Does sucrose space exist?

J. theor. Biol. (1981) 90,317-336

Does Sucrose Space Exist? V. SITARAMAM

AND M. K. JANARDANA

SARMA

National Institute of Nutrition, Indian Council of Medical Research, Jamai Osmania (P.O.), Hyderabad 500 007, A.P., India (Received

15 May 1980)

Subcellular organelles, on isolation in sucrose media, have sucrose in the inner space. The paradoxical observations of osmotic behaviour in the presence of external sucrose and rapid kinetics of entry of sucrose in mitochondria have led to the postulation of a two-compartment model, i.e. sucrose space hypothesis. A critical reappraisal of earlier work has suggested the alternative hypothesis of altered permeability of biological membranes to sucrose during centrifugation. Based on the robust physical theory of osmosis, direct experimental strategies are evolved to evaluate the sucrose space hypothesis. Some of the theoretical and experimental consequences of gravity-mediated entry of sucrose are explored. 1. Introduction

Sucrose entry into subcellular particles has remained an unsolved riddle in biochemical methodology pertaining to subcellular fractionation, for over two decades. Sucrose being the most commonly employed solute in the preparation of dense media for differential and gradient centrifugation, entry of sucrose into subcellular particles engaged the attention of the earlier workers (for reviews vide de Duve, 1965; Tedeschi, 1971). The concept of sucrose space was specifically formulated to overcome the anomalous experimental observations in mitochondria (Werkheiser and Bartley, 1957), which behave as true osmometers with regard to external sucrose concentrations (Tedeschi and Harris, 1955), and yet exhibit rapid kinetics of sucrose entry (Tedeschi, 1965; Jackson and Pace, 1956; Amoore and Bartley, 1958). Sucrose space, once hypothesized, has come to stay though the central premises of the hypothesis have not been fully tested with appropriate experimental logic. A detailed evaluation of sucrose space hypothesis by Tedeschi (1971) suggests that single space models would fit most of the experimental data though not conclusively. A solution to the sucrose space puzzle would be of great importance in terms of our understanding of the permeability of biological membranes. As a first step, it is necessary to 317

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@ 1981 Academic

Press Inc. (London)

Ltd.

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.I.

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develop an experimental logic that critically evaluates the sucrose space hypothesis. In this paper, we trace the history and the experimental logic employed thus far in studies related to sucrose space. Based on a critical evaluation of these, we further evolve an experimental logic to falsify the hypothesis. The need for such an exercise emerges from our own experimental work and from literature analysis, which support our claim that biological membranes become permeable to sucrose when subjected to gravitational forces. We restrict the scope of this paper to a theoretical analysis and the corroborative experimental evidence will be published elsewhere. Such data, when obtained from different laboratories independently, would consolidate the view that sucrose space does not exist.

2. History of Sucrose Space The advent of centrifugation in biochemical methodology necessitated the identification of proper media of centrifugation. In one of the earliest comparisons of such media, the mitochondrial integrity was seen to be best preserved, when these are isolated in sucrose, as opposed to isotonic NaCl, KC1 solutions or distilled water (Hogeboom et al., 1948). The high solubility of sucrose in water, its relative inertness, lack of charge, availability in reasonable purity in bulk, low cost, low partition in the oil phase were some of its virtues that contributed to its popularity among biochemists as a medium of choice for differential and gradient centrifugation. The deciding factor was, naturally, the good quality of the organelles, and that indeed was the case (Wainio, 1970). Electron microscopy of mitochondria contributed significantly in the choice of the methods of isolation (Pallade, 1953; Wainio, 1970) as also the integrity of oxidative phosphorylation, a singularly most important function of the mitochondrial membranes. Sucrose is foreign to the tissuesand is metabolically inert, with the exception of the brush border of intestinal villi, which possesssucrase (Malathi.et al, 1973; Ulshen and Grand, 1979). Colligative properties of sucrose, specifically its osmotic property, received much attention, as it was realized that sucrose would affect the membranous structures due to its osmotic activity. The most detailed studies of its kind were performed by Tedeschi and Harris (1955), wherein they employed light scattering to detect changes in the organelle volume in the presence of varying concentrations of external sucrose. These studies showed that mitochondria behave asperfect “osmometers” in the presence of external sucrose indicating their relative impermeability to sucrose. It was, therefore, puzzling that sucrose can penetrate biological membranes,

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as also shown by other workers (Jackson and Pace, 1956; Amoore and Bartley, 1958). The problem of sucrose entry was specifically investigated in considerable depth by Werkheiser and Bartley (1957); here the term “sucrose space” was coined for the first time. Amoore and Bartley (1958) have demonstrated the presence of sucrose in different populations of mitochondria, in which the internal sucrose concentration varied inversely with the internal potassium concentration. Since it would be unlikely that mitochondria would accumulate sucrose against a concentration gradient, it was necessary to postulate the presence of “sucrose space” as a sub-space of the mitochondrial volume. Fortuitously, mitochondria indeed were demonstrated to have two membranes by electron microscopy (Pallade, 1953) and the intermembranous space was considered, provisionally, to be the possible site for mitochondrial sucrose. It was, therefore, necessary to postulate that the outer membrane is leaky to small molecular weight compounds, whereas the inner membrane is impermeable to sucrose, to account for its simultaneous osmotic behaviour (Fig. 1).

Qt 12

34

FIG. 1. Sucrose space in mitochondria. The upper figure represents the relative proportions of compartments as visualized by Werkheiser and Bartley (1957). Note the large intermembranous space required to accommodate sucrose at an isotonic concentration. The lower figure is a diagramatic representation of the mitochondrial cross-section as usually seen by electron microscopy. 1: outer membrane, 2: intermembranous space, 3: inner membrane, 4: matrix space.

An important consequence of sucrose entry was immediately perceived as reflected in its effect on the sedimentation properties of mitochondria (de Duve et al., 1959). When particles are sedimented by gradient (isopycnic) centrifugation, in the absence of solute exchange between the particle and the medium, the following relationships hold good at osmotic equilibrium . . . r$=f#&i(l+E)

bbm

320

V.

SITARAMAM

AN11

Pp

= pdpw,

M.

-vdff

N + m -.

li

.I.

SARKli\

(3)

+&m

where, 4, CL, pP are the initial volume, the mass and the density of the particles; C$d,pd, pd the volume, the mass and the density of the particle entirely deprived of the solvent; (Y the amount of osmotically active substances inside the particle, in millimoles per unit mass of the particle entirely deprived of the solvent; p,,. the density of the solvent; p,, the density of the solutes in solution; V the average partial molal volume of the solutes, cm3 mmolee’ ; m the total molal concentration of the solutes in mole kg ’ of solvent; and, c their total molar concentration in mole 1-l of solution. Given the presence of a “sucrose space” which equilibrates readily with external medium, an additional volume can be defined as #J,, = fiC$d of density pm, i.e. the density of the medium. Thus equations (l)-(3) become 4 =&(l+g+E)

pm

(1’)

(2'1 (3') The importance of the experiments of Tedeschi and Harris (1955) comes from the observation that mitochondria from rat liver obey equation (l), or its more general form equation (l’), and can be fitted to yield the values for parameters such as CX, +d, ,.& pd. It must, however, be noted that (Y is assumed to be constant under both normal and high gravitational fields. Further, Tedeschi’s data do not distinguish between equations (1) and (l’), as an analysis of osmotic behaviour resolves only the “dead space”, which may or may not include sucrose space. Entry of sucrose into other subcellular organelles such as lysosomes and microbodies was soon established and the concept of sucrose space was extended to these organelles as well, even though these particles have a single limiting membrane. The evidence was, at best, partial on a morphological plane for the presence of subspaces in these organelles (cf. de Duve, 1965). The presence of sucrose is also observed in synaptosomes which are pinched off and resealed nerve ending particles and not strictly subcellular organelles (Sitaramam and Sarma, unpublished observations).

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The historical accident of the demonstration of sucrose space in mitochondria, however hypothetical, was further supported by the observation that the outer membrane of the mitochondria is indeed permeable to several small molecular weight substances (O’Brien and Brierly, 1965). The suggested leakiness of outer membranes is reinforced by experimental observations that only the inner membrane appears to be the real barrier to substances. This leakiness would be difficult to visualize in pure lipid membranes and recent evidence appears to indicate the presence of voltage sensitive anion dependent channels in the outer membranes of mitochondria (Colombini, 1979). The universality of sucrose permeation across biological membranes was shrouded by the overpowering mitochondrial example. For several reasons that we shall presently explore, the most common denominator in all these studies, namely centrifugation of the particles, was not identified as the causal agent of sucrose entry, though speculated briefly in the pioneering work of Amoore and Bartley (1958). In order to examine the validity of sucrose space hypothesis, we shall first delineate the inconsistencies in the existing evidence for sucrose space. It must be clearly understood that the sucrose space, as postulated, was not a kinetic convenience, but indeed a physical entity. Identification of experimental strategies to falsify this hypothesis, therefore, must center around the physical nature of sucrose space. 3. inconsistencies

in Sucrose Space Hypothesis

It is tedious and even unnecessary to discuss in detail various experiments in support or against the hypothesis. Reviews by de Duve (1965) and Tedeschi (1971) address themselves to this task quite exhaustively. Here we restrict the analysis of pertinent literature to identify and evaluate the central premises inherent in the hypothesis. 1. Experimental demonstration of sucrose space is typified by highly inconsistent data, wherein the reported values vary from 34 to 82%, from the same laboratory and even in a single publication (Amoore and Bartley, 1958; Tedeschi, 1971). 2. By and large, the intermembranous space in mitochondria in situ or on isolation appears to be much smaller compared to that projected from sucrose space data (Fig. 1) (cf. Pallade, 1953). 3. Kinetics of sucrose penetration were suggested to be rapid initially and considerably reduced, subsequently. Sucrose being both foreign and inert, it is unlikely that specific proteinaceous channels exist to facilitate its entry. In the absence of specific pores, the entry of sucrose may

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.I.

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be considered to occur by diffusion across the lipid membrane, without directional selectivity. Thus, one would expect to demonstrate rapid kinetics of exit of mitochondrial sucrose as well, which indeed was observed by Amoore and Bartley (1958). Together with their observation that the sucrose concentration within the mitochondria was always less than that of the medium, (the authors uniformly employed centrifugation to wash the pellet in non-sucrose media, and mitochondrial sucrose was estimated in the pellet) the rapid kinetics of permeation of sucrose could only support the two-compartment (sucrose space) hypothesis. The shortest time demonstrated in which the mitochondrial sucrose equilibrated with the medium was of the order of 2 min! However, in these experiments, the amount actually penetrating the membrane was highly variable. At the time of these experiments, the structure of biological membranes was much less known than today. No reference was made by the authors, theoretically or experimentally, to the component of the cell membrane that sucrose actually traverses (lipid or protein space?). 4. Studies on osmotic behaviour of mitochondria necessarily involve exposure of the particles to varying concentrations of external solutes (e.g. sucrose, NaCl, KCl, etc.) over a wide range. Permeability studies with specific solutes were also carried out under similar conditions, especially in relation to sucrose space hypothesis (Tedeschi and Harris, 1955; de Duve, 1965). Implicit in such studies was the assumption that such experimental manipulations would not perturb the membrane. Actually it is a hazardous assumption as repeatedly confirmed in literature and also in our own experience (vi& infra). The activities of several membrane bound enzymes, particularly those which are integral proteins, are sensitive indices to any perturbations of membranes. For instance the activity of the externally faced NAD(P)H oxidase of the plasma membrane shows marked dependence on the solute concentrations in the extra-cellular medium (Takanaka and O’Brien, 1975). The 2,4-dinitrophenol stimulated ATPase activity in mitochondria shows marked dependence on external solute concentrations, (Cereijo-Santalo, 1972) which is, at least in part, due to osmotic effects. Presence of cations such as Na’ and K’ alters the Donnan potentials acrossmembranes, which may selectively influence permeability of membranes. For these reasons, it is best to avoid conditions other than the isotonic, in the determination of permeability to solutes across biological membranes. This is not often adhered to in literature and therefore such studies are difficult to evaluate. Further, in studies related to sucrose space, a variety of aqueous and

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also non-aqueous media were employed and the effects of these agents on membranes are ill-detined. 5. The origin of the sucrose space hypothesis lies in studies on mitochondria and the single most important test for the hypothesis rests on the physical identification of sucrose space by direct experimentation. Clearly, presence or absence of sucrose in the matrix space (Fig. l), which is forbidden on osmotic grounds, is the deciding experiment to falsify this hypothesis. Such an experiment was never carried out! Given the demonstration of sucrose in matrix space which is enclosed by two membranes, sucrose space hypothesis in otherwise patently single membrane systems (e.g. lysosomes, synaptosomes) hardly requires repudiation by experiments. Experimental demonstration of sucrose in mitochondrial matrix space is fraught with difficulties. The problem is one of selective removal of the outer membrane, without disturbing the inner membrane. Normally inner membrane particles are prepared from intact mitochondria by detergent treatment (right side out) or by sonication (inside out) (Malviya et al., 1968). Sonication, being destructive, would be inconclusive as sucrose would leak out of motochondria. Detergents are known to alter permeability characteristics of membranes. Presence of sucrose in matrix space, if demonstrated, would directly be in conflict with the reported rapid entry-exit kinetics of sucrose, unless one postulates altered permeability of membranes exclusively during centrifugation. It is inconceivable otherwise that the same membrane system would at once be permeable (in terms of rapid entry kinetics) and also impermeable (in terms of osmotic behaviour). A trivial explanation for sucrose entry during centrifugation would be breakage and resealing of mitochondria during centrifugation. As Tedeschi (1971) points out, such a situation would be reflected in sucrose density gradients, where one can expect to find separation of intact and damaged mitochondria. Distribution of mitochondria, by and large, does not appear to be bimodal under gradient centrifugation conditions (Beaufay and Berthet, 1963). In the differential centrifugation method normally used, the gravitational forces employed are usually much smaller (-8000g) as opposed to density gradient centrifugation (-50 000 to 300 000 g, cf. Wattiaux et al., 1974). Though damage cannot be critically ruled out, universality of sucrose entry and the low g’s commonly employed do not appear to warrant the postulation of centrifugationmediated damage to mitochondria. Thus, the presence of sucrose in the matrix space would argue for gravity-mediated entry of sucrose across membranes. Experimental demonstration of sucrose in the matrix space would further be complicated

324

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Ai’iD

M.

K.

I.

SARhl\

as centrifugation cannot be employed to harvest the pellet from washes, iest redistribution of the internal sucrose should occur. Instead, one should USC some sieving technique to separate the membranes from the medium, e.g. millipore filtration. This would necessarily mean that the amount of protein that can be filtered would be the limiting factor and methods of detection of solutes would have to be highly sensitive. One may conceive a prototype experiment to determine the localization of sucrose using digitonin. Mitochondria may be subjected to progressive solubilization in varying concentrations of digitonin, which is known to solubilize the outer membrane somewhat selectively in a concentrationdependent manner (Hoppel and Cooper, 1968; Levy, Toure and Andre. 1966). Such suspensions may be washed on millipore filters and one can estimate enzymes and solutes in the unsolubilized material retained on the filters. Figure 2 represents the data one would expect to find in such an experiment using monoamine oxidase (MAO) as a marker for the outer membrane and succinate dehydrogenase as a marker for the inner membrane or fumarase as a marker for the matrix space, plotted against digitonin concentration in the medium. If sucrose is also estimated, it may be expected to follow the solubilization curve of either MAO or fumarase, suggesting that it is localized in the outer or inner compartments, respectively (Fig. 2). 100

FIG. 2. Effect of digitonin on the solubilization of mitochondrial membranes. As digitonin is known to solubilize the outer membranes selectively, monoamine oxidase (--) would disappear from the membranes first, followed by succinic dehydrogenase (inner membrane marker) or fumarase (matrix space marker) (--I. As the active site of succinic dehydrogenase faces the interior of the inner membrane, its osmotic activity is equivalent to that of fumarase, a matrix enzyme. Digitonin concentration in the medium is plotted against the projected specific activities of marker enzymes retained on the millipore (cf. Wojtczak and Zaluska. 1969).

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EXIST

Such an experiment would be quite difficult, as only about 150-200 kg of total mitochondrial protein can be loaded on standard millipore filters (0.45 Frn pore size, 25 mm diameter), which makes it rather difficult to assay sucrose spectrophotometrically (e.g. Kulka, 1956). Further, if sucrose is present in the matrix space as well as the intermembranous space, results would be quite ambiguous. Thus alternative strategies need to be evolved for the localization of sucrose in the mitochondrial compartments. The osmotic behaviour of occluded enzyme activity is employed in the demonstration of the integrity of biological membranes (Appelmans and de Duve, 1955; Marchbanks, 1967). We shall now formalize the theory of such experiments in order to investigate the nature of sucrose space. 4. Osmotic

Behaviour (A)

THE

of Occluded ERYTHROCYTE

(Particulate)

Enzymes

EXAMPLE

Under standard conditions of pH and temperature, exposure of erythrocytes to varying concentrations of NaCl (O-l-0.9%) results in a typical curve of osmolysis (Fig. 3). The normal erythrolytic curve exhibits two sharp

External

NoCl

concentrotlon

(% w/v)

FIG. 3. Standard osmolysis curve of erythrocytes. Under standard conditions of pH and temperature, erythrocytes are incubated in the presence of varying concentrations of NaCI, centrifuged and the amount of hemoglobin (Hb) in the supernatant is measured spectrophotometrically. Per cent hemolysis is plotted against the ambient NaCl concentration. ISO: the isotonic concentration of NaCI; broken line, the inflection point (I.P.) corresponding to -50% lysis.

326

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SlTARAhlAM

ANI)

hl.

K

.I

SAKXl,\

bends in the hypotonic region corresponding to the beginning and completion of lysis as a function of ambient NaCl concentration, the inflection point corresponds to approximately half the isotonic NaCl concentration. With progressive hypotonicity, the initial swelling and the catastrophic lysis arc the expected consequences of the relationship: +l...

v<

\;.,

where V is the cell volume as 7’0 of its value in isotonic solution, Vc is the critical cell volume beyond which the cell lyses as the pressure within the cell would surpass the limit of the elastic modulus of the cell membrane, W is the water content of cells at 9 = 1, and 9, the tonicity (osmolarity of the medium as % of the osmolarity of an isotonic medium) and R, an empirical constant proposed by Ponder (1948). When the volume increase, due to water imbibition, is beyond the critical volume, V,, the cell bursts releasing hemoglobin. Though permeability of cell membranes to small molecular weight substances varies, soluble enzymes would not be able to traverse the cell

FIG. 4. Enzyme osmometry. Specific activity of the enzyme is plotted against external solute concentration. ISO: isotonic concentration of external solute. In the hypertonic region, the membrane and its permeability characteristics may be unaffected (2), permeability to substrate may increase (1) or the permeability and/or the activity may be retarded (3). Activity of the free enzyme in the presence of external solute at varying concentrations, -. -. Vertical broken lines represent break-points; upper: B.P.o and lower: B.P.,.:obtained by extrapolation of corresponding slopes and projecting the points of intersection on to the ordinate.

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membrane. Using a relatively impermeable substrate, one can readily monitor the enzyme release in suspensions by measuring the enzyme activity. By titrating the activity of the released enzyme against osmotically active external solute concentrations, one obtains osmolytic curves comparable to the erythrolysis curves (Fig. 4), independent of the low substrate concentrations employed (Appelmans and de Duve, 1955). (B)

ENZYME

OSMOMETRY

AND

ITS

LIMITATIONS

Enzyme osmometry can be used to determine the integrity of the cell membrane, the internal solute concentrations (osmotically active), the kinetic constants of occluded enzymes, and also the permeability of membranes to specific molecules. For example, let there be three species A, B and C to which the membrane is permeable in the order A
O) IL A

Osmolorlty

B

I-

b)

c

of medum

-

External

solute

concentrotlon

+

FIG. 5. The influence of (a) the permeability to external solutes and (b) the concentration of internal solute, on occluded enzyme activity. (a) Specific activity of the occluded enzyme is plotted against osmolarity of the medium (=concentration of each solute multiplied by isotonic coefficient, i; i = n& + (l-G), where n is the number of ions the solute dissociates into, 6, degree of ionization). Permeability of the membrane to the solutes is of the order: A < B < C. (b) The intra-particular concentration of solute A is varied such that A 1-c A2 i A3. Specific activity is measured in the presence of the same external solute.

contain three concentrations of the same solute, A. Whichever solute is used for osmometric titration, the resulting relationships would be as in Fig. 5b. In such comparisons, one can use either of the break-points (i.e. upper or lower) for comparison and obtain direct relationship between breakpoints and either the permeability to or the internal concentrations of the solutes.

328

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AND

bl.

K.

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Si\Khl

\

Since most enzyme assays require appreciable amounts of buffer and substrates, it is necessary that these contribute as little as possible to the net osmolarity of the external medium. It is for this reason that one cannot always obtain the osmolytic curve in its entirety and hence the necessity to compare the break-points, usually the lower. Also, cell membranes ot macrophages and leukocytes, etc., are capable of withstanding treatment with even distilled water and therefore such analysis cannot be carried out in all cases. Interpretation of enzyme osmometry curves, these being but an indirect reflection of the enzyme protein released, must be done with certain caution and it is worth mentioning two specific assumptions that need to be experimentally verified for meaningful conclusions. Assumption I. The permeability to substrate is monotonically related to the volume changes in the particles. Consider a single membrane vesicle enclosing an enzyme, E, that catalyzes the simple reaction A *B. Let the rate constant of the reaction be K and the permeability of the membrane to species A be P and to the enzyme, zero (Fig. 6). A, represents the concentration of the substrate outside the vesicle and A;, concentration inside. Under initial velocity conditions, the rate of the reaction is Jr = KA,,/( 1 + K/P).

(5)

The rate of reaction is directly related to A, Ai = A,,/( 1 f K/P).

(6)

Near isotonic conditions, the slope of the activity plotted against external solute concentrations is entirely dependent on substrate permeability. Beyond the critical solute concentration corresponding to V, (equation 4).

t P A,A AO\ \ R GFIG. 6. Kinetics of occluded enzyme activity. Enzyme, E, is enclosed in a membranous vesicle. The rate constant of the enzyme, is K = K, = K ,, and the permeability of the membrane to substrate A is P.

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EXIST

the reaction velocity primarily depends on the availability of free enzyme. The slope of the curve is essentially determined by heterogeneity of the particles, it being infinite if the population is absolutely homogeneous. Marked non-linearities in permeability, P, in relation to changes in volume (i.e. stretching of the membrane) could significantly affect the baseline value of activity and obscure the break-point. The osmotic curve, however, should be independent of substrate concentration. Assumption II. The kinetic constants of the enzyme do not vary with external solute concentration (permeability to substrate being near zero).

Membrane-bound enzymes, with the active sites exposed to the interior, are particularly prone to this problem. The activity of membrane-bound enzymes is subject to viscotropic and interfacial regulations (Sandermann, 1978) and therefore can contribute to osmotic curves, without the membrane actually bursting. This phenomenon becomes patently obvious when other occluded enzymes are also simultaneously monitored, as in the case of plasma membrane NAD(P)H oxidase (Takanaka and O’Brien, 1975). Thus, demonstration of actual release of the enzyme, similar to the release of hemoglobin on erythrolysis, becomes necessary to be confident that one is truly dealing with osmolysis. In our experience, millipore technique is a valuable adjunct to osmometry experiments. If the activity A, is a function of the external solute concentration(s), i.e. A = f(S), the activity retained on the millipore filter on washing should be, A = 1 -f(S). It is perhaps necessary to comment that though much of this discussion is fairly simple, inadequate attention was paid to this physical approach in literature. Shift of osmotic curves of lysosomal acid hydrolases in lysosomes isolated in varying concentrations of external sucrose was repeatedly observed and was not interpreted to mean varying concentrations of sucrose acquired during centrifugation (Appelmans and de Duve, 1955; Reijngoud and Tager, 1977). Similarly, in mitochondrial studies, Wattiaux (1974) found enhanced basal activity of occluded enzymes in particles sedimenting at higher densities of ambient sucrose and concluded that the integrity of mitochondria was affected at high gravitational fields. Enhanced internal concentration of sucrose in mitochondria, isolated at higher ambient sucrose concentrations, would necessarily result in higher basal activity, due to osmolysis during fixed conditions of enzyme assay. (‘3

ADDITIONAL

EXPERIMENTAL

APPROACHES SPACE

TO

EVALUATE

SUCROSE

HYPOTHESIS

What are the factors that determine the external solute concentration at which the break-point occurs? The major determinants are: (a) internal

330

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M.

K.

.I

StiRh1.l

solute concentration, (b) elastic modulus of the membrane, Cc) heterogeneity of the particles (in terms of both (a) and (b)), and (d) permeability to external solutes. By appropriate experimental design, all these possibilities can be evaluated. If particles are isolated at various centrifugal forces in varying external sucrose concentrations, existence of direct relationship between the break-points and external sucrose concentrations would indicate enhanced permeability of the membranes to sucrose under conditions of gravity. (D)

LOCALIZATION

OF OF

SUCROSE

IN THE

MATRIX

SPACE

MITOCHONDRIA

The foregoing type of experiments would suffice to determine sucrose entry across single membrane systems such as synaptosomes, as also direct estimation of sucrose in these particles by millipore technique or by measuring the entry or exit of radioactive sucrose across these particles under isotonic conditions. In mitochondria, however, the presence of two compartments complicates the type of evidence required. True osmotic behaviour of the activity of an occluded enzyme reflects the localization of osmotically active solutes in the same compartment as that of the enzyme. Judicious use of density gradient centrifugation, enzyme osmometry and sucrose estimations would readily solve this riddle. Mitochondria are routinely isolated by differential centrifugation in 0.25 M sucrose and also in approximately I.4 M sucrose by density gradient centrifugation. If membranes become permeable to sucrose during centrifugation, the external isopycnic concentration of sucrose should match the

(:;I---E 0

10

20 External

0 sucrose

10

20

(Ml

FIG. 7. Localization of sucrose in mitochondrial matrix Mitochondria are isolated in 0.25 M and 1.4 M sucrose by projected activities of MAO and fumarase are titrated against in the assay medium. Figures represent the activity profiles, if A. Mitochondria isolated in 0.25 M sucrose; B. Mitochondiria activity of MAO; 2: activity of fumarase. Note the shift corresponding to sucrose concentration used for isolation.

space by enzyme osmometry. centrifugal sedimentation. The varying sucrose concentrations sucrose enters the matrix space. isolated in 1.4 M sucrose. 1: in the fumarase activity curve

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internal concentration of sucrose. By screening various enzymes differentially localized in mitochondria, one can identify enzymes that show corresponding shifts in osmometry profiles. Localization of sucrose in the matrix space is achieved when matrix enzymes show the expected osmotic behaviour, as in Fig. 7. As Tedeschi (1971) rightly points out, a single space model would fit most data on sucrose entry and new experimental approaches are necessary to solve the problem. The experimental approaches outlined here are straightforward utilizing sensitive enzymatic measurements and the interpretation is based on robust physical theory. Sucrose being an inert molecule, a more direct demonstration of the presence of sucrose by microelectrodes is not possible. In principle, one may use specially fabricated enzyme electrodes (e.g. with sucrase) for this purpose. Enzyme osmometry, on the other hand, yields the same answers in a much simpler form. 5. Conclusions

Presence of sucrose in matrix space of mitochondria falsifies the sucrose space hypothesis totally and the discussion, thus far, focussed on relevant experimental strategies. Given such data, the brute fact of sucrose entry forces a revision of several of the existing concepts in relation to the nature of biological membranes, permeability and the theory and practice of the isolation of subcellular organelles. The real “proof” for gravity-mediated sucrose entry would be the development of an adequate theory that could reconcile various aspects of the problem, since sucrose entry as a proven fact would be but an instance of a generality (Cohen and Nagel, 1968). It must be realized that altered permeability to sucrose during centrifugation would mean relatively instantaneous equilibration of the external sucrose with the internal space. It would mean that subcellular organelles isolated in, say 1.4 M sucrose, would have the same molarity of sucrose within! Thus when we discuss the mechanism of altered permeability under gravitational fields, the problem is not one of miniscule changes in true diffusion of sucrose, but a quite dramatic quantitative equilibration across the membrane. The primary concern regarding the entry of sucrose across the lipid bilayer, which is typically considered to be a “slab” of hydrocarbon, is thermodynamic. Hydrophilic solutes such as sucrose would require a very high energy of activation to traverse the lipid bilayer. For instance, the activation energy of erythritol, which is more permeable than sucrose (Carvalho and Carvalho, 1979), has an activation energy of about 2025 kcal mole-’ (De Gier et al., 1971). Considering the ubiquity of sucrose

332

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AND

M.

I(.

.I

S,\RM.\

entry through a variety of organelles, there is no compelling reason tcl postulate proteinaceous “pores”. The possibility of large water-filled pores through the lipid membrane is no longer considered likely, both on experimental and theoretical grounds (Hirsch, 1967). The high energy requirement is both interfacial and diffusional to overcome the frictional forces of the lipid milieu. In principle, as work needs to be done to displace mass across a distance, any work-related term would suffice to explain the passage of a solute across a membrane. For instance, at a single interface, the equilibrium distribution of a molecule in a gravitational field would be c, = c,, e TGxfKl

(7)

where C is the concentration of the solute in phases i and O, m; the mass, g, the acceleration due to gravity; x, the distance; K, the Boltzman constant: and T, the temperature. The problem, however, is one of overcoming the activation energy, as the gravitational forces employed may not be adequate. A useful approach appears to be that of Zwolinski et al. (19491, who assumed that the molecules would dissolve in the bilayer and move across by diffusion. The molecules are considered to flow by jumps between equilibrium positions. Figure 8 illustrates the energy profile for a typical homogeneous lipid membrane. If the length between potential energy

-K,,,-

FIG. 8. Potential energy See text for explanation.

profile

of a small molecule

across a homogeneous

lipid membrane.

DOES

minima

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is A and the number of jumps, m: mh =S

where S is the thickness of the membrane. given by 1

2

~=k,,h+k,h(k,,lk,,,)’

(8) The permeability m

constant, P. is

(9)

where k,, is the rate constant at the interface for diffusion into membrane, k,, is the rate constant of exit from the membrane and k, is the rate constant of diffusion in the lipid phase. The partition coefficient, B, for the molecule would be k,,/k,,. In relation to sucrose entry across biological membranes, the problem is one of evaluation of the resistance encountered at the interface as well as the solubility and rate of diffusion in the lipid phase. For hydrophilic solutes like sucrose we expect that k,, 2 k,,. Further, the membrane is not homogeneous in that the microviscosity in the fatty acid chain region decreases several fold resulting in a variable k,. Lee (1976), therefore, suggested that the permeability coefficient for the membrane would be better described as (10) where R represents the resistance of the membrane-water interface to solute flow, and D,(x) is the diffusion coefficient (=k,h*) and B(x), the partition coefficient, at a distance, x, from the interface. If sucrose entry is demonstrated across biological membranes under conditions of gravitational force, the activation energy for sucrose entry compounded with high hydrostatic pressure may require energization levels much higher than that provided by the g’s employed; one is severely constrained by the existing models of biological membranes (with only two degrees of freedom in the plane of the membrane) to explain this apparent contradiction. Further, as gravitational force is exerted on the membrane, simultaneously, hydrostatic pressure also acts on these membranes, given by the relation (11) where, w, is the angular velocity in radians per second; p, the density of the medium; rl, the distance from the meniscus; r,, the radius at the meniscus: and PO is the hydrostatic pressure at the meniscus. Hydrostatic pressure is

334

‘. \I I ,i x A\?,‘\ \1 .\ v ! 1 \I

h

.> .. ,; ?,i

known to retard the permeability of molecules across membranes IJohnson, Miller and Bangham, 1973). which is reversed by tcrnper’aturts. l‘hus entrx of sucrose due to gravity should be considered asan effect distinct from the effect of hydrostatic pressure. During density gradlent ientrifugation. hydrostatic pressures of several hundred atmospheres ,LI’V commonly encountered. The effects of gravity and hydrostatic pj cssure. being opposite and simultaneous, would be expected to interact Lvith the sedimentinp particles in a complex non-linear mode and need to be critically accounted for in a qualitative analysis of their relative influences. These considerations lead us to a re-evaluation of the so called “deleterious effect” of hydrostatic pressure on subcellular particles under conditions of centrifugation (Wattiaux, 1974; Wattiaux et al., 1971 1. Wattiaux et (11. have shown that mitochondria of rat liver sediment at a higher isopycmc concentration of sucrose when subjected to high temperatures (lS”C), than those centrifuged at 0-4°C. By estimating the occluded enzyme activities of sulfite-cyt. I’ reductase and malate dehydrogenase, these authors have found that the particles isolated at low temperature have high basal activity and that high temperature (15°C) “protected” mitochondria against the high basal activity. Parallel evidence for damage at high centrifugal forces and low temperature was obtained by electron microscopy by the sameauthors. The implicit assumption in Wattiaux’s work was that sucrose does not enter mitochondria and that all fractions of mitochondria have the same isotonic requirements, be it occluded enzyme activity or be it fixation for electron microscopy. Gravity-mediated entry of sucrose, that is inhibited at high temperatures, would offer a radically different explanation for this important methodological work, in that, mitochondria are not significantly damaged by the high hydrostatic pressure created by centrifugation and the high basal activity and altered morphology of these particles was due to inadequate attention to proper isotonic conditions. The choice of right centrifugation medium for the isolation of subcellular organelles has occupied the attention of biochemists for a long time. The criteria for the selection of medium are essentially two-fold: (a) preservation of the structure as encountered in situ, (b) preservation of appropriate functions (such as tight respiratory coupling in mitochondria). During centrifugation, if mitochondria become permeable to sucrose and not to the internal solutes, then the external osmotic pressure becomes near zero and mitochondria would swell and even burst. Such not being the case, mitochondrial permeability to the dominant internal solutes also should alter, lowering the value of cy (equation ( 1i). If sucrosedoes not enter during centrifugation, as is conventionally believed, mitochondria would only shrink as they penetrate sucrose concentrations of higher osmolarity during

DOES

SUCROSE

SPACE

EXIST

335

centrifugation. In practice, the in viuo appearance of mitochondria is not always preserved on isolation in 0.25 M sucrose media though claims vastly differ in literature (Wainio, 1970). If mitochondria become permeable to both internal and external solutes on centrifugation, water imbibition would occur due to the presence of soluble protein which contributes to colloidal pressure. Thus it is not surprising that several authors recommended the inclusion of colloidal substances in sucrose media, such as glycogen, polyvinylpyrrolidone, and ficoll (cf. Wainio, 1970). In view of permeability to sucrose during centrifugation, inclusion of high molecular weight substances in preparative media could be logical. The mitochondrial example would serve well to illustrate the import of this line of reasoning. Oxidative capacity of mitochondria was found to be more susceptible to changes in physical state than P : 0 ratios (Slater and Cleland, 1953). Respiratory indices greater than 30 obtained by workers using heparin and albumin (e.g. Dow, 19671, as opposed to the usual values of 4-12 (Wainio, 1970) deserve close attention, in the evaluation of the integrity of isolated mitochondria. Gravity-mediated entry and exit of solutes requires special attention in the analysis of stoichiometric relations which are of cardinal importance in bioenergetics. Similarly, in uptake studies, centrifugation of particles should be viewed with suspicion in view of possible equilibration of solutes across the membrane during centrifugation. We have thus far outlined a theoretical analysis of sucrose space hypothesis and identified experimental strategies for the falsification of this hypothesis. Demonstration of sucrose entry under conditions of gravity opens up Pandora’s box, and many theoretical and experimental problems surface. We have experimentally evaluated the sucrose space hypothesis, based on the logic developed in this paper and have come to the conclusion that sucrose space does not exist. A mechanistic explanation for the altered permeability to sucrose appears to require a modified view of the current models of biological membranes, as a consequence of energization. The experimental data and the theoretical consequences of sucrose entry will be published elsewhere. The authors are grateful to Prof. D. Balasubramanyam, Dr. M. W. Pandit, Dr. N. J. Rao, Prof. A. N. Radhakrishnan and Prof. T. Ramasarmafor reading the manuscriptand for helpful discussions. REFERENCES AMOORE, J. E. & BARTLEY, W. (1958). Biochem. J. 69,223. APPELMANS,F.& DE DUVE, C. (1955). Bi0chem.J. 59,426. BANGHAM, A.J. SCLEE,E.J. A. (1978). Biochem. biophys. Acta 511,388.

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