein itself undergoes a conformational change on incorporation into the ternary PE-LPS enzyme complex, and that the conformation is sensitive to the nature of the lipid present [140]. So not only does the lipid activator modulate the properties of the high M, LPS substrate, but the lipid-LPS complex modulates the conformation, and possibly hence the activity, of the enzyme. (e) Cytochrome b, and the cytochrome bs-linked microsomal electron transfer chain

Cytochrome bs is a haemoprotein which is present in high concentrations in the endoplasmic reticulum of liver cells and also in outer mitochondrial, nuclear and peroxisomal membranes. In the endoplasmic reticulum it is involved in an electron transfer chain responsible for the desaturation of saturated fatty acids [141-143].

204

R.B. Freedman NADH~,".cyt. bs

NAD +

~

reductase

~

cyt.

b

S ~

d

esaturase

~02

~ ~

+ fatty acyl-CoA

unsaturated fatty acyl-CoA

The flavoprotein, cytochrome bs reductase, and the cytochrome itself have both been extensively characterised and their interaction has been studied in a variety of reconstituted and model systems; by contrast, very little is known about the desaturase component except that it is cyanide-sensitive and contains a non-haem iron group. In the endoplasmic reticulum membrane, cytochrome bs is present in considerable molar excess (lO-fold to 30-fold) over its reductase. The active sites of both these enzymes are located on the cytoplasmic face of the membrane; there is good evidence for this from the fact that these enzymes show no latency in intact microsomes, that their activity in intact microsomes is inhibitable by added antibodies, that the enzymes are easily released with proteases (see next paragraph) and from electron microscope studies on the location of ferritin-labeled antibodies [6,94]. The aspect of cytochrome bs and its reductase which has generated the greatest interest is their clearly amphipathic structure. Both consist of a globular N-terminal domain with a relatively polar amino acid composition and containing the haem or the flavin group, and a smaller distinctly non-polar C-terminal domain. The domains can be separated and isolated because the linking regions are particularly susceptible to proteases. The earliest work on cytochrome bs used treatment with trypsin or impure lipase preparations to release the protein from microsomal membranes. The product was a soluble non-aggregating protein of M, 11000 which did not readily rebind to membranes. Subsequently cytochrome bs was solubilised with detergents and it became clear that the detergent-sol ubilised material differed in several properties from that previously characterised and had a higher M, (l6700). Brief treatment with trypsin converted the detergent-solubilised material into a form very similar to that solubilised by lipase or proteases, indicating that this was in fact a proteolytic degradation product of the intact cytochrome bs corresponding to the hydrophilic catalytic domain. Similar evidence showed that cytochrome bs reductase had the same basic organisation [144-149]. The clear-cut amphipathic structural organisation of these proteins suggests that the domains can be assigned distinct functional roles. The soluble hydrophilic domains can function catalytically with soluble model substrates. However, they do not bind to microsomal or other natural membranes or to phospholipid dispersions, and have no affinity for non-ionic detergents [146,150-153]. Intact cytochrome b., by contrast, binds to microsomal membranes and to other sub-cellular fractions [154], can easily be incorporated into phospholipid vesicles and interacts strongly with detergents. Studies on the isolated non-polar C-terminal domain (residues 91-133) confirm the conclusion that it is this region which is responsible for the membrane, lipid and detergent interactions of the intact cytochrome [155]. It is therefore reasonable to claim that cytochrome bs and its reductase both consist of a domain responsible for catalysis and a domain responsible for membrane-attachment. The conclusion that the non-polar domain of cytochrome bs has the specific

Membrane-bound enzymes

205

function of anchoring this protein to the endoplasmic reticulum membrane has focused attention on the nature of this interaction. Several points are noteworthy. Firstly, the binding is not to specific sites; exogenous cytochrome bs can be bound to microsomal membranes in great quantity until it comprises 20% of the total microsomal protein and is in 100-fold excess over cytochrome b, reductase [146,150]. The cytochrome can also bind to other sub-cellular membranes, including inner mitochondrial and Golgi membranes which do not normally contain cytochrome bs [154]. Secondly, the intact cytochrome is readily incorporated into liposomes [156,157]. Work with spin-labeled lipids implies that a small number of lipid molecules interact strongly with membrane-bound cytochrome bs and are immobilised [151], but in vesicles containing a low ratio of cytochrome: lipid there is no gross effect of the protein on bulk lipid properties, such as the phase transition temperature [157]. A study with various carboxypeptidase-treated derivatives of bovine cytochrome bs was carried out to determine the residues of the non-polar domain which are "essential" for membrane-attachment [158]. Surprisingly, derivatives in which residues 128-133 and 116-133 had been removed bound to dimyristoyl-lecithin vesicles at the same rate and to the same extent as native cytochrome b., However, a derivative in which residues 107-133 had been removed did not bind to such vesicles at all. In this last derivative, the "C-terminal domain" is reduced to only 16 residues, of which 7 are polar. It is not clear whether residues 107-115 playa specific role in membrane attachment or whether the requirements are less specific but a certain minimum size and hydrophobicity are required for a functional membrane-binding domain. In view of the fact that the solubilised catalytic domains of cytochrome bs and its reductase are fully active with soluble substrates what is the function of the non-polar domains? It is unlikely that these domains lead to formation of specific long-term complexes between cytochrome bs and cytochrome bs reductase molecules. Several lines of evidence argue instead for free mobility of these species in microsomal membranes [159]. Firstly, in experiments where exogenous cytochrome bs is added to microsomes, all the added molecules are reducible by endogenous cytochrome bs reductase and there is no evidence for slow and fast phases of reduction, so that endogenous and added cytochrome must have equally free access to reductase molecules. Secondly, when most of the reductase in microsomes is selectively inactivated the fraction remaining is still capable of reducing all the cytochrome molecules present. The temperature dependence of interaction between cytochrome bs and its reductase in microsomes at temperatures above OCC is also consistent with a model in which the two components diffuse freely and independently in the lateral plane of the membrane [160]. Some of the most striking evidence on the role of the membrane-binding domain has come from studies on reconstituted systems involving cytochrome bs and the reductase bound to phospholipid vesicles. These studies show that attachment of the component proteins of the cytochrome bs -linked electron transfer chain to a membrane ensures appropriate orientation of the components for efficient electron transfer from reductase to cytochrome to the terminal desaturase. This was first

206

R.B. Freedman

hinted in crude experiments which showed that electron transfer from reductase to cytochrome in delipidated microsomes was much slower than that in native membranes and could be stimulated by lipid addition [161]. In more sophisticated reconstitution experiments it was first shown that this electron transfer required a fluid lipid phase. This was deduced from studies on the temperature dependence of the reaction

>

NADH NAD +

cyt.

s, reductase ~ cyt. bs

-
and of reactions representing the individual electron transfer steps [156,162]. The overall reaction in dimyristoyl-lecithin vesicles showed a break in the Arrhenius plot

Fig. 12. Influence of orientation and disposition on rate of electron transfer between cytochrome bs and its reductase.

Membrane-bound enzymes

207

at a temperature corresponding to the bulk phase transition in these vesicles, while the individual enzyme activities showed no such break. While the individual enzymes can function below the bulk phase transition, the overall reaction apparently requires a fluid lipid phase permitting lateral diffusion to bring the component enzymes together. An important finding from such reconstitution experiments is that rapid reaction between reductase and cytochrome requires that they be present in the same membrane vesicle [163]. Free cytochrome bs and free catalytic domain are reduced much less readily (4.5% and 13% respectively) by vesicle-associated reductase than is cytochrome bs incorporated together with reductase in a vesicle. But reaction is even slower between reductase and cytochrome when they are incorporated into separate vesicles; most of the reduction observed in such a case depends on protein movements from one vesicle to another (Fig. 12). So membrane binding of these proteins not only increases their local concentrations but ensures optimal spatial orientation for electron transfer. In the context of the physiological electron transfer chain it should also be stressed that only membrane-bound cytochrome bs can interact with the desaturase component which is very hydrophobic and is apparently more deeply "buried" in the membrane than either cytochrome bs or the reductase [141,164]. So although this electron transfer chain does not appear to exist as an organised stoichiometric membrane-bound multi-enzyme complex, the membrane nevertheless has a very significant organisational role in the function of the multi-enzyme sequence: it facilitates interactions between the components by restricting their movement to lateral diffusion in two dimensions, it orients the catalytic domains of the reductase and bs for efficient electron transfer at this step, and it enables reduced cytochrome bs to interact favourably with the non-polar desaturase component. (f) Mitochondrial ATPase complex and other coupling A TPases

Membrane-bound enzymes involved in the coupled processes of respiration (or photosynthetic electron transfer), and ATP synthesis have been among the most intensively studied of all membrane-bound enzymes, because of their immense cellular significance and their structural complexity. The multi-component enzymes responsible for ATP synthesis in many cell types are remarkably similar; so that It is possible to give an outline of the structure and function of "energy-coupling ATPase complexes" which refers to the complexes in mitochondria of respiring eukaryotes, in aerobic bacteria and in chloroplasts of green plants. Because of the complexity of these systems, the large numbers of groups working in the field and the variety of systems studied and techniques used to isolate them, many details of these enzyme systems are still controversial. Nevertheless, recent reviews make it clear that the area of established knowledge is now substantial and increasing [165-168]. Intact ATPase complexes are multi-component vectorial transmembrane enzymes which, in intact functional systems, couple an exergonic membrane process such as a transmembrane flow of H + ions to the synthesis of ATP from ADP and phosphate. The complete complexes can be extracted from membranes with retention of

208

R.B. Freedman

relevant activities (see below) by treatment with bile salts or non-ionic detergents such as Tritons. These solubilised preparations cannot carry out the complete process of coupled ATP synthesis although they can be reconstituted into comparatively well-defined systems active in coupled ATP synthesis. The key property of the solubilised intact complexes is ATPase activity which is inhibited by characteristic inhibitors of coupled ATP synthesis, namely oligomycin, dicyclohexylcarbodiimide (DCCD) and their analogues. A component of the ATPase can be comparatively easily displaced from energytransducing membranes by mechanical agitation, sonication or the use of mild chaotropic agents. This fragment, known as F] (or CF] in the case of the chloroplast enzyme), is a water-soluble active ATPase which is not sensitive to DCCD and oligomycin. The structure and organisation of F I , the extrinsic component of the complete ATPase complex, are better understood than those of the intrinsic membrane components which are collectively referred to as the Fo component. In addition to the difference in sensitivity to inhibitors, F]-ATPase differs from the ATPase of intact mitochondrial membranes and from the complete, detergentsolubilised ATPase complex in its nucleotide specificity, its metal ion requirement, and in its instability at low temperatures (0- 10°C). Pure preparations of F]-ATPase contain no phospholipid and show no phospholipid requirement for activity. F) can obviously be regarded as a typical extrinsic protein, and, as would be expected, it is located exclusively on one surface of energy-coupling membranes, namely the matrix face (M-face) of the inner mitochondrial membrane, the inner face of the bacterial cytoplasmic membrane and the outer face of chloroplast thylakoid membranes. Evidence for this is provided by all the techniques dependent on accessibility to reagents, substrates, enzymes and antibodies but it also emerges clearly from electron microscopy. In negatively stained preparations of energycoupling membranes, spheres with a diameter of 10 nm are seen lining the matrix surface; these disappear when F 1 is displaced from the membrane and reappear when it is rebound. Isolated F] is a large spherical molecule approx. 10 nm in diameter. F] is usually said to be connected to the body of the membrane by a "stalk" but this feature may be an artefact of the preparative procedure for electron microscopy. F]-ATPase comprises five distinct types of polypeptide chain, known as a, /3, y, B, e and with approximate Mrs of 60000, 55000, 35000, 17000 and < 10000, respectively. In addition, some preparations of mitochondrial F]-ATPase contain an additional small polypeptide known as the F)-inhibitor protein. The M, of F] is 350000-380000 but there is sufficient uncertainty in these Mrs for the stoichiometry of the individual polypeptides in F] to be a subject for continuing debate. The stoichiometries (a/3)3yl)f. and (a/3hy(l)f.h are both claimed to be most consistent with data on relative amounts of the individual polypeptides and cross-linking, and of course it is possible (if unlikely) that enzymes from different sources have different stoichiometries. Arrangements of the subunits within F) have been proposed on the basis of data on accessibility to chemical reagents and cross-linking; possible arrangements are indicated in Fig. 13. Chemical modification and other

Membrane-bound enzymes

209

or

Membrane sector top view

"""""_<1 F, sector ..-'--->--,Stalk sector

1>-....,..--<1 Membrane

~~j sector

L

5 types of firmly-bound SUbunits.

1!F, - ~~6~~~r

probably oscp possibly Fc 2

1-f

possibly both

4 types of subunits + phospholipids

Fig. 13. Models of the disposition of component polypeptides in the energy-transducing ATPase complex. Adapted from [6] (a), [168] (b), [165] (c), and [114] (d).

data indicate that the ATPase catalytic sites are located on f3 subunits, although other significant nucleotide-binding sites may be located on a chains [169]. The functions of the other subunits are not clear. Isolated F] is fully water-soluble and has no affinity for phospholipids but it wil, rebind to membranes depleted of F 1; this rebinding depends on other extrinsic proteins of the membrane, which, in the case of the mitochondrial system, are known as oligomycin-sensitivity-conferring protein (OSCP) and factor 6 (Mr 18000 and 8000, respectively). The fact that these proteins function to link F 1 to the membrane sector of the complete ATPase complex has led to the suggestion that they form the "stalk" visible in electron micrographs, but this is not certain. Analogous proteins have been recognised in some other coupling ATPase complexes but not all. The membrane sector of the complete ATPase complex (Fo) is less wellcharacterised. In mitochondrial ATPase it comprises 4 or 5 proteins, but because of their non-polar nature and insolubility many of their properties are not definitely established. In the thermophile PS3 [114] and in chloroplasts [168] only 2 or 3 proteins make up this sector. All complete ATPase complexes contain a small (M, 8000) extremely non-polar polypeptide (often referred to as subunit 9) which binds the inhibitor of oxidative phosphorylation, DCCD. This polypeptide is soluble in non-polar solvents, and so is properly described as a proteolipid. There appear to be multiple (possibly 6) copies of this proteolipid in the ATPase complex, and it has been proposed that these form an oligomeric pore which mediates H + -ion translocation through the coupling membrane. Of the other components of Fo, one or two

210

R.B. Freedman

have Mrs in the range 20000-30000; the remainder are of the order of 10000. The structural organization of the component polypeptides of Fo and their locations in the membrane are not known. Intact, detergent-solubilised ATPases have Mrs of the order of 450000 to 500000 and contain all of the components referred to above, together with some phospholipid. The phospholipid is essential for their oligomycin- and DCCD-sensitive ATPase activity. The complexes in free solution cannot carry out energy-transducing functions. These functions can be regained either by spontaneous aggregation of the solubilised preparations to form membrane structures or by incorporation of the ATPase complex into a model membrane vesicle. The ATPase complex, incorporated into a vesicle together with complexes representing one span of the mitochondrial electron transfer chain, can bring about the coupled process of oxidative phosphorylation [113-115]. Similarly, when the ATPase complex is incorporated into vesicles, together with the light-energy transducing protein bacteriorhodopsin, a functional light-dependent ATP-synthesising system is formed (see refs. in [115]). Such reconstituted systems are of course only models for the natural energytransducing membranes. In the natural membranes there may be structural or stoichiometric relationships between the ATPase and proteins of the electron-transfer chain or the adenine nucleotide translocator protein, but these have not been definitely established. The ATPases must be regarded as complicated multicomponent membrane-bound enzymes which form part of a vectorial multi-enzyme sequence.

References I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18

Roodyn, D.B. (1967) Enzyme Cytology, Academic Press, London. Coleman, R. (1973) Biochim. Biophys. Acta 300, 1-30. Salton, M.RJ. (1974) Adv. Microb. Physiol. 11,213-283. Sandermann, H. (1978) Biochim. Biophys. Acta 515, 209-237. Gennis, R.B. and Jonas, A (1977) Annu. Rev. Biophys. Bioeng. 6, 195- 238. DePierre, J.W. and Ernster, L. (1977) Annu. Rev. Biochem. 46, 201-262. Martonosi, A, ed. (1976) The Enzymes of Biological Membranes, Vols. 1-4, Plenum, New York. Dahl, J.Land Hokin, L.E. (1974) Annu. Rev. Biochem. 43, 327-356. Jergensen. P.L. (1975) Quart. Rev. Biophys. 7, 239- 274. Schwartz, A, Lindenmayer, G.E. and Allen, J.e. (1975) Pharmacol. Rev. 27, 3-134. McLennan, D.H. and Holland, P.e. (1975) Annu. Rev. Biophys. Bioeng. 4, 377-404. Tada, M., Yamamoto, T. and Tonomura, Y. (1978) Physiol. Rev. 58, 1-78. Arion, W.J., Wallin, B.K., Lange, AJ. and Ballas, L.M. (1975) Mol. Cell. Biochem. 6, 75-83. Arion, W.J., Lange, AJ. and Ballas, L.M. (1976) J. BioI. Chern. 251, 6784-6790. Stetten, M.R. and Burnett, F.F. (1967) Biochim. Biophys. Acta 139, 138-147. Roth, S., McGuire, E.J. and Roseman, S. (1971) J. Cell BioI. 51, 536-547. Shur, B.D. and Roth, S. (1975) Biochim. Biophys. Acta 415, 473- 512. Freedman, R.B. and Hawkins, H.e. (1980) The Enzymology of Post-translational Modifications of Proteins, Academic Press, London. 19 Reed, L.J. (1974) Ace. Chern. Res. 7,40-46.

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20 Kirschner, K. and Bisswanger, H. (1976) Annu. Rev. Biochem. 45, 143-166. 21 Hucho, F. (1975) Angew. Chern. 14,591-601. 22 Gaertner, F. (1978) in P.A Srere and R.W. Estabrook (Eds.), Microenvironments and Metabolic Compartmentation, Academic Press, New York, pp. 345-353. 23 Welch, G.R. and De Moss, JA (1978) Ibid., pp. 323-344. 24 Srere, P.A and Estabrook, R.W. (Eds.) (1978) Microenvironments and Metabolic Compartmentation, Academic Press, New York. 25 Masters, c.r. (1978) Trends Biochem. Sci. 3, 206-208. 26 Wikstrom, M. and Krab, K. (1979) Biochim. Biophys. Acta 549, 177-222. 27 Kimura, K., Endou, H., Sudo, J.-I. and Sakai, F. (1979) J. Biochem. 85, 319- 326. 28 Stegemann, J.J. and Klotz, AV. (1979) Biochem. Biophys. Res. Commun. 87,410-415. 29 Singer, S.J. (1974) Annu. Rev. Biochem. 43, 805-833. 30 Shnitka, T.K. and Seligman, AM. (1971) Annu. Rev. Biochem. 40, 375-396. 31 Dodge, J.T., Mitchell, e. and Hanahan, D.J. (1963) Arch. Biochem. Biophys. 100, 119-130. 32 Duchon, G. and Collier, H.B. (1971) J. Memb. BioI. 6, 138-157. 33 Tillmann, W., Cordua, A and Schroter, W. (1975) Biochim. Biophys. Acta 382, 157-171. 34 McDaniel, c.r., Kirtley, M.E. and Tanner, M.JA (1974) J. BioI. Chern. 249, 6478-6485. 35 Kant, JA and Steck, T.L. (1973) I. BioI. Chern. 248, 8457-8464. 36 Shin, B.e. and Carraway, K.L. (1973) J. BioI. Chern. 248, 1436-1444. 37 McDaniel, e.F. and Kirtley, M.E. (1975) Biochem. Biophys. Res. Commun. 65, 1196-1200. 38 Steck, T.L. (1974) I. Cell. BioI. 62, 1-19. 39 Marchesi, V.T., Furthmayr, H. and Tomita, M. (1976) Annu. Rev. Biochem. 45, 667-698. 40 Letko, G. and Bohnesack, R. (1974) FEBS Lett. 39, 313-316. 41 Wilson, J.E. (1978) Trends Biochem. Sci. 3, 124-125. 42 De, B.K. and Kirtley, M.E. (1977) I. BioI. Chern. 252, 6715-6720. 43 Parker, r.c and Hofmann, J.F. (1967) J. Gen. Physiol. 50, 893-916. 44 Schrier, S.L. (1966) Am. J. Physiol. 210, 139-145. 45 Fossell, E.T. and Solomon, AK. (1978) Biochim. Biophys. Acta 510,99-111. 46 Fossell, E.T. and Solomon, AK. (1979) Biochim. Biophys. Acta 553, 142-153. 47 Tipping, E., Ketterer, B. and Christodoulides, L. (1979) Biochem. J. 180,319-326. 48 Parry, G., Palmer, D.N. and Williams,·D.J. (1976) FEBS Lett. 67,123-129. 49 Gatt, S. and Bartfai, T. (1977) Biochim. Biophys. Acta 488, 13-24. 50 Gatt, S. and Bartfai, T. (1977) Biochim. Biophys. Acta 488, 1-12. 51 Skou, J.C. (1975) Quart. Rev. Biophys. 7,401-434. 52 Cunningham, e.e. and Hager, L.P. (1971) J. BioI. Chern. 246, 1575-1582. 53 Roelofsen, B. (1978) in P. Nicholls, J.V.M. M~ler, P.L. Jorgensen and AJ. Moody (Eds.), Membrane Proteins, Pergamon, Oxford, pp. 183-190. 54 De Pont, J.J.H.H.M., van Prooijen-van Eeden, A and Bonting, S.L. (1978) Biochim. Biophys. Acta 508, 464-477. 55 Tanford, C., Reynolds, JA, McCaslin, D.R., Rizzolo, L.J. and Dean, W.L. (1978) in P. Nicholls, J.V. Moller, P.L. Jorgensen, AJ. Moody (Eds.), Membrane Proteins, Pergamon, Oxford, pp. 3- II. 56 Raison, J.K. (1973) J. Bioenerg. 4, 285-309. 57 Linden, CD, and Fox, CF. (1975) Acct. Chern. Res. 8,321-327. 58 Meichior, D.L. and Steim, J.M. (1976) Annu. Rev. Biophys. Bioeng. 5,205-238. 59 Overath, P., Thilo, L. and Trauble, H. (1976) Trends Biochem. Sci. I, 186-189. 60 Kimelberg, H.K. (1977) in G. Poste and G.L. Nicolson (Eds.), Dynamic Aspects of Cell Surface Organization, North-Holland, Amsterdam, pp. 205-293. 61 Chapman, D. (1975) Quart. Rev. Biophys. 8,185-235. 62 Shimshick, E.J. and McConnell, H.M. (1973) Biochemistry 12,2351-2360. 63 Esfahani, M., Limbrick, AR., Knutton, S. and Wakil, SJ. (1971) Proc. Natl. Acad. Sci. USA 68, 3180-3184. 64 Mavis, R.D. and Vagelos, P.R. (1972) I. BioI. Chern. 247,653-659.

212

R.B. Freedman

65 Lenaz, G., Sechi, AM., Parenti-Castelli, G., Landi, 1. and Bertoli, E. (1972) Biochem. Biophys. Res. Commun. 49, 536- 542. 66 Rottem, S., Cirillo, V.P., DeKruyff, B., Shinitzky, M. and Razin, S. (1973) Biochim. Biophys. Acta 323,509-519. 67 DeKruyff, B., van Dijk, P.W.M., Goldbach, R.W., Demel, R.A. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 330, 269-282. 68 Watson, K., Bertoli, E. and Griffiths, D.E. (1973) FEBS Lett. 30, 120-124. 69 Wisnieski, RI., Parkes, J.G., Huang, Y.O. and Fox, e.F. (1974) Proc. Natl. Acad. Sci. USA 71, 4381-4385. . 70 Morrisett, J.D., Pownall, H.J., Plumlee, R.T., Smith, L.e., Zehner, Z.E., Esfahani, M. and Wakil, S.I. (1975) I. BioI. Chern. 250, 6969-6976. 71 Kimelberg, H.K. and Papahadjopoulos, D. (1974) I. BioI. Chern. 249, 1071-1080. 72 Law, I.H. and Snyder, W.R. (1972) in CF. Fox and AD. Keith (Eds.), Membrane Molecular Biology, Sinauer, pp. 3-26. 73 Cossins, AR. (1977) Biochim. Biophys. Acta 470,395-411. 74 Nozawa, Y. and Kasai, R. (1978) Biochim. Biophys. Acta 529, 54-66. 75 Raison, J.K. and Lyons, J.M. (1971) Proc. Natl. Acad. Sci. USA 68,2092-2094. 76 Lyons, J.M. and Raison, J.K. (1970) Plant Physiol. 45, 386-391. 77 Lyons, J.M. and Raison, J.K. (1970) Compo Biochem. Physiol. 37, 405-411. 78 Raison, I.K., Lyons, J.M., Mehlhorn, R.I. and Keith, AD. (1971) I. BioI. Chern. 246,4036-4040. 79 Houslay, M.D. and Palmer, R.W. (1978) Biochem. J. 174,909-919. 80 Klingenberg, M. and Buchholz, M. (1970) Eur. J. Biochem. 13,247-252. 81 Weiner, J.H. (1974) J. Memb. BioI. 15, 1-14. 82 Racker, E. (1970) Essays Biochem. 6, 1-22. 83 Harmon, H.J., Hall, J.D. and Crane, F.L. (1974) Biochim. Biophys. Acta 344, 119-155. 84 Kenny, AJ. and Booth, AG. (1976) Biochem. Soc. Trans. 4, 1011-1017. 85 Michell, R.H., Coleman, R. and Lewis, B.A. (1976) Biochem. Soc. Trans. 4, 1017-1020. 86 Evans, W.H. (1976) Biochem. Soc. Trans. 4, 1007-1011. 87 Freedman, R.B. (1979) Trends Biochem. Sci. 4, 193-197. 88 Ji, T.H. (1979) Biochim. Biophys. Acta 559, 39-69. 89 H6chli, M. and Hackenbrock, C.R. (1979) Proc. Natl. Acad. Sci. USA 76, 1236-1240. 90 Lee, AG. (1977) Trends Biochem. Sci. 2, 231-233. 91 Chapman, D., Gomez-Fernandez, J.C. and Goni, F.M. (1979) FEBS Lett. 98, 211-223. 92 Svensson, H., Dallner, G. and Ernster, 1. (1972) Biochim. Biophys. Acta 274, 447- 461. 93 Winquist, 1. and Dallner, G. (1976) Biochim. Biophys. Acta 436, 399-412. 94 DePierre, J.W. and Dallner, G. (1975) Biochim. Biophys. Acta 415, 411-472. 95 Mull, R.H., Voigt, T. and Flemming, K (1975) Biochem. Biophys. Res. Commun. 64, 1098-1106. 96 Peterson, J.A, O'Keeffe, D.H., Werringlocs, J., Ebel, R.E. and Estabrook, R.W. (1978) in P.A. Srere and R.W. Estabrook (Eds.), Microenvironments and Metabolic Compartmentation, Academic Press, New York, pp. 433-447. 97 Yang, c.s. (1975) FEBS Lett. 54,61-64. 98 Seidegard, J., Moron, M.S., Eriksson, L.e. and DePierre, J.W. (1978) Biochim. Biophys. Acta 543, 29-40. 99 Stasiecki, P., Waechter, F., Bentley, P. and Oesch, F. (1979) Biochim. Biophys. Acta 568, 446, 453. 100 Green, D.E., Tisdale, H.D., Criddle, R.S., Chen, P.Y. and Bock, R.M. (1961) Biochem. Biophys. Res. Comun. 5, 109-114. 101 Senior, AE. and McLennan, D.H. (1970) J. BioI. Chern. 245, 5086. 102 Helenius, A and Simons, K (1975) Biochim, Biophys. Acta 415, 29- 79. 103 Tanford, e. and Reynolds, J.A. (1976) Biochim. Biophys. Acta 457, 133-170. 104 Weber, KA and Osborn, M.-J. (1975) in H. Neurath and R.L. Hill (Eds.), The Proteins, 3rd ed., Academic Press, New York, Vol. I, pp. 179-223. 105 Cuatrecasas, P. (1974) Annu. Rev. Biochem. 43, 169-214.

Membrane-bound enzymes

213

106 Haugen, DA and Coon, M.J. (1976) 1. BioI. Chern. 251, 7929- 7939. 107 Coon, MJ., van der Hoeven, TA, Dahl, S.B. and Haugen, DA (1978) Methods Enzymol. 52, 109-117. 108 Nielsen, N.C and Fleischer, S. (1973) 1. BioI. Chern. 248, 2549-2555. 109 Nielsen, N.C, Zahler, W.L. and Fleischer, S. (1973) J. BioI. Chern. 248, 2556-2662. 110 Gazzotti, P., Bock, H.G. and Fleischer, S. (1974) Biochem. Biophys. Res. Commun. 58, 309-315. III Bock, H.-G. and Fleischer, S. (1975) 1. BioI. Chern. 250, 5774-5781. 112 Gazzotti, P., Bock, H.-G. and Fleischer, S. (1975) J. BioI. Chern. 250, 5782-5790. 113 Kagawa, Y. (1976) in A. Martonosi (Ed.), The Enzymes of Biological Membranes, Plenum, New York, VolA, pp. 125-142. 114 Kagawa, Y. (1978) Biochim. Biophys. Acta 549, 31-53. 115 Racker, E. (1979) Methods Enzymol. 55, 699- 711. 116 Montal, M. (1976) Annu. Rev. Biophys. Bioeng. 5, 119-175. 117 Kundig, W. (1976) in A. Martonosi (Ed.), The Enzymes of Biological Membranes, Plenum, New York, VoU, pp. 31-55. 118 Postma, P.W. and Roseman, S. (1976) Biochim. Biophys. Acta 457,213-257. 119 Saier, M. (1977) Bacteriol. Rev. 41, 856- 871. 120 Razin, S. (1972) Biochirn. Biophys. Acta 265, 241- 296. 121 Birnbaumer, L. (1973) Biochim. Biophys. Acta 300, 129-158. 122 Avruch.T. and Pohl, S.L. (1973) in D. Chapman and D.F.H. Wallach (Eds.), Biological Membranes, Academic Press, New York, Vol. 2, pp. 185-219. 123 Rubalcava, B. and Rodbell, M. (1973) 1. BioI. Chern. 248, 3831-3837. 124 Jaeons, S. and Cuatrecasas, P. (1977) Trends Biochem. Sci. 2, 280- 282. 125 Orly, J. and Schramm, M. (1976) Proc. Natl. Acad. Sci. USA 73, 4410-4414. 126 Schulster, D. (1979) Biochem. Soc. Trans, 7, 310-314. 127 Houslay, M.D., Ellory, I.C, Smith, GA, Hesketh, T.R., Stein, I.M., Warren, G.B. and Metcalfe, I.C (1977) Biochim. Biophys. Acta 467,208-219. 128 Schlegel, W., Kempner, E.S. and Rodbell, M. (1979) 1. BioI. Chern. 254, 5168- 5176. 129 Levitzki, A. and Helmreich, E. (1979) FEBS Lett. 101, 213-219. 130 Cassel, D. and Selinger, Z. (1977) Proc. Natl. Acad. Sci. USA 74, 3307-3311. 131 Tolkovsky, A.M. and Levitzki, A. (1978) Biochemistry 17, 3795- 3810. 132 Hanski, E., Rimon, G. and Levitzki, A. (1979) Biochemistry 18,846-853. 133 Gennis, R.B. and Strorninger, I.L. (1976) in A. Martonosi (Ed.), The Enzymes of Biological Membranes, Plenum, New York, Vol. 2, pp. 327-341. 134 Gennis, R.B. and Stromirtger, I.L. (1976) J. BioI. Chern. 251,1264-1269. 135 Gennis, R.B. and Sinensky, M. and Strominger, J.L. (1976) J. BioI. Chern. 251, 1270-1276. 136 Gennis, R.B. and Strorninger, J.L. (1976) J. BioI. Chern. 251, 1277-1282. 137 Rothfield, L.I., Romeo, D. and Hinckley, A. (1972) Fed. Proc. 31, 12-17. 138 Muller, E., Hinckley, A. and Rothfield, L.I. (1972) 1. BioI. Chern. 247, 2614-2622. 139 Hinckley, A., Muller, E. and Rothfield, L.I. (1972) 1. BioI. Chern. 247, 2623-2628. 140 Beadling, L. and Rothfield, L.I. (1978) Proc. Natl. Acad. Sci. USA 75,3669-3672. 141 Strittmatter, P., Spatz, L., Corcoran, D., Rogers, M.J., Setlow, B. and Redline, R. (1974) Proc. Natl. Acad. Sci. USA 71, 4565-4569. 142 Mathews, F.S. and Czerwinski, E.W. (1976) in A. Martonosi (Ed.), The Enzymes of Biological Membranes, Plenum, New York, Vol.a, pp. 143-197. 143 Enoch, H.G. and Strittmatter, P. (1979) J. BioI. Chern. 254, 8976-8981. 144 Ito, A. and Sato, R. (1968) J. BioI. Chern. 243,4922-4923. 145 Spatz, L. and Strittmatter, P. (1971) Proc. Natl. Acad. Sci. USA 68, 1042-1046. 146 Strittmatter, P., Rogers, M.J. and Spatz, L. (1972) 1. BioI. Chern. 247, 7188-7194. 147 Mihara, K. and Sato, R. (1972) J. Biochem. 71, 725-735. 148 Spatz, L. and Strittmatter, P. (1973) 1. BioI. Chern. 248, 793- 799. 149 Ozols, J. (1974) Biochemistry, 13,426-434.

214

R.B. Freedman

150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166

Enomoto, K. and Sato, R. (1973) Biochem. Biophys. Res. Commun. 51,1-7. Dehlinger, P.J., Jost, P.e. and Griffiths, O.H. (1974) Proc. Natl. Acad. Sci. USA 71, 2280-2284. Robinson, N.e. and Tanford, e. (1975) Biochemistry 14, 369-378. Dufourcq, J., Bemon, R. and Lusson, C. (1976) Biochim. Biophys. Acta 433,252-263. Remade, J. (1978) J. Cell. BioI. 79, 291-313. Fleming, P.J. and Strittmatter, P. (1978) J. BioI. Chern. 253, 8198-8202. Rogers, M.J. and Strittmatter, P. (1975) J. BioI. Chern. 250, 5713-5718. Holloway, P.W. and Katz, J.T. (1975) J. BioI. Chern. 250, 9002-9007. Dailey, H.A and Strittmatter, P. (1978) J. BioI. Chern. 253, 8203-8209. Rogers, M.J. and Strittmatter, P. (1974) J. BioI. Chern. 249, 895-900. Leon, M., Bonfils, e. and Debey, P. (1978) Arch. Biochem. Biophys. 191,216-223. Rogers, M.J. and Strittmatter, P. (1973) J. BioI. Chern. 248, 800-806. Strittmatter, P. and Rogers, MJ. (1975) Proc. Natl. Acad. Sci. USA 72, 2658-2661. Enoch, H.G., Fleming, P.J. and Strittmatter, P. (1977) J. BioI. Chern. 252, 5656-5660. Enoch, H.G., Catala, A and Strittmatter, P. (1976) J. BioI. Chern. 251, 5095-5103. Senior, AE. (1973) Biochim. Biophys. Acta 301,249-277. Tzagoloff, A (1976) in A Martonosi (Ed.), The Enzymes of Biological Membranes, Plenum, New York, VolA, pp. 103-124. Pedersen, P.L., Amzel, L.M., Soper, J.W., Cintron, N. and Hullihen, J. (1978) in G. Schafer and M. Klingenberg (Eds.), Energy Conservation in Biological Membranes, Springer, Berlin, pp. 159-194. Baird, B.A and Hammes, G.G. (1979) Biochim. Biophys. Acta 549, 31-53. Harris, D.A (1978) Biochim. Biophys. Acta 463,245-273. Papa, S. et al. (1978) in P. Nicholls, J.V., Mjtller, P.L., Jjlrgensen and AJ. Moody (Eds.), Membrane Proteins, Pergamon, Oxford, pp. 37-48. Wikstrom, M., et al. (1978) Ibid., pp. 85-94. Desnuelle, P. (1979) Eur. J. Biochem. 101, I-II.

167 168 169 170 171 172