Inhibitors of the adenine nucleotide translocase

Pharmac.Ther.Vol. 7, pp. 329-349

0163-7258/79/1101-0329/$5.00/0

C) PergamonPress Ltd. 1979. Primedin Great Britain

Specialist Subject Editors: MARIA ERECII~SKAand DAVID F. WILSON

INHIBITORS

OF THE ADENINE TRANSLOCASE

NUCLEOTIDE

MARION STUBBS

Metabolic Research Laboratory, Radcliffe Infirmary, Oxford OX2 6HE, U.K.

1. INTRODUCTION Cellular compartments and their role in cellular energy metabolism have been a subject of intensive investigation in biological research for many years. The mammalian cell consists of a cytosolic compartment, bounded by a plasma membrane, which contains various organeUes including mitochondria (see Fig. 1). Compartmentation within the mitochondria was revealed by the classic electron microscopic studies (Palade, 1953) and by the separation techniques of Werkheiser and Bartley (1957). Further understanding of these compartments and the membranes that separate them has been due to the use of specific inhibitors. The outer mitochondrial membrane is readily penetrated by molecules, either charged or uncharged, up to a mol. wt of about 5000. Consequently the outer membrane space (i.e. the space between the inner and outer mitochondrial membranes) is in equilibrium with the cytosol as far as the low mol. wt (< 5000) substances are concerned. However, the inner mitochondrial membrane which encloses the matrix space (inner mitochondrial compartment) has limited permeability properties: it is freely permeable only to such substances as H20, CO2, 02, ethanol, NH3 and acetate but is selectively permeable to some other small ions. This selective permeability is achieved by special carrier or translocating systems for facilitating transport of such metabolites (for Review see Chappell (1968) and Meijer and Van Dam (1974)). These translocator systems are obviously a key area for the study of the regulation of mitochondrial energy metabolism. Many such transport systems have been discovered by the use of specific inhibitors. In fact the study of one particular inhibitor, atractyloside, permitted the identification of the adenine nucleotide translocase.

Mitochondria mitochondr|ai

matrix

Cytoplasm

inter-membrane space Outer mitochon drial membrane ;nner mit ochondrial

Cell membrane

Endoplasmic reticulum

membrane

Nucleus

FIG. 1. A scheme of a typical mammalian cell showing mitochondria and their compartments. 329

MARION STUBBS

330

1.1. DISCOVERY AND NATURE OF THE ADENINE NUCLEOTIDE TRANSLOCASE

The adenine nucleotide translocase is perhaps the most important carrier in the inner mitochondrial membrane, in that it transports ATP,* the major form in which energy is transported throughout the cell. ATP formed in the mitochondria during oxidative phosphorylation is exchanged by the translocator in a stoichiometric (1:1) fashion for ADPt produced by cytosolic energy-consuming reactions. The inhibitor of this reaction, atractyloside, was first described in 1868 by Lefranc who isolated and crystallized it, and called it atractylic acid because of its occurrence in rhizomes of Atractylis gummifera, a thistle found in the southern Mediterranean. This plant was of interest at the time because animals had been known to die after eating the rhizomes. Many years later after an incident in which a class of school children were poisoned, some fatally, investigations showed that in some small mammals atractyioside caused a decrease in oxygen uptake, hyperglycaemia followed by hypoglycaemia with convulsions and eventually death (Santi, 1958). Further studies showed that atractyloside inhibited competitively the oxidative phosphorylation of ADP added to isolated rat liver mitochondrial suspensions (Bruni and Contessa, 1961 ; Bruni et al., 1962; Vignais et al., 1961, 1962; Vignais and Vignais, 1964) and in 1964 Kemp and Slater stated that 'the site of action (of atractyloside) appears to be on the mitochondrial membrane where it inhibits the reaction between intramitochondrial ATP and extramitochondrial ADP'. Their statement was based on the fact that Bruni et al. (1964) had shown an inhibition by atractyloside of ADP 'binding' to the mitochondria. Similar conclusions were reached by Chappell and Crofts (1965), Heldt et al. (1965) and Klingenberg and Pfaff (1966). Heldt (1966) stated more specifically that the inhibition by atractyloside of the phosphorylation of exogenous ADP, and not of endogenous ADP, may be due to a specific protein which was called translocase. Translocase is the name given to a protein catalysing a translocation--hence the name adenine nucleotide translocase or translocator may be used synonymously. Characterisation of the exchange was first attempted by Pfaffet al. (1965) who showed that the penetration of the nucleotides was extremely rapid (i.e. faster than the minimum sampling time of 1 sec) and that ADP was exchanged in preference to ATP. They also confirmed with others (Du6e and Vignais, 1965; Brierley and O'Brien, 1965; Winkler et al., 1968) that atractyloside acts specifically on the adenine nucleotide translocase which is present in the inner mitochondrial membrane. The nonspecific permeation of the outer mitochondrial membrane by the nucleotides was unaffected by the inhibitor. The normal function of the adenine nucleotide translocase is to transport ADP into the mitochondria and ATP out during oxidative phosphorylation. The carrier is specific for ADP and ATP and does not transport AMP~ or any other ribonucleoside di- or triphosphates (Winkler et al., 1968 ; Pfaff and Klingenberg, 1968 ; Du6e and Vignais, 1969). Although phosphate is also essential for the oxidative phosphorylation of ADP it is not transported by the adenine nucleotide translocase but by one of two separate carriers--either in exchange for dicarboxylate or for hydroxyl ions (see Meijer and Van Dam, 1974 for review). The adenine nucleotide translocase can be considered a catalyst in that it speeds up an exchange reaction: the exchange of ATP for ADP. An enzyme, however, is defined as 'any of a class of complex organic substances that cause chemical transformations in plants and animals' (Oxford English Dictionary). Thus although the adenine nucleotide translocase catalyses a chemical exchange it does not catalyse a chemical transformation in that the chemicals ATP and ADP are unchanged. Nevertheless, the translocase has been assumed to behave in a similar manner to an enzyme in that the mechanism of translocation requires the formation of a carrier substrate complex, similar to an enzyme-substrate complex, which can * ATP: Adenosine 5'-triphosphate. t ADP: Adenosine 5'-diphosphate. AMP: Adenosine 5'-monophosphate.

Inhibitors of the adenine nucleotide translocase

331

accept and release substrate on either side of the membrane. As such, classical kinetics, such as those of Michaelis and Menten have been applied to its study. Since the identification of the adenine nucleotide translocator as the site of action of inhibition by atractyloside Vignais' group in Grenoble and Klingenberg's group in Munich have, among others, studied the translocase extensively using atractyloside, carboxyatractyloside, and bongkrekic acid as major tools to help to elucidate its mechanism of action and its regulation (see Vignais (1976) and Klingenberg (1976a) for review). The inhibitors have proved invaluable in furthering the understanding of the translocase and in Sections 4-6 the major inhibitors will be dealt with individually. Other inhibitors, namely various derivatives ofatractyloside, isobongkrekic acid and agaric acid will also be briefly reviewed in Section 7. Another Section (8) will be concerned with the effects of physiological inhibitors and effectors of the translocase such as long-chain fatty acyl-CoAs, thyroid hormone, and glucagon. The penultimate Section (9) will deal with proposed mechanisms of adenine nucleotide transport and finally Section 10 will be a discussion on the regulation of the translocator and its role in cellular energy metabolism. 2. EXPERIMENTAL METHODS USED IN STUDIES OF THE ADENINE NUCLEOTIDE TRANSLOCASE Most of the studies on the translocase have been performed on isolated mitochondria and there are several problems associated with their use. Firstly, it is not known whether mitochondria, when isolated, behave in the same way as when they are in intact cells. Secondly, mitochondria from some tissues exhibit very high metabolic activity, particularly in oxidative metabolism, often one to two orders of magnitude faster than those in whole cells, which makes determination of transport rates correspondingly more difficult. Thirdly, the substances to be studied (in our case ATP and ADP) are themselves rapidly metabolised. Therefore it has been necessary to develop special techniques to overcome some of these practical difficulties. Adenine nucleotides labelled with 3H, 14C and 32p are available and this allows sophisticated experiments to be performed. In essence there are two methods used to assay the rate of adenine nucleotide transport, namely direct exchange where radioactively labelled ATP or ADP are added to the mitochondria, and reverse or back exchange where unlabelled ATP or ADP are added to the mitochondria which have been previously loaded with labelled adenine nucleotides. In both cases the reaction is terminated by the addition of atractyloside or carboxyatractyloside followed by rapid centrifugation through a layer of silicone into a fixative in what has become known as the 'inhibitor stop' method (Vignais and Du6e, 1966; Klingenberg and Pfaff, 1967 ; Pfaff and Klingenberg, 1968). This involves incubation of the mitochondria at low temperatures to minimise the exchange rate for the required time period, addition of atractyloside* at a final concentration of 10-4 M,immediately followed by centrifugation at 25,000 x g for 5 rain with the maximum centrifugal force being reached within 30 sec (Du6e and Vignais, 1969). This method allows incubation periods as short as 5 sec. An alternative method consists of rapid filtration of the mitochondrial suspension through a Millipore filter and then analysis of the labelled products in the filtrate and in the pellet fraction (Brierley and O'Brien, 1965; Winkler et al., 1968). Another method devised for allowing extremely short periods of contact between the mitochondria and substance of interest is achieved by superimposing several layers in a centrifuge tube with a density gradient increasing towards the bottom of the tube. The mitochondria are incubated on the top layer and on centrifugation migrate through the lower layers. The exposure time is determined by the depth of the layer and speed of centrifugation and can be calibrated by measuring a metabolic function of known rate such as oxidation of 3-hydroxybutyrate (Klingenberg et al., 1964). Klingenberg (1976a) has reported data using an even more rapid sampling device where it is claimed that the kinetics of the adenine nucleotide exchange can be resolved down to * Or carboxyatractyloside (which is commercially available) at a final concentration of 5 x 10-6 M. J.p.~'. 7/2--1

332

MARION STUBBS

80 msec. This device is named a 'rapid automated mixing and sampling apparatus' (RAMSA or RAMPRESA; Klingenberg, 1979). A 'Quench flow' method (Nohl and Klingenberg, 1978) has also been reported. 3. PROPERTIES OF THE ADENINE NUCLEOTIDE EXCHANGE (SEE TABLE 1) The amount of endogenous adenine nucleotide present in the mitochondria is constant. This is because the exchange of ATP and ADP is strictly coupled in a 1 : 1 manner, i,e. for one ATP translocated from the mitochondria to cytosol one ADP is translocated from cytosol to mitochondria or vice versa (Pfaff et al., 1965; Du6e and Vignais, 1965). All the intramitochondrial (endogenous) nucleotides are exchangeable (Du6e and Vignais, 1969). The translocase is highly specific for ATP and ADP, with the virtual exclusion of AM P; free ATP and ADP appear to be the true substrates rather than their magnesium complexes (Verdouw and Bertini, 1973; Duszyfiski and Wojtczak, 1975). 3.1. K s VALUES The apparent K,, values for external ADP and ATP depend on the energy state of the mitochondria. When the mitochondrion is functioning normally, i.e. as it is assumed to function in the intact cell and oxidatively phosphorylating ADP, the Km for ADP is between I and 12 #M (Du6e and Vignais, 1969 ; Pfaff et al., 1969 ; Souverijn et al., 1973) depending on the intramitochondrial ATP/ADP ratio (Vignais et al., 1973b) and the K mfor ATP is 150 #M. However when the mitochondria are uncoupled the K m for ATP decreases to 1 #M. This dependence of the K,, on the energy state of the mitochondria led investigators to assume an effect on the conformation of the translocator protein or its environment (see Vignais and Vignais, 1972 and below). 3.2. THE PREFERENTIAL UPTAKE OF A D P

ADP is translocated preferentially to ATP in coupled mitochondria. This phenomenon is as yet not fully understood but experiments in isolated mitochondria (Klingenberg et al., 1969; Davis and Lumeng, 1975) and in isolated hepatocytes (Zuurendonk and Tager, 1974; Siess and Wieland, 1976) show that the ATP/ADP ratio is 5-10 times higher outside the mitochondria than inside. According to Klingenberg (1970) this difference is a result of the preferential uptake of ADP by the mitochondria and corresponds to the membrane potential. Only in the presence of uncoupler, when the ATP/ADP ratios are greatly perturbed and the membrane potential abolished, are ATP and ADP taken up at a similar rate.

TABLE 1. The adenine nucleotide translocase. General properties Property Catalyses the exchange of I mol ATP for I tool ADP Excludes AMP and all nucleotides with other bases, e.g. guanine, cytosine, etc. At 30°C the rate of exchange is ~ 36/~mol/min/g wet wt. or 600/~mol/min/g mitochondrial protein Substrates are free ATP and ADP rather than Mg 2+ complexes Under the following conditions: coupled mitochondria uncoupled mitochondria (energy-rich) (energy-poor) ADP is translocated ADP and ATP translocated in preference to ATP with equal preference KmADP 1-12/~M 1-12 #M K,~ATP 150 #M 1-2/~M

Reference

(1) (2,8) (3)

(4)

(5) (5,6,7) (5,6)

(1) Du6e and Vignais, 1965 ; (2) Winkler et al., 1968 ; (3) Klingenberg, 1976a; (4) Verdouw and Bertina, 1973 ; (5) Souverijn et al., 1973 ; (6) Du6e and Vignais, 1969 ; (7) Pfaff et al., 1969 ; (8) Pfaff and Klingenberg, 1968.

Inhibitors of the adenine nucleotide translocase

333

At an intracellular pH of 7.4 most of the ADP and ATP would be present as ADP a- and ATP 4-. This raises a problem in an exchange mechanism of the sort described since exchange of the two nucleotides brings about an imbalance of charges. According to Wulf et al. (1978) about 40-50 per cent of the A D P - A T P exchange is electroneutral in the physiological direction (ADPi~-ATPo~t). In experiments with isolated mitochondria H ÷ is released (along with ATP) when ADP is added and this partly balances the charge difference. More recently La Noue et al. (1978) claim that the exchange is 'fully electrical' and that the H ÷ movements on the addition of ADP or ATP are not 'an inherent part of the carrier mechanism but move in a passive manner to balance the charge separation'. Thus according to Klingenberg and coworkers the A T P - A D P exchange is 'electrical', the preferential uptake of ADP causes a difference in the internal and external ATP/ADP ratio and this difference corresponds to, and is driven by, the membrane potential (Klingenberg, 1970; Klingenberg and Rottenberg, 1977). This means that energy generated by the respiratory chain is used for both the synthesis and transport of ATP, the amount of energy required for the transport being the difference between the internal and external ATP/ADP ratios, i.e. 2-4 kcals (Klingenberg, 1970). Other mechanisms have been proposed including local increases in proton concentrations (see Vignais, 1976) and conformational changes at the substrate site of the translocator (Souverijn, 1974). In the latter case it is proposed that in the presence of uncoupler the conformation of the translocase would be such that ATP and ADP were transported with equal preference. However, in the energy-rich state an energy dependent conformational change would take place that would increase the affinity of the translocase for extramitochondrial ADP one to two hundred times. In this mechanism the balance of charges would be effected through Pi exchange for a hydroxyl ion as follows: ADP 3- + H P O , 2- ~ ATP*- + O H 3.3. TEMPERATURE DEPENDENCE

The adenine nucleotide exchange is extremely sensitive to temperature as shown in Fig. 2 (Pfaffet al., 1969; Du6e and Vignais, 1969; Klingenberg, 1976a). In rat liver mitochondria the activation energy is very high, about 30 kcal between 0 and 18°C and about 11 kcal above 18°C. The high activation energy is interpreted by both Vignais (1976) and Klingenberg (1976a) to indicate that a phase transition in the membrane phospholipid is important for the translocation process.

363024¢=

12:L

60 0

I

IC)

'

20 '

'

3'0

oc

Flo. 2. Temperature dependence of ADP exchange. From Klingenberg (1976a). Measurements in rat liver mitochondria prelabelled with 14C adenine nucleotides. The exchange started by addition of 200 pM A D P and measurements obtained by RAMSA (Klingenberg, 1979). ~tmol/min/g wet wt

calculated assuming 60 mg mitochondrial protein/g wet wt (Scholz and B/icher, 1965).

MAPdONSTUBBS

334

4. ATRACTYLOSIDE 4.1. MOLECULAR STRUCTURE Atractyloside is a glycoside, the carbohydrate portion of which consists of a simple glucose unit containing two residues of potassium sulphate and one residue of isovaleric acid. The aglycone portion of the molecule is a diterpene (see Fig. 3). Atractyloside, in common with ADP (and carboxyatractyloside and bongkrekic acid--see 5 and 6) has 3 ( - ) charges which are postulated to be a minimum requirement for the translocase since AMP 2- is excluded (Klingenberg et al., 1970). 4.2. ACTIVE MOIETY A search for the active part of the atractyloside molecule led to the finding that it is probably the aglycone, atractyligenin, that is the active moiety (Vignais, 1969 ; Vignais et al., 1966). In order to determine which functional groups of the atractyligenin molecule are involved in the inhibition of translocation, Vignais (1969) tested a number of analogues and found that a small change of some functional groups, for instance, an additional methyl group at C-4 position, led to a significant change of the inhibitory effect. In other words, the atractyligenin moiety of atractyloside is very specific for the translocase site but it has a much lower efficiency than atractyloside (see also Vignais et al., 1973a). 4.3. KINETICS OF THE ATRACTYLOSIDEINHIBITION Atractyloside is generally believed to be competitive with ADP for the translocator binding site (Vignais et al., 1973a). However, Klingenberg (1976a) claims it is far from a clear competitive relationship in that his results show that in spite of a 10-fold increase in I-ADP], the apparent inhibition constant only increases 2-fold. The binding sites for atractyloside are not saturable but it is thought that there are two classes of sites, one with low affinity with a dissociation constant of 0.3/.IM and one with high affinity with a dissociation constant of 0.01 #M (Vignais et al., 1970; Vignais and Vignais, 1971). However, binding sites for carboxyatractyloside (see 5) are saturable and it has been found that the number of high affinity sites for atractyloside (and for bongkrekic acid) is the same as the number of carboxyatractyloside binding sites (Vignais et al., 1973a). 4.4. SITE OF ACTION Atractyloside does not penetrate the inner mitochondrial membrane and is therefore assumed to exert its effect by occupying a site on the outside of the inner membrane. The evidence for this comes from experiments with so-called 'inside-out' sonic particles where atractyloside is ineffective (Shertzer and Racker, 1974). The exact location of the atracty-

%s-o

c. oH o

O=C

:

CHz

~"

,

CHz ,

-

-

CH CH3 CHa

FIG. 3. Atractyloside and related compounds. (i) atractyloside: R = H, (ii) carboxyatractyloslde: R = COOH, (iii) epi-atractyloside: R = H, C O O H at C4 position in equatorial rather than axial position, (iv) apocarboxyatractyloside: R = C O O H and isovaleric acid removed, (v) succinylatractyloside: succinyl group attached to CH2OH of glucose disulphate.

Inhibitors of the adenine nucleotide translocase

335

loside binding site is still unclear. As a competitive inhibitor of adenine nucleotide transport it might be thought that the inhibitor competes with ADP for its binding site. However, addition of/ZM amounts of ADP or ATP can enhance the number of atractyloside binding sites in inner mitochondrial membrane vesicles (Vignais et al., 1973a; Vignais and Vignais, 1971). These effects can be explained by the unmasking of specific carrier sites when the functioning of the carrier is induced by ADP (see Vignais et al., 1976b). 4.5. ATRACTYLOSIDEAS AN EXPERIMENTAL TOOL

Atractyloside competitively inhibits ADP transport in all mammalian cells tested (except possibly foetal liver cells ; van Lelyveld and Hommes, 1978) but is virtually ineffective in plant mitochondria (Passam et al., 1973) or requires a much higher concentration than mammalian mitochondria (Jung and Hanson, 1973; Vignais et al., 1976a). Effective inhibition of adenine nucleotide transport by atractyloside is achieved at a concentration of 10 -7 M (Vignais et al., 1966) in mammalian mitochondria, at a concentration of 10 -4 M in plant mitochondria (Jung and Hanson, 1973) and at 10-SM in isolated rat hepatocytes (Stubbs et al., 1978) where a higher concentration is needed to allow penetration of the plasma membrane. The competitive nature of atractyloside and the fact that it can be radioactively labelled with high specific activity (Brandolin et al., 1974) has proved useful in assessing which fraction of the total ADP that apparently 'binds' to the mitochondrial membrane is, in fact, bound to the carrier site, which portion is bound non-specifically to the membrane and which portion is actually exchanged with ATP. In mitochondria there are 10-30 times more endogenous adenine nucleotides than translocase sites, therefore for such experiments it is necessary to deplete the mitochondria of all endogenous nucleotides prior to assessing the ADP (or ATP) binding by displacement with atractyloside. This can be achieved by lubrol treatment of the mitochondria (Winkler and Lehninger, 1968), phosphate swelling (Wiedemann et al., 1970) or by preparations of matrix-free inner membrane vesicles (Vignais et al., 1973a). Such experiments have shown that there are 1-1.5 mol of adenine nucleotide translocase per mol cytochrome a (Wiedemann et al., 1970; Winkler and Lehninger, 1968). Brandolin et al. (1974) have partially purified an atractyioside binding protein from rat liver having an apparent mol. wt of 50,000-60,000 which is probably identifiable with the translocase. The purification procedure is based on affinity chromatography using succinylatractyloside linked to Sepharose (see Section 7.2).

5. CARBOXYATRACTYLOSIDE 5.1. MOLECULAR STRUCTURE

Carboxyatractyloside is very similar in structure to atractyloside with the exception of one extra carboxyl group at the C4 position of the diterpene nucleus (see Fig. 3). It is also called gummiferin and was first isolated as such (Stanislas and Vignais, 1964) and later identified as carboxyatractyloside (Defaye et al., 1971). 5.2. ACTIVE MOIETY

It has been suggested that it is not the additional carboxyl group (R) (see Fig. 3) that gives carboxyatractyloside its more powerful inhibitory effect on the adenine nucleotide translocase, but rather its configuration. Riccio et al. (1973) found that epi-atractyloside, which has similar inhibitory properties to carboxyatractyloside, has its carboxyl group in an equatorial position, whereas in atractyloside it is in the axial position. Klingenberg (1976b) rationalizes this by postulating that the greater proximity of the equatorial carboxyl group to the sulphate group favours binding since in ADP the three anion groups are also in the same region. However, there is a significant difference in the size of the molecules (see Vignais et al., 1973b) which casts some doubt on this interpretation.

336

MARIONSTUBBS

5.2. KINETICS OF THE CARBOXYATRACTYLOSIDE INHIBITION Carboxyatractyloside binds selectively and with very high affinity to the inner mitochondrial membrane. It is functionally different from atractyloside in that it inhibits the translocase non-competitively (Vignais et al., 1973a). The inhibition is irreversible, since the bound carboxyatractyloside cannot be displaced by ADP. This is in contrast to atractyloside where the bound inhibitor can be displaced by ADP (Vignais et al., 1971). The noncompetitiveness of the carboxyatractyloside binding is essentially due to its very high affinity for its binding sites on the membrane. The dissociation constant is 5-10 nM (Vignais et al., 1973a). 5.3. SITE OF ACTION

Like atractyloside, carboxyatractyloside is a non-penetrant inhibitor and therefore assumed to inhibit at a point on the outer surface of the inner mitochondrial membrane. The binding sites for the inhibitor are saturable and similar in quantity to those found with atractyloside, i.e. 1.5-2.0 mol adenine nucleotide translocase/mol cytochrome a in rat liver mitochondria (Vignais et al., 1973a, 1973b). 5.4. CARBOXYATRACTYLOSIDE AS AN EXPERIMENTAL TOOL Mammalian, yeast and plant mitochondria all exhibit a high affinity for carboxyatractyloside. Effective inhibition of the adenine nucleotide translocase is obtained at a concentration of 10-TM carboxyatractyloside in mammalian mitochondria (Vignais et al., 1973a) and 10 -5 M in isolated hepatocytes (Stubbs et al., 1978). Because of the very tight binding of carboxyatractyloside to the translocase the inhibitor has proved an invaluable tool in the further study of the translocase and particularly in its attempted isolation and purification. Klingenberg et al. (1974) prepared a soluble protein retaining the carboxyatractyloside binding after loading beef heart mitochondria with [3SS]carboxyatractyloside. This protein had a mol. wt of 29,000 (Riccio et al., 1975a,b). Bojanovski et al. (1976) have also isolated a carboxyatractyloside binding protein of similar mol. wt which on analysis consisted of 85 per cent protein, 14 per cent phospholipids and 1 per cent triglycerides. More recently a further purification has been achieved (Klingenberg et al., 1978) and a mol. wt of 60,000 was calculated from the carboxyatractyloside binding indicating that the carboxyatractyloside protein complex consists of two 30,000 subunits. From this can be calculated that the proportion of protein associated with the adenine nucleotide translocase in beef heart mitochondria is about 10 per cent of the total mitochondrial protein. 6. BONGKREKIC ACID 6.1. MOLECULAR STRUCTURE The antibiotic bongkrekic acid is a long-chain unsaturated fatty acid with three carboxylic groups (see Fig. 4). It is one of the toxic principles produced by the organism Pseudomonas cocovenenans and was first shown to inhibit the adenine nucleotide exchange by Henderson and Lardy (1970). 6.2. ACTIVE MOIETY AND SITE OF ACTION

Bongkrekic acid is only effective in inhibiting the adenine nucleotide exchange at acidic pH and at temperatures above 20°C (Erdelt et al., 1972). Its inhibitory effect is not immediate, the lag-time depending on pH (Kemp et al., 1971). It can be radioactively labelled with tritium (Lauquin and Vignais, 1976; Babel et al., 1976). Experiments with [aH] bongkrekic acid indicate that the protonated acid diffuses through the lipid phase of the inner mitochondrial membrane and interacts with the translocase on the matrix side of the membrane (Lauquin and Vignais, 1976). It is the lipophilic nature of bongkrekic acid that enables it to penetrate

Inhibitors of the adenine nucleotide translocase

337

~H3 CH=CH

c.~

.CH~ .c.=c/.

! CH__/~ N, / _ /C\/ -- H CHz HOOC ~H ~--'CHi~iCH2 CH~-~,

~

COOH ,-,u ,./

'CH r'C--C"H e l l / -CH3

\

CH3

/CH z" COOH

FIG. 4. Bongkrekic and isobongkrekic acid. Bongkrekic acid and isobongkrekic acid are isomers with the trans and cis configuration respectively at the dicarboxylic end of the molecule.

the membrane. Thus the main difference between bongkrekic acid and the two atractylosides is that bongkrekic acid is a penetrant inhibitor and acts at the inner surface of the inner mitochondriai membrane, whereas the atractylosides are non-penetrant and act at the outer surface. 6.3. KINETICS OF BONGKREKIC ACID INHIBITION Bongkrekic acid exhibits behaviour that is very different from the behaviour of the two atractylosides. For instance, bongkrekic acid, rather than removing ADP from the carrier binding sites as might be expected of an inhibitor of the translocase, significantly increases the binding of 14C ADP to the translocase (Erdelt et aL, 1972; Klingenberg and Buchholz, 1973). This bound ADP cannot be removed either by an excess of (unlabelled) ADP or by atractyloside. Not only does bongkrekic acid enhance the binding of ADP to the mitochondrial membrane but the addition of ADP increases the number ofbongkrekic acid sites (Vignais et aL, 1976b). Energisation of the membrane also increases the number of high affinity sites for bongkrekic acid. The inhibition of the adenine nucleotide exchange by bongkrekic acid is of a mixed type unless the mitochondria are pre-incubated with the inhibitor and 0.5/~M ADP. The subsequent inhibition of the exchange (on addition of 14C ADP) is increased 10-fold and becomes typically uncompetitive (Lauquin and Vignais, 1976). These findings suggest the formation of a ternary bongkrekic acid-translocase-ADP complex. In this interpretation it is supposed that the bongkrekic acid binds to a site different from the ADP-binding site and that the increase of ADP binding by bongkrekic acid is due to an increase in affinity of the translocase sites for ADP. Klingenberg (1976a; 1976b), however, interprets the findings in terms of a 'reorientation' mechanism for the translocase, the details of which are discussed in Section 9. Bongkrekic acid binding sites amount to 0.15-0.3 nmol/mg mitochondrial protein in rat liver mitochondria and have a dissociation constant of about 2 x 10- s M(Klingenberg et ai., 1970; Lauquin and Vignais, 1976). The binding sites are similar in number to the high affinity atractyloside and carboxyatractyloside binding sites. 6.4. BONGKREKIC ACID AS AN EXPERIMENTAL TOOL

Bongkrekic acid is active at a concentration of 1 0 - 6 M in rat liver mitochondria (Henderson and Lardy, 1970; Klingenberg et aL, 1970) and at a similar concentration in plant mitochondria (Passam et al., 1973; Vignais et al., 1976a). / Such specific inhibitors of the translocase provide ideal tools for investigating its mechanism of action. The fact that bongkrekic acid binds to the matrix side of the carrier has permitted isolation of a bongkrekate-binding protein, extracted from the inner side of the mitochondrial membrane (Aquila et al., 1978) that can be compared directly with a carboxyatractylate-binding protein isolated from the outer side of the mitochondrial membrane (Klingenberg et al., 1978). There are similarities in the two proteins,in mol. wt, amino acid composition and isoelectric point but differences in susceptibility to proteolytic attack. The significance of these findings are discussed in Section 9.

338

MARIONSTUBBS

7. OTHER INHIBITORS (SEE TABLE 2) 7.1. APOATRACTYLOSIDEAND APOCARBOXYATRACTYLOSIDE Apoatractyloside and apocarboxyatractyloside (Vignais et al., 1973a) are the derivatives of atractyioside and carboxyatractyloside caused by the removal of the isovaleric group at the C-2 position of the glucose molecule (see Fig. 3). They inhibit adenine nucleotide translocation but apocarboxyatractyloside behaves as a competitive inhibitor with respect to ADP compared to carboxyatractyloside which is non-competitive (see Section 5). The binding of apocarboxyatractyloside is inhibited competitively by atractyloside and noncompetitively by carboxyatractyloside and bongkrekic acid. 7.2. Succl NYLATRACTYLOSIDE

Succinylatractyloside is atractyloside with a succinyl group attached to the primary alcohol of the glucose disulphate moiety (see Fig. 3). It retains a high affinity for mitochondria and can be covalently linked to sepharose. It has, therefore, been used in attempts to purify the atractyloside binding protein by affinity chromatography (Brandolin et al., 1974). 7.3. EPI-ATRACTYLOSIDE

Epi-atractyloside is an isomer of atractyloside but an uncompetitive inhibitor with respect to ADP, similar to carboxyatractyloside in its action. It is assumed that the carboxyl group is in the equatorial position compared to atractyloside where it is in the axial position (Riccio et al., 1973). 7.4. ISOBONGKREKICACID Isobongkrekic acid is an isomer of bongkrekic acid and differs from it by the two carboxyl groups at the dicarboxylic end of the molecule (Fig. 4) having the cis configuration rather than the trans configuration (Lauquin et al., 1976a). Isobongkrekic acid is an uncompetitive inhibitor of ADP translocation provided the mitochondria are pre-incubated in the presence of the inhibitor and a low concentration (0.5 #M) of ADP. Isobongkrekic and bongkrekic acids compete for the same site in mitochondria but the affinity of bongkrekic acid is 2-4 times higher than that of isobongkrekic acid. 7.5. AGARIC ACID

Agaric acid or ~-cetyl citric acid is citrate covalently bound to a saturated alkyl chain of 16 carbons (Chavez and Klapp, 1975). It competitively inhibits the adenine nucleotide exchange at a concentration of 40/tM. 7.6. SPIN-LABELLEDACYL ATRACTYLOS1DE A number of spin-labelled acyl derivatives of atractyloside have been prepared (Lauquin et al., 1977a) to use as probes for the adenine nucleotide translocase. They inhibit the exchange of adenine nucleotides with the same efficiency as unlabelled acyl atractylosides and the inhibition is of the mixed competitive and non-competitive type. Results obtained from experiments using these probes have been helpful in gaining knowledge about the nature of the lipid environment in which the translocator works. 7.7. ARYL-AZIDO ATRACTYLOSIDE A photoactive aryl-azido atractyloside has been synthesised (Lauquin et al., 1976b) which can be used to covalently label the translocase in mitochondria. It competitively inhibits ADP transport with the same efficiency as atractyloside and competes with atractyloside for binding to mitochondria. Such compounds have been used in attempts to identify the atractyloside binding protein in rat liver and heart mitochondria.

Inhibitorsof the adenine nucleotidetranslocase

339

7.8. N-ETHYL MALEIMIDE

N-ethyl maleimide which is a lipophilic and therefore penetrant inhibitor, only inhibits the adenine nucleotide exchange when the mitochondria have been pre-incubated with ADP (Vignais and Vignais, 1972, 1973) or have been energised by respiration (Vignais et al., 1976b). This is because a small proportion of SH groups are unmasked by interaction of the ADP with the membrane and these are then susceptible to the sulphydryl reagent. Atractyloside inhibits unmasking of the SH groups. 8. PHYSIOLOGICAL EFFECTORS OF THE ADENINE NUCLEOTIDE TRANSLOCASE 8.1. LONG-CHAIN ACYL-CoA In isolated rat liver mitochondria the CoA esters of long-chain fatty acids inhibit adenine nucleotide exchange in a competitive manner with respect to ADP. Palmitoyl-CoA is the most efficient inhibitor of ADP transport and has an inhibition constant in the order of lo-’ M (Morel et al., 1974). The physiological significance of the inhibition by long-chain acyl-CoAs is unclear since the metabolism of a fatty acid (oleate) with a concomitant increase in fatty acyl-CoA does not affect adenine nucleotide transport in isolated rat hepatocytes (Akerboom et al., 1977). It may be that the acyl-CoAs are bound to protein and therefore not free to act on the translocase in vivo. Although long-chain acyl-CoAs probably have no physiological significance as inhibitors of the translocase in liver (see Section 10) it has been postulated from experiments with fat cell mitochondria that they may play a role in adipose tissue in relation to the control of pyruvate dehydrogenase (Loffler et al., 1975). Their ability to bind and inhibit the translocase from both sides of the mitochondrial membrane (in heart; Chua and Shrago, 1977) and the fact that they can be spin-labelled, have made them useful tools with which to probe the translocase and its environment (Devaux et al., 1975). 8.2. THYROID HORMONE It has been shown that the rate of ADP transport in rat liver mitochondria is decreased after thyroidectomy and that the rate is restored to normal after treatment with thyroxine (Portnay et al., 1973 ; Babior et al., 1973 ; Hoch, 1977). 8.3. GLUCAGON Glucagon causes an increase in the intramitochondrial adenine nucleotide content of hepatocytes (Siess et al., 1977 ; Bryla et al., 1977) and in mitochondria isolated from glucagon treated animals (Bryla et al., 1977; PrpiC et al., 1978). According to Bryla et al. (1977) this increase is accompanied by an increased rate of ADP uptake by mitochondria although in similar experiments PrpiC et al. (1978) find no effect of glucagon on the ATP-ADP exchange. The significance of these findings are as yet unclear. 9. PROPOSED MECHANISMS FOR THE ADENINE NUCLEOTIDE TRANSLOCASE Advantage has been taken of the specific and natural inhibitors of the adenine nucleotide translocase in attempts to elucidate the mechanism by which it transports ADP and ATP across the mitochondrial membrane. Several approaches have been made including (a) the proposal of a model based on the interaction of the translocase with its inhibitors (from Klingenberg’s group, the details of which will be described below), (b) the development of indicators such as photoaffinity labelled modified substrates and inhibitors to probe the translocase and its environment (from Vignais’ group-see below) and (c) the use of the inhibitors to isolate the carrier protein, an approach that complements the other two (Brandolin et al., 1974; Egan and Lehninger, 1974; Riccio et al., 1975a,b; Bojanovski et al., 1976; Shertzer and Racker, 1976; Klingenberg et al., 1978; Aquila et al., 1978).

340

MARIONSTUBBS

Various models for the translocase have been considered (see Vignais, 1976 and Klingenberg, 1976a for review). The models for transport across biological membranes fall into two general classes, the mobile carrier and the fixed pore. The mobile carrier is defined as a macromolecule that can shuttle by rotational or translational movement across the membrane, exposing its binding site alternatively to the outer side and inner side of the membrane but not to both sides at the same time. The fixed pore model consists of a channel that spans across the membrane, and which can exist in two different conformational states, open to the outside and closed to the inside or alternately closed to the outside and open to the inside, and an intermediate state when the substrate is being translocated from one side to the other. According to Klingenberg et al. (1976), however, the mobile carrier concept encompasses both mobile and fixed carrier mechanisms which introduces some confusion. The first model proposed by the group of Klingenberg (Erdelt et al., 1972; Klingenberg and Buchholz, 1973), a so-called 'mobile-reorientating mechanism' was put forward to explain some unusual features of the bongkrekic acid inhibition of the translocase. In contrast to atractyloside, bongkrekic acid was found (a) to have a lag phase in its inhibitory effect in isolated mitochondria (Kemp et al., 1971), (b) to be a lipophilic substance and therefore able to penetrate the lipid membrane and (c) to increase the binding of ADP in mitochondria. These characteristics, along with atractyloside binding data, led the investigators to assume that the carrier can exist in two states, oriented either to the inside or to the outside of the mitochondrial membrane. The carrier binds to bongkrekic acid only when it is facing the inside (m-state) and to the atractylosides only when it is facing the outside (c-state). The increased binding of ADP under the influence of bongkrekic acid was explained as follows: in the absence of external ADP the empty carrier accumulates on the outer side of the membrane and when ADP is added the carrier-ADP complex moves to the inside. The subsequent addition of bongkrekic acid removes the ADP from the carrier because of its higher affinity and the bound ADP is released into the matrix space (see also Klingenberg, 1976c). The major assumption in this model that has been disputed is that bongkrekic acid competes from the same site as ADP whereas Lauquin and Vignais (1976) claim that bongkrekic acid is an uncompetitive inhibitor and that it binds to another site to form a ternary translocase: ADP: bongkrekic acid complex. If the reorientation of the binding sites in the mobile reorientating mechanism is achieved by rotational movement of the carrier, then the substrate would bind from both sides in the same manner, e.g. head first (Fig. 5). However, in a fixed channel mechanism the substrate would bind head first in one orientation state, and tail first in the other. The extreme asymmetry seen from the binding of bongkrekic acid and the atractylosides has led Klingenberg (1976b) and his group (Klingenberg et al., 1976, 1977) now to favour a model of the fixed pore type--a so-called 'gated pore' mechanism. In this model which is essentially a channel spanning the membrane, the protein remains fixed, the substrate is squeezed through the channel and at any one time the substrate binding site is facing either the inside, or the outside (see Fig. 6). It is still a reorientating mechanism in that the substrate site is orientated to one or other side of the membrane. The binding sites are activated for translocation only

FIG.5. A mobilereorientatingcarrier model.In this modelthe bindingsite switchesto the other side of the membraneby rotational movementof the carrier protein.

Inhibitors of the adenine nucleotide translocase

341

FIG. 6. An asymmetric gated pore model (after Klingenberg et al., 1977).(A) c-state; when the binding site faces the outside or inter-membrane space. (B) activated c-state: when the substrate is bound to the carrier and a conformational change takes place. (C) activated m-state : when the substrate : carrier complex faces the matrix. (D) m-state; when the substrate is released and the carrier faces the matrix. According to Klingenberg et al. (1977) the binding site has a region common to both orientation states, and that for each state, additional specific regions are operative in binding the substrate.

by the simultaneous binding of the substrates, ADP on one side of the membrane and ATP on the other, thus allowing a counter exchange. It is proposed that the substrate binding site consists of a central binding area and outer binding areas which are specifically active in either the c- or m-states (see Fig. 6). Since the reorientation model postulates identical binding sites for substrate and inhibitors it has to be assumed that this site is in the central binding area that is common to both orientation states and that for each state additional specific regions are operative in binding the substrate. In the absence of ADP the carrier is expected to be mainly in the c-state and requires ADP to catalyse the transition to the m-state for bongkrekic acid binding (Klingenberg et al., 1977). To explain the asymmetry of the translocase, the model requires that there are conformational differences between the two carrier-substrate complexes. Evidence for this comes from two types of experiment ; in sonic particles (inside-out vesicles) (Klingenberg, 1977b; Lauquin et al., 1977b) where ADP transport is inhibited by bongkrekic acid, but not by the atractylosides unless the vesicles have been preloaded with these inhibitors; and from antibody evidence (Buchanan et al., 1976) where both the.carboxyatractyloside binding protein (Klingenberg et al., 1978) and the bongkrekic acid binding protein (Aquila et al., 1978), supposedly the outer facing carrier complex (c-state) and inner facing carrier complex (m-state) respectively, have been shown to have similar molecular weights, amino acid compositions and isoelectric points but differ in antibody specificity and reactivity to -SH

342

MARION STUBBS

groups. The conclusion is drawn that the two proteins represent different conformational states of the carrier. Other evidence to support the asymmetric gated pore model comes from studies on the isolated inhibitor binding proteins (Riccio et al., 1975a,b; Klingenberg et al., 1978) where it is indicated that the translocase is a dimer consisting of two subunits each of 30,000 mol. wt. Further studies, such as, the reconstitution experiments of Kramer and Klingenberg (1977a) and Kramer et al. (1977) have shown that it is possible to incorporate the isolated carrier proteins into liposomes and to reconstitute carboxyatractyloside and bongkrekic acid binding. An ADP catalysed transition between the c-state and m-state of the carrier which is an integral part of the reorientating model has also been confirmed in reconstitution studies (Kramer and Klingenberg, 1977b). The other approach to the elucidation of a mechanism for the translocase has been the use of modified inhibitors, such as spin-labelled acyl atractyloside (Lauquin et al., 1977a), photoaffinity labelled aryl-azido atractyloside (Lauquin et al., 1976b) and spin-labelled acylCoA (Devaux et al., 1975) and modified substrates such as photoaffinity labelled analogues of ADP (Sch/ifer et al., 1976; Lauquin et al., 1978). Experiments using such probes have revealed much information about the carrier in situ. The studies with spin-labelled acyl atractyloside (Lauquin et al., 1977a) have shown that the carrier, when inhibited by atractyloside, penetrates the mitochondrial membrane only partially, and this finding could be accommodated in a mechanism of the asymmetric gated pore type. However, alternative models are proposed for both the spin-labelled acyl atractyloside findings and for the results from submitochondrial particle experiments (Lauquin et al., 1977b). Thus, although several possibilities still exist the asymmetry of the carrier is agreed implicating two different conformational states (Lauquin et al., 1977b; Klingenberg et al., 1978) and a gated pore mechanism appears at the present state of knowledge to be a satisfactory model for the translocase. However, details of whether the inhibitors and substrates bind to the same or different sites is still unclear (Vignais, 1976; Lauquin et al., 1977b; Klingenberg, 1976b; Klingenberg et al., 1977). 10. DISCUSSION 10.1. CONTROVERSIALISSUES CONCERNING THE REGULATION OF THE TRANSLOCASE The experimental material used most commonly for studies on the adenine nucleotide translocase and its inhibitors has been isolated mitochondria, complemented by some experiments on sub mitochondrial particles, inside-out vesicles and more recently on the isolated atractyloside, carboxyatractyloside and bongkrekic acid-binding proteins. More intact systems, such as isolated perfused liver (Tager et al., 1973) and isolated hepatocytes (Akerboom et al., 1977; Stubbs et al., 1978) have been studied only rarely. The problem in such intact systems is the interpretation of the effects of the inhibitors when so many different metabolic pathways are operative. However, such experiments attempt to give insight into the functioning of the adenine nucleotide transl9case in vivo. A real understanding of its nature, its mechanism of action and its regulation can only be achieved by a wide variety of experiments that complement each other. For example, taken in isolation the fact that fatty acyl-CoAs inhibit the translocase in isolated liver mitochondria looks interesting--but taken with a view to the role of the translocator in liver metabolism in vivo an inhibition by acylCoAs makes little physiological sense since in situations such as starvation and long term exercise where fats are an important fuel, the translocase needs to be working at high capacity in order to deliver the ADP to the respiratory chain and the ATP to the energy requiring sites in the cytosol. In fact, no inhibition of the translocase by long chain acyl-CoAs is observed in isolated hepatocytes (Akerboom et ai., 1977; see also Morel et al., 1974). One area of controversy concerning the regulation of the translocase concerns the interpretation of data from isolated mitochondria on the one hand and from isolated hepatocytes on the other hand. The question being asked is whether the translocase is rate-

Inhibitors of the adenine nucleotidetranslocase

343

limiting for oxidative phosphorylation and bearing on this question is whether the rate of respiration is dependent on the cytosolic [ATP]/[ADP] ratio or the cytosolic [ATP]/[ADP] x [Pi] ratio. Davis and Lumeng (1975), Kiister et al. (1976) and Davis and Davis-van Thienen (1978) claim that respiration is dependent on the [ATP]/[ADP] ratio in isolated mitochondria and therefore that the translocase is rate-limiting. This finding is disputed by Holian et al. (1977) who found that 02 uptake was dependent on the [ATP]/[ADP] x [Pi] ratio over a range of [Pi] from 1.5 to 8.5 mu in isolated mitochondria and therefore that the translocase was not rate-limiting. The reasons for the differences between the groups are not clear although neither Davis and Lumeng (1975) nor Kiister et al. (1976) varied [Pi] in their experiments. In isolated hepatocytes, however, the 02 uptake remained relatively constant over a 10-fold change in [ATP]/[ADP] ratio, suggesting that respiration is not dependent on the [ATP]/[ADP] ratio (Erecifiska et al., 1977), thus supporting the data of Holian et al. (1977). One of the problems in trying to reconcile these different points of view is that in the more intact systems (isolated hepatocytes or perfused organ) the highest cytosolic [ATP]/[ADP] x [Pi] ratio achieved in rat liver hepatocytes is about 3000 M- 1 (Akerboom et al., 1978 ; see also Wilson et al., 1974b). This value is found with a cytosolic Pi concentration of about 3 mu. In isolated mitochondria much higher values can be attained--as high as 46,000 u with succinate as substrate (Erecifiska et al., 1974). The reasons for these differences need to be considered. In vivo there is a constant supply of ADP to the mitochondria formed by cytosolic ATP consuming biosynthetic processes, e.g. glucose or urea synthesis in liver. However, when mitochondria are isolated and incubated in the presence of substrate, ADP and Pi, no such ATP consuming systems are present unless either a glucose-hexokinase trap (Kiister et al., 1976) or purified mitochondrial ATPase* are added as an ADP regenerating system (Davis and Lumeng, 1975). Even under these conditions the [ATP]/[-ADP] x [Pi] ratios attained are still very much higher than those actually measured in the more intact systems. Davis and Lumeng (1975) report a value of 15,000M -1 at physiological concentrations of Mg 2 ÷ where oxygen uptake was 70 per cent of the state 3 (see Chance and Williams, 1956) respiration and Davis and Davis-van Thienen (1978) report values between 10,000 and 60,000 M- 1 at physiological Pi concentrations. Only when the Pi is 27 mM (a very unphysiological concentration for most tissues) is the [ATP]/[ADP] x [Pi] ratio similar to that measured in hepatocytes and when [Pi] is in the physiological range (3 raM) then Davis and Davis-van Thienen (1978) show that respiration could be correlated to the [ATP]/[ADP] x [Pi] ratio but exclude the possibility by stating that at low Pi concentrations the Pi transport becomes limiting. Akerboom et al. (1977) concluded from experiments with isolated hepatocytes where atractyloside inhibited the synthesis of glucose from lactate, a process which depends upon the availability ofcytosolic ATP and therefore on translocation, that the translocase is ratelimiting in vivo. However, this argument is not valid since in any sequence of reactions (where every step depends on the substrate supply by the preceding step) inhibition of any single step is liable to cause an inhibition of the overall process. Hence the parallelism between the inhibition of an intermediary step and the overall rate is not necessarily evidence of rate limitation by the individual step under normal conditions. So is the translocator rate-limiting for oxidative phosphorylation in vivo? The following pieces of evidence, together with the arguments put forward above, suggest that it is not. It may well become rate-limiting under certain conditions in isolated mitochondria but the following points suggest that at least under physiological conditions it does not limit the rate of oxidative phosphorylation of ADP (Stubbs et al., 1978): (a) the K s for ADP for the translocator is low, < 12/.tM whereas the substrate concentration is relatively high, at least 115/~M (Akerboom et al., 1978) so that it is not substrate limited; (b) the rate of translocation at 37°C (measured in isolated mitochondria; see Fig. 2 and Table 1) is as high as, if not higher than, the maximum rate of oxidative phosphorylation seen in isolated hepatocytes (Stubbs et al., 1978); (c) near-equilibrium exists between the first two sites of oxidative phosphorylation * ATPase: Adenosine5'-triphosphatase.

344

MARIONSTUBB$

and the external (cytosolic) [ATP]/[ADP] x [Pi] ratio according to the following equation : K =

[NAD+][cytochome C2+] 2 x [ATP] 2 [NADH][cytochrome C3+] 2 x [ADP] 2 x [Pi] 2

where ATP, ADP and Pi are cytosolic values and NAD and NADH are free mitochondrial values. Translocation which is an obligatory intermediate in the reaction sequence cannot therefore be rate-limiting (Wilson et al., 1974a,b) and (d) as stated above the rate of respiration in hepatocytes is not dependent on the ATP/ADP ratio (Erecifiska et al., 1977). For further discussion, see Stubbs et al. (1978). If the translocase is not rate-limiting for oxidative phosphorylation and it is just an intermediate step in an overall equilibrium between the cytosolic phosphorylation state and mitochondrial respiratory chain (Wilson et al., 1974a,b) then the proposals of Kemp and Out (1975) that there is a direct interaction between the translocase and the oxidative phosphorylation machinery that does not involve the intramitochondrial adenine nucleotides, is very attractive. Experimental evidence from Vignais et al. (1975), Kemp and Out (1975) and Out et al. (1976) suggest that the translocase interacts with the F1ATPase* in such a way that the ATP formed during oxidative phosphorylation of external ADP is transported to the cytosol by the transiocase without first being released into the matrix. In other words, the translocase and ATPase are functionally linked to catalyse phosphorylation or dephosphorylation of extramitochondrial ADP or ATP without participation of the intramitochondrial adenine nucleotides( see also Vignais, 1976). Such a direct link between the respiratory chain and the external adenine nucleotides would remove the necessity for an electrogenic exchange such as that proposed by Klingenberg (1970), The preferential uptake of ADP could be explained by an increase in affinity of the translocator for ADP by an intrinsic conformational change in the mitochondrial membrane (Souverijn et al., 1973; Souverijn, 1974). This view is, however, disputed by Klingenberg (1977a) who claims that external ADP mixes freely with the endogenous pool after it has been translocated, and similarly that the ATP formed from ADP phosphorylation mixes with the endogenous (matrix) ATP before being translocated to the cytosol. Klingenberg's view is that the preferential uptake of ADP (in energy rich conditions) causes the external ATP/ADP ratio to be higher than the internal ATP/ADP ratio (see also Klingenberg, 1970) and that this difference corresponds to the membrane potential. Thus the energy from the respiratory chain is divided into two portions, one part (70-80 per cent) for the phosphorylation of ADP and one part (20-30 per cent) for the translocation of adenine nucleotides from a low internal ATP/ADP ratio to a high external ratio.

10.2. CONCLUDING REMARKS

Such controversy is stimulating indeed and only more experiments can answer the three most important questions concerned with the adenine nucleotide translocator: (a) is the translocase rate-limiting for oxidative phosphorylation, (b) is there a direct link between the translocase and the F1ATPase and (c) does the transiocase operate by a so-called 'reorientating mechanism' or some other? One very recent proposal which raises even more questions about the translocase and its regulation is the finding of Reynafarje and Lehninger (1978) that there is another transport system that is atractyloside-insensitive and electroneutral and functions only when ATP is being transported from the cytosol into the mitochondria as follows: ATP 4- (out) - - ~ ADP 3- (in) + 0.5 Pi 2- (in) Its biological role would be in ATP dependent reversed electron transport. * F1ATPase: Adenosine 5'-triphosphatase containing coupling Factor I.

1Y

Uncompetitive

Aryl-azido atractyloside Spin labelled acyl atractyloside Agaric acid N-ethyl maleimide Bongkrekic acid

Isobongkrekic acid

Not known Not known

Thyroid hormone Glucagon

Penetrant and non-penetrant Not known Not known

Penetrant

Non-penetrant Penetrant Penetrant

Non-penetrant Non-penetrant

Non-penetrant Non-penetrant Non-penetrant Non-penetrant

Non-penetrant

Penetrant’ or non-penetrantt

Can be spin labelled. Acts at both inner and outer sides of the mitochondrial membrane Causes increase in intramitochondrial adenine nucleotide content

Active only after preincubation with ADP Active only at acidic pH and at temperatures above 15-20°C

-

(3) (4)

COOH in equatorial rather than axial position Can be covalently linked to sepharose for affinity chromatography Photo affinity labelled

(10) (11917)

(9913714)

(7)

(8) (12) (15716)

(5) (6)

(1) (1)

(42)

Reference

Uncompetitive according to (2)

Comment

of inhibitors and effecters of the adenine nucleotide translocase

-_.

1

.

.

-..

_

-

.1 _ _

_ _

-.

._

^_

.--_

* Penetrant : generally lipophilic, act at matrix side of inner mitochondrial membrane. t Non-penetrant: generally hydrophilic, act at outer surface of inner mitochondrial membrane. (1) Vignais et al., 1973a; (2) Klingenberg, 1976a; (3) Riccio et al., 1973; (4) Brandolin et a/., 1974; (5) Lauquin et al., 1976b; (6) Lauquin et al., 1977a; (7) Lauquin et al., 1976a; (8) Chavez and Clapp, 1975; (9) Morel et al., 1974; (10) Babior et al., 1973; (11) Bryla et al., 1977; (12) Vignais and Vignais, 1972,1973; (13) Devaux et a[., 1975; (14) Chua and Shrago, 1977; (15) Erdelt et al., 1972; (16) Lauquin and Vignais, 1976; (17) PrpiC et al., 1978).

Competitive

Long-chain acyl CoA

Physiological

Competitive Mixed competitive and non-competitive Competitive Synergistic Uncompetitive, synergistic

Carboxyatractyloside Apocarboxyatractyloside Epi-atractyloside Succinylatractyloside

Type of inhibition (with respect to ADP)

Competitive or partially competitive Non-competitive Competitive Uncompetitive Competitive

Atractyloside

Chemical

Inhibitor or effe-ctor

TABLE 2. Summary

346

MARIONSTUBBS SUPPLEMENTARY

READING

1. VIGNAIS, P. M., VIGNAIS, P. V. a n d DEFAYE, G. (1978) S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p o f atractyloside and diterpenoid derivatives on oxidative phosphorylation and adeninen u c l e o t i d e t r a n s l o c a t i o n in m i t o c h o n d r i a . I n : A t r a c t y l o s i d e : C h e m i s t r y , B i o c h e m i s t r y a n d T o x i c o l o g y , pp. 3 9 - 6 8 . SANTI, R. a n d LUCCIANL S. (eds.). P i c c i n M e d i c a l B o o k s , P a d o v a . 2. VIGNAIS, P. i . , BRANDOLIN, G., LAUQUIN, G. J. M. a n d CHABERT, J. (1979) (3H)- o r (asS)l a b e l e d a t r a c t y l o s i d e a n d c a r b o x y a t r a c t y l o s i d e , a t r a c t y l o s i d e d e r i v a t i v e s u s e d for affinity c h r o m a t o g r a p h y , p h o t o a f f i n i t y l a b e l i n g a n d s p i n l a b e l i n g , a n d (3H)- o r ( t * C ) - l a b e l e d b o n g k r e k i c acid. I n : M e t h o d s in E n z y m o l o g y , Vol. LV. A c a d e m i c Press, N e w Y o r k , U.S.A. Acknowledoements--My thanks are due to Madge Barber for secretarial help, to Derek Williamson for critical reading of the manuscript, and to Pierre and Paulette Vignais for helpful advice.

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