ascorbate/N,N,N' ,N' -tetramethyl-p-phenylenediamine/ cytochrome c, a high proton-motive force (>130 mV), inside negative and alkaline, can be generated in ...
Proc. Nati. Acad. Sci. USA Vol. 82, pp. 7555-7559, November 1985 Biochemistry
Incorporation of beef heart cytochrome c oxidase as a proton-motive force-generating mechanism in bacterial membrane vesicles (membrane fusion/secondary solute transport)
ARNOLD J. M. DRIESSEN, WIM
DE
VRIJ,
AND
WIL N. KONINGS*
Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
Communicated by L. N. M. Duysens, July 19, 1985
ABSTRACT Membrane vesicles derived from the strictly fermentative lactic acid bacterium Streptococcus cremoris have been fused with proteoliposomes containing the beef heart mitochondrial cytochrome c oxidase by means of a freeze/thawsonication technique. Evidence that fusion has taken place was obtained by freeze-etch electron microscopy, showing a lessdense intramembranous particle distribution in the 'fused membranes than in the bacterial membranes, and by sucrose gradient centrifugation, indicating a buoyant density of the majority of the membranes after fusion that was between the buoyant densities of the starting membrane preparations. In the fused membranes, 55-60% of the cytochrome c oxidase molecules are oriented with the cytochrome c binding site at the outer surface of the membrane. With the electron-donor system ascorbate/N,N,N' ,N' -tetramethyl-p-phenylenediamine/ cytochrome c, a high proton-motive force (>130 mV), inside negative and alkaline, can be generated in the fused membrane, and this proton-motive force can drive secondary transport of several amino acids. The procedure described can be used for incorporating a proton-motive force-generating system in isolated membrane vesicles from bacterial or eukaryotic origin that lack a suitable primary proton pump.
Many biological membranes, such as the cell membranes of eukaryotic cells or fermentative bacteria, however, do not contain such proton-translocating electron-transfer systems. The only proton pump in many of these membranes is the proton-translocating ATPases. In membrane vesicles with the same polarity as the cytoplasmic membrane of whole cells, these ATPases are located at the inner side of the membrane and, therefore, are not accessible for ATP. Some procedures are available to generate artificially a Ap or one of its components in these systems. For instance, an electrical potential A4 can be generated by valinomycin-mediated potassium movement. However, all of these procedures lead to the generation of gradients with a transient character, which severely limits the applicability ofthese procedures for detailed studies of membrane functions (4, 5). To overcome these experimental difficulties, we have developed a procedure for the incorporation of a powerful Ap-generating system into biological membranes by fusion of membrane vesicles with liposomes in which this Ap-generating system has been incorporated. We have applied this procedure to incorporate beef heart cytochrome c oxidase in membrane vesicles of the homofermentative bacterium Streptococcus cremoris. With the electron donor ascorbate/ N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)/cytochrome c, a high Ap, inside negative and alkaline, can be generated in these fused membranes, and this Ap can drive secondary transport of several amino acids.
Studies of the role of the proton-motive force (Ap) on membrane-bound proteins are often hampered by the lack of good model systems. Ideally the protein(s) under study should be functionally incorporated together with a Apgenerating system in membranes that form closed structures and can maintain a Ap for some length of time. If the protein is isolated and purified, it can be incorporated together with a Ap-generating system into proteoliposomes. This advanced stage has been reached for the lactose permease (protein M) of Escherichia coli, which has been coreconstituted in liposomes with cytochrome o oxidase (1) and for the alanine carmer of the thermophilic bacterium PS3 which has been coreconstituted with beef heart mitochondrial cytochrome c oxidase (2). Most membrane proteins, which depend on the Ap for activity, have not been isolated, and the'activity of these proteins can only be studied in their natural membranes. Prbcedures have been developed for the isolation of membrane vesicles with defined polarity. These membrane vesicles are excellent model systems for studies of energytransducing processes, if a Ap of the desired magnitude and polarity can be generated. This is, for instance, the situation in membrane vesicles from bacteria that contain protontranslocating electron-transfer systems. In the presence of a suitable electron donor and acceptor, a Ap can be generated across the membrane that forms the driving force for many
MATERIALS AND METHODS Growth of S. cremoris and Isolation of Membrane Vesicles. S. cremoris Wg2 (prtr) was grown anaerobically on MRS (22) broth at a controlled pH of 6.4 in a 5-liter fermenter. Membrane vesicles of S. cremoris were prepared as described (4), suspended in 50 mM potassium phosphate (pH 7.0) containing 10 mM MgSO4 at a concentration of 10-15 mg of protein per ml and stored in liquid nitrogen. Incorporation of Cytochrome c Oxidase in Liposomes. Cytochrome c oxidase was isolated from beef heart as described by Yu et al. (6). The heme content was determined spectrophotometrically using an absorption coefficient of 13.5 mM-1 cm-1 for reduced minus oxidized heme at the wavelength couple 605 - 630 nm (7). The heme content was found to be 10.4 nmol/mg of protein. Cytochrome c oxidase was incorporated' into liposomes by a modification of the procedure of Hinkle et al. (8). Acetone-washed asolectine (40 mg) dispersed in 2 ml of 10 mM K-Hepes (pH 7.0) containing Abbreviations: Ap, transmembrane electrochemical potential; Adi, transmembrane electrical potential; ApH, transmembrane pH gradient; N-NBD-PtdEtn, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine; Ph4P', tetraphenylphosphoniumr; TMPD,
bacterial solute transport systems (3). The publication costs of this article were defrayed in part by page charge
N,NN',N'-tetramnethyl-p-phenylenediamine; S-13, 5-chloro-3-tert-
payment. This article must therefore be hereby marked "advertisement"
butyl-2'-chloro-4'-nitrosalicylanilide. *To whom reprint requests should be addressed.
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Biochemistry: Driessen et al.
45 mM KC1 and 30 mM octyl /-D-glucopyranoside (octyl glucoside) was sonicated to clarity under a constant stream of nitrogen gas at 40C using a probe-type sonicator (MSE Scientific Instruments, West Sussex, U.K.). After the addition of 9 nmol of heme a, the solution was shaken and dialyzed at 40C for 4 hr against a 500-fold volume of 10 mM K-Hepes (pH 7.0) supplemented with 45 mM KCl and subsequently was dialyzed against the same medium supplemented with 5 mM Ca42. Dialysis was continued overnight at 40C against a 500-fold volume of this buffer without CaCl2 to remove excess calcium. Under these conditions, about 35 mmol of Ca2+ per mol of phospholipid remained attached to the liposomes. The diameter of the liposomes obtained with this procedure varied between 40 and 300 nm, as observed in electron micrographs of negatively stained liposomes. The liposomes showed a respiratory control index of 2.5-3.0 (7). Fusion of Cytochrome c Oidase Proteoliposomes with S. cremoris Membrane Vesicles. S. cremoris membrane vesicles, 75 1.d containing 1.2 mg of protein, and 500 ,u1 of cytochrome
Proc. Natl. Acad. Sci. USA 82
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c oxidase proteoliposomes containing 1.125 nmol of cytochrome c oxidase were mixed and rapidly frozen in liquid nitrogen. The material was thawed for 20 min at room temperature, mixed with a Vortex, and subsequently sonicated for 30-60 sec in a plastic tube with a bath-type sonicator (Sonicor 50 Watt, Sonicor Instruments, New York). Determination of Ai# and ApH. & (interior negative) was determined from the distribution of tetraphenylphosphonium ion (Ph4P+) across the membrane as measured with a Ph4P+selective electrode (9). Reaction mixtures contained (final concentrations) 10 mM K-Hepes (pH 7.0), 45 mM KCl, 2 AM Ph4P', 5 mM MgCl2, and membranes as indicated in a total volume of 1.0 ml. The accumulation of Ph4P+ was calculated from the amount of Ph4P+ that disappeared from the external medium. The Ai was calculated with the Nernst equation. A correction for concentration-dependent binding of Ph4P+ to the membranes, according to the model of Lolkema et al. (10), was applied. ApH (interior alkaline) was determined
FIG. 1. Freeze-fracture images of the external fracture (EF) (A, C, and E) and protoplast fracture (PF) (B, D, and F) sites of S. cremoris membrane vesicles (A and B) proteoliposomes containing cytochrome c oxidase (C and D), and freeze/thaw-sonication vesicles (E and F). (Bar = 0.1 ,um.)
Biochemistry: Driessen et al. from the distribution of ['4C]acetate by using automated flow dialysis as described (11). For calculation of Aqi and ApH, the internal volume of the cytochrome c oxidase proteoliposomes was estimated from the trapped amount of calcein (12). This internal volume was found to be 5 1.l per mg of phospholipid. The internal volume of the S. cremoris membrane vesicles was found to be 4.3 ptl/mg of protein (4). A value of 8 ul/mg of protein was determined from the trapped amount of calcein and from the equilibration of [14C]leucine under nonenergized conditions for the fused membranes. Sucrose Gradient Centrifugation. Discontinuous sucrose gradients were formed in Beckman ultracentrifugation tubes on top of a 1-ml 65% (wt/wt) sucrose cushion. The gradients were formed from the following sucrose concentrations in 10 mM Tricine (pH 8.0) containing 100 mM KCl and 1 mM EDTA: 15% (1.5 ml), 30% (1 ml), 34% (1 ml), 38% (1 ml), 42% (i ml), and 46% (1 ml). Fused membranes or a mixture of cytochrome c oxidase proteoliposomes (0.042 nmol of oxidase) and S. cremoris membrane vesicles (0.52 mg of protein) were layered on top of the gradient. The gradients were centrifuged for 19 hr at 145,000 x g in a SW 41 rotor (180C). After fractionation, protein was determined by using the Bradford procedure (13), and liposomal phospholipid was determined by assaying N-(7-nitro-2,1,3-benzoxadiazol-4yl)phosphatidylethanolamine (N-NBD-PtdEtn) fluorescence using an excitation and emission wavelength of 475 and 530 nm (14). N-NBD-PtdEtn was incorporated into the cytochrome c oxidase proteoliposomes during detergent dialysis with a concentration of 0.5 mol% of total phospholipid. Other Analytical Procedures. Uptake of [U-14C]leucine by the fused membranes was measured by filtration as described (15). Cytochrome c oxidase activity was measured at room temperature by following the decrease in absorbance of the peak of cytochrome c and using an absorption coefficient (reduced minus oxidized) of 19.5 mM-1 cm-1 (550 - 540 nm) (7). The orientation of cytochrome c oxidase was determined as described by Casey et al. (16). For freeze-fracture electron microscopy, concentrated suspensions of membranes in 10% glycerol were rapidly frozen in liquid Freon 12. Freezeetched replicas were prepared by the method of Moor (17) with a Balzers BA 360 freeze-etching unit (Balzers, Liechtenstein). Protein (18) and phospholipid phosphorus (19) were assayed as described. Materials. Octyl glucoside, crude asolectine, and horse heart cytochrome c were purchased from Sigma. Asolectine was prepared from crude asolectine as described (20). NNBD-PtdEtn was kindly provided by J. Wilschut (Dept. of Physiological Chemistry, University of Groningen, The Netherlands).
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appeared to be lost upon fusion with cytochrome c oxidase proteoliposomes (compare Fig. 1 E and F with A and B). Concomitant with the dilution of intramembranous parti-
cles from the S. cremoris membrane vesicles into the lessparticle-dense liposomal membrane, intermixing of membrane phospholipids occurred as indicated by the dilution of nonexchangeable fluorescent phospholipids originally present in the bacterial or liposomal membranes (not shown). Analysis of the fused membranes on a sucrose gradient revealed the appearance of a band with a buoyant density intermediate between those of the cytochrome c oxidase proteoliposomes and S. cremoris membrane vesicles (Fig. 2). At the ratio of cytochrome c oxidase proteoliposomes to S. cremoris membrane vesicles (0.07 nmol of oxidase per mg of S. cremoris protein) that was used in this experiment, the band contained all of the liposomal phospholipids and S. cremoris membrane vesicle proteins. The buoyant density of this band decreased with increasing liposome-to-bacterial membrane ratios (not shown). At the ratio of cytochrome c oxidase proteoliposomes to S. cremoris membrane vesicles (0.96 nmol of oxidase per mg of S. cremoris protein) that was used for further experiments, approximately 5% of the liposomes were recovered at their original buoyant density (not shown). All S. cremoris membrane vesicles moved to lower densities, indicating that all membrane vesicles had fused. The cytochrome c binding sites were exposed to the outer
200
210110
RESULTS Fusion of S. cremoris Membrane Vesicles with Cytochrome c Oxidase Proteoliposomes. To incorporate beef heart cytochrome c oxidase in membrane vesicles of S. cremoris,
asolectine proteoliposomes containing beef heart cytochrome c oxidase were fused with S. cremoris membrane vesicles by rapid freezing followed by slow thawing and a brief sonication step. Evidence for fusion has been supplied by freeze-etch electron microscopy (Fig. 1). Membrane vesicles from S. cremoris contain a high density of particles on the extracellular fracture phase (Fig. IA), while proteoliposomes have a less-dense particle distribution (Fig. 1C). Upon freeze/thaw-sonication treatment, the particle density became intermediate between these extremes (Fig. 1E), indicating a dilution of the particles of S. cremoris in the plane of the liposomal membrane. The original asymmetrical distribution of particles in the extracellular and protoplast fracture phases of S. cremoris vesicles
10
15 Fraction number
FIG. 2. Sucrose gradient centrifugation of a suspension of S. cremoris membrane vesicles mixed with cytochrome c oxidase proteoliposomes (A) and freeze/thaw-sonication vesicles (B). Sucrose gradient centrifugation was performed as described. After fractionation of the gradients, fractions were assayed for protein (e), liposomal phospholipid (o), and refractive indices. The liposomal phospholipid content was determined from the fluorescence (in arbitrary units, a.u.) of N-NBD-PtdEtn, which was incorporated into the cytochrome c oxidase proteoliposomes at a concentration of 0.5 mol% total phospholipid. Densities (i) were estimated from the
refractive indices.
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Table 1. Quantitation 'of A /i (interior negative) and ApH (interior alkaline) generated in cytoclirome c oxidase proteoliposomes (COVs) and in fused membranes (FTS) upon addition of ascorbate (20 mM) and cytochrome c (20 ,uM) FTS COVs A*, -Z&pH, , A A*/i, -ZApH, Apj Additions mV mV mV mV mY mW ND ND ND -49 -83 ND None TMPD (0.4 mM) -92 -46 -138 -68 -67 -135 TMPD (0.4 mM)/ valinomycin (4 -76 -76 -54 0 -54 0 AM) TMPD (0.4 dtM)/ nigericin (40 -76 0 -101 -76 0 -101 nM) Cytbchrome c oxidage proteoliposomes with a cytochrome c oxidase-to-phospholipid ratio of 1:5500 were prepared as described and fused with S. cremoris membrane vesicles at a cytochrome c oxidase-to-S. ctermoris membrane protein ratio of 0.96 (nmol/mg of protein). Membrane quantities containing 0.23 nmol of cytochrome c oxidase were used for At determinations. Flow dialysis was performed with fused membranes containing 0.39 nmol of cytochrome c oxidase and with proteoliposomes containing 0.68 nmol of cytochrome c oxidase. ND, not determined. A Z-value of 5 g was used to convert ApH units into mV.
surface of the membrane in the proteoliposomes for 62-64% and in the fused membranes for 55-60%. Generation of a Ap in the Fused Membranes. The energyconserving properties of the fused membranes were investigated by measurements of the Ap generated upon addition of the electron donor cytochrome c (20 ,4M) in the presence of ascorbate (20 mM). The magnitude of the A/vi, estimated from the accumulation of the lipophilic cation P1hP1, and the ApHi estimated from the accumulation of the weak acid acetate, in the fused membranes and the cytochrome c oxidase proteo-
(1985)
liposomes are summarized in Table 1. The A/i generated by the addition of ascorbate/cytochrome c was significantly stimulated by the electron mediator TMPD (400 AM). Similar Ati values were obtained when high concentrations of ascorbate (up to 50 mM) without TMPD were used. Under those conditions, the turnover of the oxidase was stimulated and the ratio of reduced to oxidized cytochrome c increased (not shown), suggesting that electron transfer between ascorbate and oxidized cytochrome c is the rate-limiting stepin electron transfer to oxygen. Low concentrations of nigericin (40 nM), an ionophore that catalyzes electroneutral exchange of protons for K+, stimulated Adi generation and dissipated the ApH. On the other hand, valinomycin (4 1uM), which mediates the influx of K', caused a rapid release of accumulated Ph4P+ and induced a small but significant increase in ApH. Ap generation was completely dependent on the presence of cytochrome c oxidase and was decreased by the cytochrome c oxidase inhibitor cyanide and by uncouplers (not shown). Generation of a Ap was only observed after brief sonication of the fused membranes. Presumably sonication decreased the ion permeability of the membrane (21). Proton-Motive Force-Driven Solute Transport in the Fused Membranes. Since the fused membranes contain secondary transport proteins and an effective Ap-generating system, it offers an excellent model system for studies of secondary transport processes. This has been demonstrated for the secondary transport system for leucine. The uptake of leucine was studied under conditions such that only a Ati could be generated (in the presence of nigericin). In the presence of the electron-donor system ascorbate/TMPD/ cytochrome c, leucine was rapidly accumulated by the fused membrane vesicles (Fig. 3A), and a steady-state level of accumulation was reached in about 3 min. Under these experimental conditions, no accumulation of leucine could be demonstrated in cytochrome c oxidase proteoliposomes or in S. cremoris membrane vesicles (Fig. 3A). The Ap generated and the rate of leucine uptake in the fused membranes
A
15_ x
E
¢10 l/
5C1
Time (min)
Time (min)
FIG. 3. Leucine transport in membrane vesicles of S. cremoris fused with cytochrome c oxidase proteoliposomes by freeze/thaw-sonication. (A) Leucine uptake induced by the addition of ascorbate/TMPD/cytothrome c in S. cremoris membrane vesicles (0.05 mg of protein) alone (e), cytochrome c oxidase proteoliposomes (0.05 nmol) (o), and fused membranes (o) (containing 0.05 mg of protein and 0.049 nmol of oxidase). For ascorbate/TMPD/cytochrome c-driven uptake of leucine, 20 ,uM cytochrome c was added to 100 Al of a solution containing 10 mM K-Hepes (pH 7,0), 45 mM KCl, 200 ILM TMPD, 10 mM ascorbate, and 40 nM nigericin. [U-_4C]leucine (12 TBq/mol) was added 30 sec after the initiation of the experiment to a final concentration of 3.8 AiM. (B) Effect of cyanide (o), nigericin (n), valinomycin (0), and S-13 (m) on ascorbate/TMPD/cytochrome c-driven leucine transport (o). The experiments were performed as described for ascorbate/TMPD/cytochrome c-driven leucine transport except that, where indicated, cyanide (1 mM), nigericin (40 nM), valinomycin (4 AM), or S-13 (10 ALM) were present.
Biochemistry: Driessen et al. Table 2. Ap and leucine transport activity in S. cremoris membrane vesicles fused with cytochrome c oxidase proteoliposomes upon addition of ascorbate (10 mM)/ TMPD (200 juM)/cytochrome c (10 AtM) Ratio CytOxase Ratio asolectine Initial rate of leucine (nmol)/S. cremoris (mg)/S. cremoris Ap,* protein (mg) protein (mg) mV uptaket 0.24 2.2 -19 0.01 4.2 0.49 -33 0.18 8.6 0.96 -62 0.37 1.45 12.9 -69 0.45 17.1 1.93 -60 0.23 Various amounts of cytochrome c oxidase proteoliposomes were fused with S. cremoris membrane vesicles, giving the indicated cytochrome c oxidase (CytOxase) to S. cremoris membrane protein ratios. Fusion was performed by freeze/thaw-sonication with a fixed volume of 0.6 ml and 0.72 mg of S. cremoris membrane protein. *Ap was estimated from the accumulation of Ph4P1 in the presence of 40 nM nigericin. tThe initial rate of leucine uptake, shown in nmol of leucine per mg of S. cremoris protein per min, was measured over the first 15 sec as described in the legend to Fig. 3.
increased when fusion of S. cremoris membrane vesicles was performed with increasing quantities of cytochrome c oxidase proteoliposomes (Table 2). The highest rate of leucine uptake was observed in the presence of ascorbate/ TMPD/cytochrome c (not shown). Uptake of leucine was almost completely blocked by cyanide (1 mM), a potent inhibitor of cytochrome c oxidase, and by the uncoupler S-13 (5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide; 10 ,M) (Fig. 3B). Valinomycin (4 ,AM) also inhibited strongly ascorbate/TMPD/cytochrome c-driven leucine uptake. Dissipation of the ApH with a concomitant increase of the A by low concentrations of nigericin (40 nM) decreased leucine uptake only to a minor extent. These findings indicate that the steady-state level of leucine accumulation is dependent on a Ap generated by cytochrome c oxidase.
DISCUSSION This paper describes a simple procedure for the functional incorporation into biological membrane vesicles of a powerful mechanism for the generation of a Ap, inside negative and alkaline. The Ap-generating system used is beef heart cytochrome c oxidase. This enzyme has almost ideal features for its use as a generator of a Ap with the right polarity in membrane vesicles: (i) it can be obtained in pure form and in relatively large quantities with simple procedures; (ii) it retains almost full activity during the isolation and the incorporation procedures; (iii) only molecules with the rightside-out orientation in the membrane vesicle will be reduced with the electron-donor system ascorbate/TMPD/ cytochrome c; and (iv) it has a high turnover rate, and the incorporation of a few molecules in the vesicular membrane is sufficient for the generation ofa high Ap. In this paper it has
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been shown that beef heart cytochrome c oxidase can generate a high Ap in membrane vesicles of the lactic acid bacterium S. cremoris and that this system can supply the energy for secondary transport of amino acids. The same procedure can also be used for the incorporation of other proton-generating systems such as bacteriorhodopsin (data not shown). The results show that this incorporation procedure of cytochrome c oxidase into biological membranes offers attractive possibilities for the energization of membrane vesicles of organisms that lack a suitable Ap-generating system. This includes membrane vesicles from fermentative bacteria and membrane vesicles derived from the cell membrane or organelle membrane from eukaryotic cells. With this system, the role of the Ap or one of its components in energy-dependent processes can be studied in these membranes. The authors thank M. Veenhuis and J. Zagers for the freezefracture electron micrographs presented in Fig. 1. This study has been made possible by the Stichting voor Biofysica, with financial support from the Netherlands Organization for the Advancement of Pure Scientific Research (Z.W.O.). 1. Matsushita, K., Patel, L., Gennis, R. B. & Kaback, H. R. (1983) Proc. Natl. Acad. Sci. USA 80, 4889-4893. 2. Hirata, H., Sone, N., Yoshida, M. & Kagawa, Y. (1977) J. Supramol. Struct. 6, 77-84. 3. Hellingwerf, K. J. & Konings, W. N. (1985) Adv. Microb. Physiol., in press. 4. Otto, R., Lageveen, R. G., Veldkamp, H. & Konings, W. N. (1982) J. Bacteriol. 149, 733-738. 5. Lancaster, J. R. & Hinkle, P. C. (1977) J. Biol. Chem. 252, 7657-7661. 6. Yu, C. A., Yu, L. & King, T. E. (1975) J. Biol. Chem. 250, 1383-1392. 7. Yonetani, T. (1965) J. Biol. Chem. 236, 1680-1688. 8. Hinkle, P. C., Kim, J. J. & Racker, E. (1972) J. Biol. Chem. 247, 1338-1339. 9. Shinbo, T., Kama, N., Kurihara, K. & Kobatake, Y. (1978) Arch. Biochem. Biophys. 187, 414-422. 10. Lolkema, J. S., Hellingwerf, K. J. & Konings, W. N. (1982) Biochim. Biophys. Acta 681, 85-94. 11. Hellingwerf, K. J. & Konings, W. N. (1980) Eur. J. Biochem. 106, 431-437. 12. Oko, N., Kendall, D. A. & MacDonald, R. C. (1982) Biochim. Biophys. Acta 691, 332-340. 13. Bradford, M. (1976) Anal. Biochem. 72, 248-254. 14. Struck, D. K., Hoekstra, D. & Pagano, R. E. (1981) Biochemistry 20, 4093-4099. 15. Kaback, H. R. (1974) Methods Enzymol. 31, 698-709. 16. Casey, R. P., Ariano, B. H. & Azzi, A. (1982) Eur. J. Biochem. 122, 313-318. 17. Moor, H. (1964) Z. Zellforsch. Mikrosk. Anat. 62, 546-580. 18. Lowry, 0. H., Rosebrough, N. J., Farr, A. J. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 19. Ames, B. N. & Dubin, D. T. (1960) J. Biol. Chem. 235, 769-775. 20. Kagawa, Y. & Racker, E. (1971) J. Biol. Chem. 246,
5477-5487. 21. Pick, U. (1981) Arch. Biochem. Biophys. 212, 186-194. 22. DeMan, J. C., Rogosa, M. & Sharpe, M. (1960) J. Appl. Bacteriol. 23, 130-135.
