Involvement of an essential arginyl residue in the coupling activity of Rhodospirillum rubrum chromatophores

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 190, No. 2, October, pp. 578-584, 1978

Involvement

of an Essential Arginyl Residue in the Coupling Rhodospirillum rubrum Chromatophores

RUBEN H. VALLEJOS, WANDA I. M. LESCANO,

AND

Activity of

HECTOR A. LUCERC

CEFOBI, Suipacha 531,2000 Rosario, Argentina, Centro de Estudios Fotosintkticos y Bioquimicos (Consejo National de Znvestigaciones Cientificas y Tknicas, Fundaci6n M. Lillo and Universidad National de Rosario) Received March 14, 1978; revised May 8, 1978 The arginine reagents phenylglyoxal and 2,3-butanedione in borate buffer completely inhibited photophosphorylation and Mg-ATPase of Rhodospirillum rubrum chromatophores. The inactivation rates followed apparent first order kinetics. Oxidative phosphorylation and the light-dependent ATP-P, exchange reactions of R. rubrum chromatophores and the Ca-ATPase activity of the soluble coupling factor were similarly inhibited by 2,3butanedione in borate buffer. The apparent order of reaction with respect to inhibitor concentrations for all these reactions gave values of near 1 suggesting that inactivation was the consequence of modifying one arginine per active site. ATP synthesis and hydrolysis by R. rubrum chromatophores were strongly protected against inactivation by ADP and ATP, respectively, and by other nucleotides that are substrates of the reactions but not by the products. Similarly, the Ca-ATPase of the soluble coupling factor was protected by ATP but not by ADP. Inactivation of chromatophores reactions by butanedione in borate buffer was more rapid in the light than in the dark. The results suggest that the catalytic sites for ATP synthesis and hydrolysis on the chromatophore coupling factor are different and both contain an essential arginine.

studies (24) that indicate that one arginine of lactate dehydrogenase interacts with the pyrophosphate bridge of NAD. Recently, Riordan and co-workers (9,10) have proposed that an arginyl residue is present in the ATP binding sites of creatine kinase, glut-amine synthetase and carbamylphosphate synthetase. The mitochondrial coupling factor or ATPase has been shown by Marcus et al. (11) to contain an arginine in its hydrolytic site. Andre0 and Vallejos (12) have found that the soluble spinach chloroplast ATPase has also one essential arginyl residue in its catalytic site that may play a role in binding ATP. Spinach chloroplast photophosphorylation, ATP-Pi exchange and Mg-ATPase activities were inhibited by phenylglyoxal and butanedione (13, 14). Inactivation followed modification of one arginine per active site and was prevented by adenine nucleotides (13). The arginine reagents behave as energy transfer inhibitors (14). In this paper we show that the arginine

Photosynthetic formation of ATP in chromatophores of the bacterium Rhodospirillum rubrum involves the participation of a coupling factor. This coupling factor has been solubilized and purified (1). It is a large molecule (d& = 350,000) with Ca-dependent ATPase activity. The molecular weight, subunit composition, sensitivity to antibiotics and other properties of the R. rubrum coupling factor are similar to those of mitochondrial (F1) and chloroplast (CFI) coupling factors (l-4). Chemical modification of enzymes with cu-dicarbonyl reagents such as monomeric 2,3-butanedione in borate buffer (5) or with phenylglyoxal (6, 7) has allowed the study of the eventual participation of arginine residues in the binding of anionic substrates or ligands to the enzymes (5, 8-23) since they are highly selective reagents for arginine under mild conditions. Contribution of arginine to the binding of nucleotides to the active site of enzymes has been shown by chemical modification (5, 8-23) and by X-rays crystallographic 578 0003-9861/78/1902-0578$02.00/O Copyright 0 1978by Academic Press, Inc. All rights of reproduction in any form reserved.

ESSENTIAL

ARGININE

IN Rhodospirillum

reagents 2,3-butanedione and phenylglyoxal completely inhibited ATP synthesis and hydrolysis and ATPaP: exchange reactions of R. rubrum chromatophores and the Ca-ATPase of their soluble coupling factor. The protection afforded by adenine nucleotides suggests that the sites for ATP synthesis and hydrolysis are different. MATERIALS

AND

METHODS

Rhodospirillum FUbFUm cells (Van Niel strain Sr) were grown anaerobically in the light in l-liter flasks at 30°C as described (25). Chromatophores were prepared as previously described (3), suspended in 250 mu sucrose, 20 mM Tris-HCl (pH 8) and 5 mM MgClz, and stored in liquid nitrogen. The concentration of Bchl’ was determined using the extinction coefficient of 140 mM-’ .crn-’ at 880 nm (26). The Ca-ATPase was solubilized from R. FUbFUm chromatophores and purified as described (1). Protein was determined according to Lowry et al. (27) with bovine serum albumin as the standard. Photophosphorylation was determined as described (3) in a reaction medium (1 ml) containing 30 mM Tris-HCl (pH 8), 5 mu MgC12, 300 PM PMS, 1 PM HOQNO, 2 mu ADP, 4 mM Pi (containing 10” cpm of 32P) and chromatophores corresponding to 10 pg Bchl. The light-dependent ATP-P, exchange reaction was determined in a similar reaction medium except that ADP was replaced by 4 mM ATP, P, was 7 mM (containing 5.10” cpm of “P) and PMS was 400 PM. Oxidative phosphorylation was determined as described (3) with NADH as substrate. The Mg-ATPase activity of chromatophores was determined in a reaction medium (1 ml) containing 50 mM Tris-HCl (pH 8), 5 mM ATP, 5 mM MgCh and chromatophores (30 pg of B&l). After 5 min at 30°C the reaction was stopped by adding 0.1 ml of trichloroacetic acid (50%, w/v) and the Pi liberated was determined calorimetrically according to Sumner (28). Ca-ATPase activity was determined in a reaction medium (0.25 ml) containing 0.1 M Tris-HCl (pH 8), 5 mM ATP, 5 mM CaCb and about 10 pg of the soluble ATPase. The Pi liberated was determined colorimetrically according to Sumner (28). Chemical modification of chromatophores and of the soluble Ca-ATPase with 2,3-butanedione or phenylglyoxal was carried out at 25°C in a medium containing 50 mu borate buffer (pH 7.8) appropriate concentrations of 2,3-butanedione or phenylglyoxal and chromatophores (0.4 mg of Bchl per ml) or CaATPase (0.2 mg of protein per ml). Samples were taken at different times and their activities assayed. ’ Abbreviations used: Bchl, bacteriochlorophyll; PMS, phenazine methosulfate; HOQNO, 2-n-heptyl-4hydroxyquinoline.

579

rubrum CHROMATOPHORES

When the reagent was phenylglyoxal the Tris-HCl buffer of the reaction media was replaced by the same concentration of borate buffer (pH 8) to avoid interference by Tris (6). Incubations of chromatophores were carried out in the dark unless otherwise stated. For these experiments chromatophores were suspended in 100 mM borate buffer (pH 8) 5 rn~ MgCh. Solutions of 2,3butanedione or phenylglyoxal were freshly prepared for each experiment. Concentrations about lo-fold higher than required were prepared in 100 mM borate buffer (pH 7.8). The pH of the solutions was adjusted with 1 N NaOH. 2,3-Butanedione was obtained from BDH Chemicals Ltd. (England). Phenylglyoxal and nucleotides were from Sigma Chemical Co. All other chemicals were of analytical grade. RESULTS

Incubation of R. rubrum chromatophores with the arginine reagents phenylglyoxal or 2,3-butanedione in borate buffer resulted in complete inhibition of the PMS-induced photophosphorylation. Figure 1 shows that

20

Lo

60

80

TIME (mm)

FIG. 1. Effect of 2,3-butanedione and phenylglyoxal on photophosphorylation in R. rubrum chromatophores. Experimental conditions were as described in the text. Control values were on average 451 pmol of ATP h-’ ‘rng-’ of B&l. Numerals on the slopes indicate concentration (mu) of butanedione (M) or phenylglyoxal (A-A) during preincubation with the chromatophores for the times stated.

580

VALLEJOS,

LESCANO,

the inactivation rates with several concentrations of both reagents followed apparent first order kinetics. Complete inhibition with the higher concentrations tested was achieved (not shown). Similar inhibitions of the Mg-dependent ATPase activity of R. rubrum chromatephores modified by both arginine reagents were also observed. The same R. rubrum chromatophores in the dark can form ATP coupled to the aerobic oxidation of NADH (29). The ATPPi exchange reaction of these chromatophores is greatly stimulated by light (30) and is considered to be a partial reaction of photophosphorylation. An inhibition of the oxidative phosphorylation and the light ATP-Pi exchange reaction of chromatephores by butanedione in borate buffer very similar to that of photophosphorylation was observed (not shown). Again the inactivation rates followed apparent first order kinetics. Johansson et al. (1) have purified from R. rubrum chromatophores a coupling fattor with Ca-ATPase activity. This coupling factor is involved in photophosphorylation and in the ATP-Pi exchange reaction and probably also in oxidative phosphorylation (31). When the coupling factor rebinds to the chromatophores, the Mg-ATPase activity is recovered (1). Figure 2 shows that incubation of the soluble coupling factor with increasing concentrations of 2,3-butanedione in borate buffer resulted in inhibition of the Ca-ATPase activity of the factor. The inactivation rates followed apparent first order kinetics. With 100 mM butanedione the ATPase activity was inhibited by 98% in 120 min. The apparent order of reaction (n) with respect to inhibitor concentration for all the reactions assayed can be calculated from a plot of the log of the pseudo-first order reaction constants (Fz’)against the log of the inhibitor concentrations as previously used by other authors (32-35) and ourselves (12,13). Figure 3 shows that such plots for butanedione inhibition of photophosphorylation (n = l.OO), Mg-ATPase (n = 0.97), oxidative phosphorylation (n = 0.93), the light-dependent ATP-Pi exchange reaction of chromatophores (n = 0.90) and

AND

LUCERO

2 50 3: E5

TlME ,m,nl FIG. 2. Effect of 2,3-butanedione on the Ca-ATPase activity of soluble coupling factor. Experimental conditions were as described in the text. Control values were on average 58 pm01 Pi. h-“ r ng-’ of B&l. Numerals on the slopes indicate concentration of butanedione (mu) during preincubation with the coupling factor

for the time stated. I

2 -

8 y : l5 ’ 1on5 075

, 1

I 15 lag IBUTANEDIONE)

2 tmM)

FIG. 3. Apparent order of reaction (n) with respect to 2,3-butanedione concentration. The values of k (pseudo-first order constant) were graphically calculated from the results of Figs. 1 and 2 and similar ones for oxidative phosphorylation, light dependent ATPP, exchange and Mg-ATPase of chromatophores and plotted as shown. The values of n obtained were 0.93 for oxidative phosphorylation (u), 0.90 for the light-dependent ATP-Pi exchange reaction (U--U), 0.91 for Ca-ATPase for the soluble coupling factor (A-A), 1.00 for photophosphorylation (H) and 0.97 for Mg-ATPase of chromatophores (A-----A).

the soluble Ca-ATPase (n = 0.91) all gave straight lines with slopes of nearly 1 suggesting that modification by butanedione of only 1 arginyl residue per active site produced the inhibition of these reactions. It is remarkable that the results obtained

ESSENTIAL

ARGININE

IN Rhodospirillum

with the soluble Ca-ATPase agree perfectly well with the inhibition of the photophosphorylation, oxidative phosphorylation, Mg-ATPase and ATP-Pi exchange activities of chromatophores shown in Figs. 1 to 3. They suggest that the modification by butanedione of only 1 arginine per active site, located in the coupling factor, is responsible of the described inhibition of the phosphorylating, ATP-Pi exchange and ATPase activities. Photophosphorylation and the Mg-dependent ATPase of chromatophores were more sensitive to phenylglyoxal than to butanedione (Fig. 1). The second order rate constants of the inhibition of these reactions calculated from plots of k’ vs. concentration of inhibitor were 3 and 1.6 M-’ - min-’ for phenylglyoxal and 1.2 and 0.6 M-’ . min-’ for butanedione. The n with respect to phenylglyoxal concentrations was also nearly 1. The complete inhibition of phosphorylating and ATPase activities of R. rubrum chromatophores by chemical modification of one arginyl residue per active site suggests that the involved arginine is in the active site. If such were the case protection by the substrate might be expected. Table I shows that indeed 25 mu ADP strongly protected photophosphorylation and oxidative phosphorylation against inactivation by butanedione while the same concentration of ATP was without effect. On the other hand, 25 I~WI ATP afforded a considerable protection of both the particulate, Mg-ATPase and the soluble Ca-ATPase while ADP did not protect at all. A similar protection by the substrates of photophosphorylation and Mg-ATPase against inactivation by phenylglyoxal is shown in Table I. The protection afforded to the chromatophores and coupling factor reactions by the substrates but not by the products against inactivation by the arginyl reagents used reinforces the suggestion that the arginine involved is in the active site. In addition to ADP, GDP, and IDP, which are also substrates of photophosphorylation, afforded a similar protection to the reaction against inactivation by butanedione (Table II) while CDP, UDP, and the

581

rubrum CHROMATOPHORES TABLE

I

PROTECTION BY ADP AND ATP AGAINST INACTIVATION OF R. rubrum CHROMATOPHORES AND SOLUBLE COUPLING FACTOR BY ARGININE REAGENTS” k’ (mini’)

Experiment Control

ADP

ATP

31.2 16.7 17.5

11.1 17.2 6.5

31.3 5.6 18.5

22.2

21.0

4.0

30.3 20.0

10.2 19.2

23.3 5.5

Chemical modification with 2,3-butanedione Photophosphorylation Mg-ATPase Oxidative phosphorylation Ca-ATPase of coupling factor

Chemical modification with phenylglyoxal Photophosphorylation Mg-ATPase

(LIn experiment 1, R. rubrum chromatophores or soluble coupling factor were incubated with 25 mM 2,3-butanedione in the absence or presence of 25 mM ADP or ATP in the experimental conditions described in the text. The activities of the reactions stated were measured in aliquots taken at different times of incubation as described in the text. The results obtained were plotted as in Figs. 1 and 2 and the stated pseudofirst order reaction constant, k’ (lOOO/half-time of inactivation) were calculated from the plots. In experiment 2, chromatophores were similarly treated with 10 mu phenylglyoxal. When present, ADP or ATP were 20 mM. TABLE II EFFECT OF NUCLEOSIDE DI- AND TRIPHOSPHATES ON THE INACTIVATION OF PHOTOPHOSPHORYLATION BY BUTANEDIONEAdditions

(25 mM)

None ADP GDP IDP CDP UDP ATP CTP GTP ITP

k’ (min-‘) 55.6 23.3 26.3 28.1 41.7 58.9 55.7 50.0 52.6 55.6

fl R. rubrum chromatophores were incubated with 50 mM 2,3-butanedione with the additions stated and photophosphorylation was measured subsequently in aliquots taken at different times as described in the text. The pseudo-first order constants k’ (lOOO/halftime of inactivation) were graphically calculated from plots similar to Fig. 1.

582

VALLEJOS.

LEXCANO,

nucleoside triphosphates ATP, CTP, GTP, and ITP afforded little or no protection at all. Light induces a conformational change in the spinach thylakoid membrane and its coupling factor (36) which results in the exposure of some of the thiols (37) vicinal dithiols (38, 39) and amino groups (40) of the factor to different chemical reagents. Similarly, a light-dependent inhibition of Rhodopseudomonas capsulata ATPase by N-ethyhnaleimide was described (41). The chemical modification of R. rubrum chromatophores by arginine reagents described above was carried out in the dark. When similar experiments were performed in the light it was observed that the rates of inactivation of photophosphorylation and Mg-ATPase reactions were more rapid. Table III shows the second order rate constants of inactivation of photophosphorylation and Mg-ATPase in the dark and in the light. It is noteworthy that the KZ of photophosphorylation inactivation in the light increased 25 times with respect to inactivation in the dark while that of the Mg-ATPase increased only about 4 times. As a consequence, the sensitivity to butanedione of photophosphorylation was 11 times higher than that of Mg-ATPase when inactivation was carried out in the light compared with only twice as high when carried out in the dark. A protection of photophosphorylation by 20 mu ADP against inactivation by 5 mM butanedione TABLE

III

EFFECT

R.

OF LIGHT ON CHEMICAL MODIFICATION OF rubrum CHROMATOPHORES BY BUTANEDIONE~

K:! (K’

min-‘)

Condition during inactivation Light Dark Photophosphorylation Mg-ATPase

1.2

0.6

30.0 2.6

n Treatment of R. rubrum chromatophores with several concentrations of 2,3-hutanedione in the dark or in saturating light was carried out as described in the text. Photophosphorylation and Mg-ATPase activities of chromatophores were determined in aliquots taken at different times from each incubation mixture and the second rate constants of inactivation (kz) stated were calculated from plots of k’ (see Table I) vs. concentration of inhibitor.

AND

LUCEHO

in the light siinilar to the experiment in the dark reported in Table I was observed (result not shown). DISCUSSION

In this paper we present evidence that chemical modification of one arginyl residue per active site in R. rubrum chromatophores by phenylglyoxal or 2,3-butanedione in borate buffer resulted in complete inhibition of the photosynthetic and oxidative phosphorylation, the Mg-ATPase and the light ATP-Pi exchange reactions. Localization of the involved arginyl residues in the soluble coupling factor is suggested by the fact that the Ca-ATPase of the factor was similarly inhibited by the arginine reagents. Previous work from this laboratory (12, 13) has shown that the spinach chloroplast coupling factor 1 has also one arginine essential for photophosphorylation, ATP-Pi exchange and ATPase activities. Arginine has been postulated to play a role in the binding of anionic ligands (5) and protection by them against inactivation may be expected. The protection afforded by adenine nucleotides (Table I) and other nucleotides that were substrates of the reactions (Table II) reinforces the suggestion that the arginine involved is in the active site and contributes to the binding of the nucleotide. Particularly striking is the fact that a strong protection of the ATPase was afforded, both in chromatophores and in the soluble coupling factor, only by ATP that is the substrate of the reaction and not by ADP, while the phosphorylating reactions were protected by ADP and not by ATP (Table I). A similar but less striking different degree of protection by adenine nucleotides of the reactions of ATP synthesis and hydrolysis in spinach chloroplasts was previously reported (13). Marcus et al. (11) and Frigeri et al. (42) have separately found that the inhibition of the ATPase and Pi-ATP exchange activities of mitochondrial preparations by arginine reagents was dissimilarly protected by adenine nucleotides suggesting that different essential arginines are involved. Inactivation of both the photophospho-

ESSENTIAL

ARGININE

IN Rhodospirillum

rylation and Mg-ATPase activities of chromatophores modified with butanedione was much more rapid in the light than in the dark (Table III). The increase in sensitivity in the light was higher for synthesis of ATP than for the backward reaction. The effect of light may be mediated by a conformational change of the membrane-bound coupling factor induced by energization by light which would facilitate the access of the reagent to the arginine(s). If the inhibition of the phosphorylating and ATPase activities of chromatophores are the consequence of the modification of only one arginyl residue per active site and if this catalytic site were the same for the forward and backward reactions, one would expect that protection by any agent against inactivation and increase of sensitivity to modification in the light should be similar for both types of reactions. On the other hand if the catalytic sites were different and both had one essential arginine it is possible that the nucleotide that is the substrate of each reaction protects only at its binding site and that modification in the light may affect differently both arginines. These are precisely the results shown in this paper. The suggestion that the sites for ATP synthesis and hydrolysis are different in chloroplast has been previously put forward by Shahak et al. (43) based on the use of 1,N6-ethenoadenosine di- and triphosphate as substrate. More recently VanderMeulen and Govindjee (44) using the same compounds have shown that the binding sites for ADP and ATP on chloroplast coupling factor are not the same. A similar suggestion for mitochondrial ATP synthesis and hydrolysis has been put forward by Penefsky (45), Leimgruber and Senior (46), and Pedersen (47) and for Mycobacterium phlei by Lee et al. (48). Thus, there are three types of results favouring different catalytic sites: (a) some adenine nucleotide analogs have different specificity as substrates (43); (b) some analogs are better inhibitors or competitive ligands for one site than for the other (43-45,48); and (c) the arginine involved in each type of reaction may be different since protection was afforded only by the respec-

rubrum CHROMATOPHORES

583

tive substrate and when modification was carried out in the light the sensitivity of photophosphorylation was higher than that of Mg-ATPase (this paper). Other explanations are possible for some of these results (49). By different catalytic sites we do not necessarily mean remote, completely separate sites. Our results and those of others (43-45) may be explained by sites partially different. For instance, the arginine involved in the binding of ADP during the synthesis of ATP may not be the one involved in the hydrolytic reaction but most of the other aminoacid residues may be the same. ACKNOWLEDGMENTS This work was supported by grants from the Consejo NacionaI de Investigaciones Cientificas y T&micas (Argentina) R.H.V. is a Career Investigator and H.A.L. is a Fellow of the same Institution. REFERENCES 1. JOHANSSON, B. C., BALTSCHEFFSKY, M., BALTSCHEFFSKY, H., BACCARINI-MELANDRI, B. A. AND MELANDRI, B. A. (1973) Eur. J. Biochem. 40,109-117. 2. JOHANSSON, B. C., AND BALTSCHEFFSKY, M. (1975) FEBS Lett. 53,221-224. 3. RAVIZZINI, R. A., LESCANO, W. I. M., AND VALLEJOS, R. H. (1975) FEBS Lett. 58,285-288. 4. LUCERO, H., LESCANO, W. I. M., AND VALLEJOS, R. H. (1978) Arch. Biochem. Biophys. 186,9-14. 5. RIORDAN, J. F. (1973) Biochemistry 12.3915-3923. 6. TAKAHASHI, K. (1968) J. Biol. Chem. 243, 6171-6179. 7. TAKAHASHI, K. (1977) J. Biochem. 81,395-403. 8. LANGE, L. G., RIORDAN, J. F., AND VALLEE, B. C. (1974) Biochemistry 13.4361-4370. 9. POWERS, J. G., AND RIORDAN, J. F. (1975) Proc.

Nat. Acad. Sci. USA 72,2616-2620. 10. BORDERS, C. L., JR., AND RIORDAN, J. F. (1975) Biochemistry 14.4699-4704. 11. MARCUS, F., SCHUSTER, S. M., AND LARDY, H. A. (1976) J. Biol. Chem. 251.1775-1780. 12. ANDREO, C. S., AND VALLEJOS, R. H. (1977) FEBS

Lett. 78,207-210. 13. VALLEJOS, R. H., VIALE, A., AND ANDREO, C. S. (1977) FEBS Lett. 84,304-308 14. SCHMID, R., JAGENDORF, A. T., AND HULKOWER, S. (1977) Biochim. Biophys. Acta 462, 177-186. 15. PAL, P. K., AND COLMAN, R. F. (1976) Eur. J.

B&hem.

68.437-443.

16. DOMAGK, G. K., DOMSCHKE, W., AND VON HINUBER, C. (1970) Hoppe-Seyler’s 2. Physiol. Chem. 351.718-720.

584

VALLEJOS,

LESCANO,

17. YANG, P. C., AND SCHWERT, G. W. (1972) Biochemistry 11,2218-2224 18. FOSTER, M., AND HARRISON, J. H. (1974) Biochem.

Biophys. Res. Commun. 68,263-267. 19. BLEILE, D. M., FOSTER, M. BRADY, J. W., AND HARRISON, J. H. (1975) J. Biol. Chem. 250, 6222-6227. 20. BLUMENTHAL, K. N., AND SMITH, E. L. (1975) J.

Biol. Chem. 260,6555-6559. 21. DAVID, M., RASCHED, I., AND SUND, H. (1976)

FEBS Lett. 62, 288-292. 22. NAGRADOVA, N. K., ASRYANTS, R. A., BANKEVICH, N. V., AND SAFRONOVA, M. I. (1976) FEBS Lett. 69,246-248. 23. NAGRADOVA, N. K., AND A~RYANTS, R. A. (1975)

Biochim. Biophys. Acta 386,365-368. 24. ADAMS, M. J., BUEHNER, M., CHANDRASEKHAR, K., FORD, G. C., HACKERT, M. L., LILJAS, A., ROSSMAN, M. G., ANIILEY, I. E., ALLISON, W. S., EVERSE, J., KAPLAN, N. O., AND TAYLOR, S. S. (1973) Proc. Nat. Acad. Sci. USA 70,1968-1972. 25. NEWTON, J. W. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. 0. eds.), Vol. 5, pp 73-77, Academic Press, New York. 26. CLAYTON, R. (1963) in Bacterial Photosynthesis (Gest, H., San Pietro, A. and Vernon, L. P. eds.), p. 495, Antioch Press, Yellow Springs, Ohio. 27. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. L. (1951) J. Biol. Chem. 193, 265-275. 28. SUMNER, J. B. (1944) Science 100,413-414. 29. YAMASHITA, J., YOSHIMURA, S., MATUO, Y., AND HORIO, T. (1967) B&him. Biophys. Acta 143, 152-172. 30. HORIO, T., NISHIKAWA, K., KATSUMATA, M., AND YAMASHITA, J. (1965) Biochim. Biophys. Acta 94.371-382. 31. LIEN, J., AND GEST, H. (1973) Arch. Biochem. Biophys. 159,730-737.

AND

LUCERO

32. LEVY, H. M., LEBER, P. D., AND RYAN, E. M. (1963) J. Biol. Chem. 238, 3643-3659. 33. SCRU’ITON, M. C., AND UTTER, M. F. (1965) J.

Biol. Chem. 240,3714-3723. 34. KEECH,

D. B., AND FARRANT, R. K.

(1968)

Biochim. Biophys. Acta X1,493-503. 35. HOLLENBERG, P. F., FLASHNER, M., AND COON, M. J. (1971) J. Biol. Chem. 246,946-953. 36. RYRIE, I. J., AND JAGENDORF, A. T. (1971) J. Biol.

Chem. 246, 582-588. 37. MCCARTY, R. E., PITTMAN, P. R., AND TSUCHIYA, Y. (1972) J. Biol. Chem. 247,3048-3051. 38. ANDREO, C. S., AND VALLEJOS, R. H. (1976) Biochim. Biophys. Acta 423, 590-601. 39. VALLEJOS, R. H., AND ANDREO, C. S. (1976) FEBS Lett. 61, 95-99. 40. OLIVER, D., AND JAGENDORF, A. T. (1976) J. Biol. Chem. 251, 7168-7175. 41. BACCARINI-MELANDRI, A., FABBRI, E., FIRSTATER, E.; AND MELANDRI, B. A. (1975) B&him.

Biophys. Acta 376,72-81. 42. FRIGERI, L., GALANTE, Y. M., HANSTEIN, W. G., AND HATEFI, Y. (1977) J. Biol. Chem. 252, 3147-3152. 43. SHAHAK, Y., CHIPMAN, D. M., AND SHAVIT, N. (1973) FEBS Lett. 33,293-296. 44. VANDERMEULEN, D. L., AND GOVINDJEE (1977)

Eur. J. Biochem. 78.585-598. 45. PENEFSKY, H. S. (1974) J. Biol.

Chem. 249,

3579-3585. 46. LEIMGRUBER, R. M., AND SENIOR, A. E. (1976) J. Biol. Chem. 251, 7103-7109. 47. PEDERSEN, P. L. (1975) J. Supramol. Struct. 3, 222-230. 48. LEE, S. H., KALRA, V. K., RITZ, C. J., AND BRODIE, A. F. (1977) J. Biol. Chem. 262,1084-1091. 49. SHAVIT, N. (1977) in Photosynthesis I (Trebst, A., and Avron, M., eds.), Vol. 5, pp. 350-357, Springer-Verlag, Berlin.