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Molecular weights of some cytochromes cc'
238
BIOCHIMICAET BIOPHYSICAACTA
BBA 35794 MOLECULAR W E I G H T S OY SOME CYTOCHROMES cc'
MICHAEL A. CUSANOVICH Department of Chemistry, University of Arizona, Tuscon, Ariz. 85721 (U.S.A.) (Received November 9th, 197o)
SUMMARY The molecular weights of seven different cytochromes cc' have been determined by sedimentation equilibrium centrifugation. Studies were conducted in a variety of solvents and it was found that 6 M guanidine promoted the dissociation of three of the cytochromes cc' into subunits. The apparent size of Chromatium cytochrome cc' was unaffected by 6 M guanidine. However, the Chromatium cytochrome was completely dissociated into subunits of approx. I2 ooo molecular weight in the presence of zoo mM NaOH. This last result suggests that the existence of a diheme peptide as previously reported should be re-evaluated.
The cytochromes cc', long implicated in bacterial photosynthesis 1-a and recently isolated from the denitrifying bacteria Pseudomonas denitrificans 4,~, have been the subject of detailed physicochemical studies. 5-s. In a number of these studies, the possibility of subunits was suggested. Available evidence (compiled in ref. 9) has indicated most of the cytochromes cc' are diheme proteins having a molecular weight of approx. 28 ooo. Further, tile amino acid sequence of a diheme peptide from Chromatium cytochrome cc' has been reported 1°. To clarify the question of subunits, the molecular weights of seven different cytochromes cc' were determined. Molecular weights were studied by the short column sedimentation equilibrium method described by YPHANTIS11 and calculated by a modification a2 of the method of VAN HOLDE AND BALDWIN 13, in which the synthetic boundary run was omitted. The cytochromes cc' used in this work were isolated as previously reportedS, 14-17. The purified proteins were diluted to the appropriate concentration (x-5 mg/mi) and dialyzed overnight against 0.02 M Tris-o.5 M NaC1, pH 7.3 (Tris NaCI), o.I M potassium phosphate-6 M guanidine HC1, pH 7-4 (6 M guanidine), or the buffer indicated. Protein concentrations were estimated assuming a molecular weight of x4 ooo per heine and the heine concentrations determined from pyridine hemochromogens5. In all eases, a 5 of o.725 was assumed and the temperature maintained at 2o °. Molecular weights were calculated with a computer program written by D. Hopkins (Department of Biology, University of California at San Diego). This proBiochim. Biophys. Acta, 236 (I97I) 238-241
239
MOLECULAR WEIGHTS OF CYI'OCHROMES CC' TABLE I MOLECULAR W E I G H T S OF CYTOCHROME
ce'
Cytochrome ~
Molecular weight × lO -3 Tris-NaCl
6 31 guanidine
R. spheroides c y t o c h r o m e cc" R. rubrum cytochrome cc" (28 840 ) P. denitrificans c y t o c h r e m e ce' (26 400) R. gelatinosa cytochrou;.~ cc' (15 ooo) R. palustris cytochrome c' (14 820) R. capsulatus cytochronle ec' (27 720) Chromatium cytochrome cc' (27 900)
23-4 25.2 27. 4 27.0 13.2 22. 5 27.6
i ± ± ± ± ± ~
19.6 1.5.1 17.3 20. 3 18.o 21. 3 29.5
± £ ± ± ± ± ±
Horse h e a r t cytochronle c
II.I
Z~ 1 , 2
II.8
~Z I . I
0.8 3.6 1.9 2.5 1. 5 3.0 2.0
4.1 1.9 1.9 1.2 2. 5 1.2 1.2
Other
27.2 ± 1.6 (A) 19.3 ± 3.0 (B) 30.0 26. 4 25.3 29.4 I2.O 12.1
± ± ± ± ~ i
0.3 2.6 0.2 1.8 I. 4 1.3
(C) (D) (E) (F) (G) (A)
All d e t e r m i n a t i o n s were made under conditions described in the text. The particular c y t o c h r o m e was in the ferri state unless noted otherwise. A density for 6 M guanidine of 1.14 was used. Protein concentrations varied between I and 5 mg/ml, in all cases determinations were made at least at two different protein concentrations. (A) Solvent o.oi M Tris, p H 7.3. (B) Solvent 6 M guanidine, R. palustris c y t o c h r o m e cc' was reduced with an excess of Na2S204. (C) Solvent Tris-NaC1, Chromatium cytochrome cc' was reduced with an excess of Na2S204. (D) Same as (C) except the protein was s a t u r a t e d with CO. (E) Solvent o.i M Tris-2 M NaC1, p H 7.3. (F) Solvent 0.025 M p o t a s s i u m p h o s p h a t e -0.025 M sodium acetate p H 5.1. (G) Solvent o.I M N a O H . * I n parentheses is the molecular weight determined from amino acid analysis (see ref. 9).
gram calculated the average molecular weight for a run and the molecular weight at various distances across the centrifuge cell. Thus, an indication of the homogenity of the sample was obtained. Table I summarizes the results obtained. Horse heart cytochrome c (Sigma Type IV) was used as a standard and gave essentially identical molecular weights in Tris-NaC1 and 6 M guanidine. Rhodospirillum rubrum, Pseudomonas denitrificans and Rhodopseudomonas spheroides cytochrome cc' are substantially dissociated in the presence of 6 M guanidine. Dissociation was indicated by the lower average molecular weight and the fact that the samples show a distribution of molecular sizes across the centrifuge cell. The distribution of molecular sizes across the centrifuge cell can be interpreted as resulting from incomplete dissociation into subunits. Thus, the presence of a small amount of dimers would cause the calculated molecular weights to increase as the distance from the center of rotation decreased. This distribution is shown in more detail in Table II where results obtained with P. denitrificans cytochrome cc' are given as an example. Rhodopseudomonas gelatinosa eytochrome cc', although dissociated as judged from the decreased molecular weight in 6 M guanidine, (as compared to Tris-NaC1), was found to be homogeneous. The molecular weight determined in 6 M guanidine was not one-half of the Tris-NaC1 molecular weight, suggesting that the hydrodynamic properties of R. gelatinosa eytochrome cc' were substantially modified. This makes it impossible at this time to determine if R. gelatinosa cytoehrome cc' is dissociated into subunits. Biochim. Biophys. Acta, 236 (1971) 238-241
240
M. A. CUSANOVICH
TABLE [ l SEDIMENTATION
l'ris-NaCl
EQUILIBRIUM
O F P . denitrificans C Y T O C H R O M E
CO"
6 M guanidine
Distance ~
M o l . wt. × ro -a D i s t a n c e
M o l . wt. × l o 3
28.o 33.6 39.2 44.8 5o.4 56.o 61.6 67.2 72.8 78.4 84.0
31.1 29.7 24.5 25.2 27.0 27.8 25. 9 27. 5 28.1 27.7 27.5
t3.2 t6-5 16.2 15. 4 15.o 16.9 r8. i 18.o 17. 4 17.8 19.3
20. 4 31.6 36.8 42.0 47.2 52.4 57.6 62.8 68.0 73.2 78.4
* The distance s h o w n is the percentage from the top of the cell to where the m e a s u r e m e u t .was made. Because of the limitations of the Schlieren optics used in this work, the top 2O°'o and b o t t o m IO'~, of the cell could not be used in the calculations presented.
Rhodopseudomonas palustris cytochrome c' has been shown to be a monoheme protein 16 with a molecular weight of approx. 14 ooo, a result confirmed in this work (Tris-NaC1). However, in the presence of 6 M guanidine the apparent molecular weight increases. This observation would suggest that in the presence of 6 M guanidine there is an increased partial specific volume. However it is not possible to make a definitive statement with the data available. The molecular weights of Chromatium cytochrome cc' and Rhodopseudomonas capsulatus cytochrome cc' were not affected by the presence of 6 M guanidine. Chromatium cytochrome cc' was studied more extensively in order to correlate molecular weight with kinetic studies 8. As shown in Table I, incubation in the solvents listed, except o.I M NaOH, have no effect on the apparent molecular weight, whereas kinetic studies of CO binding in the same solvents suggest the presence of two molecular species 8. However, o.I M NaOH completely dissociates Chromatium cytochrome cc' into subunits having a molecular weight approximately one-half of the Tris NaC1 value. The effect of o.I M NaOH on tile partial specific volume of Chromatium cytochrome cc' is unknown, but it is unlikely that the variation of~ would be large enough to account for the apparent decrease in molecular weight (see discussion below). Thus, it can be concluded that Chromatium cytochrorne co' is made up of at least two subunits. Summarizing the results reported, R. spheroides, R. rubrum, P. denitrificans and Chromatium cytochromes cc' consist of subunits which can be dissociated. The effect of guanidine on R. gelatinosa cytochrome cc' is unclear at this time, but neither R. capsulatus cytochrome cc' nor Chromatium cytochromes cc' can be dissociated in this solvent. It should be pointed out the number of heroes per molecule is not known in the case of R. capsulatus cytochrome cc' since the reported molecular weight based on amino acid analysis (13 86o per heme) is not consistent with the results obtained in this study (II 25o per heme or 22 5oo per 2 hemes). The results obtained in Tris-NaC1 for the various cytochromes, other than R. capsulatus and R. sflheroides B i o c h i m . B i o p h y s . A c t a , 236 (I97 I) 238-241
MOLECULAR WEIGHTS OF CYTOCHROMES CC'
241
cytoehromes cc' which have not been previously reported are in good agreement with estimates from other sources 9. The major problem in the interpretation presented is the effect of guanidine on the partial specific volmne of the proteins studied. A number of studies have been made on a variety of proteins and ~ was only slightly, if at all, affected by 6 M guanidine17, is. Further, as shown in Table I, 6 M guanidine has no apparent effect on the hydrodynamic properties of horse heart cytochrome c. Nothing can be said at this point in regard to the role of dimerization in the function of the cytochromes cc'; indeed, tile existence of a naturally occurring monoheine cytochrome cc' (R. palustris cytochrome c') suggests that dimerization is not important in determining the physico-chemical properties of purified cytochromes cc'. The results reported here with Chromatium cytochromes cc' suggest that further investigation into the subunit structure is important as the results presented here are contradictory to the existence of a diheme peptide as previously reported 1°. I would like to thank Dr. M. D. Kamen, in whose laboratory a portion of this work was done. REFERENCES i J. M. OLSON AND B. CHANCE, Arch. Biochem. Biophys., 88 (196o) 28. 2 S. MORITA, M. EDWARDS AND J. GIBSON, Biochim. Biophys. Acta, lO8 (1965) 45. 3 M. A. CUSANOVICH, R. G. BARTSCH AND M. D. KAMEN, Biochim. Biophys. Acta, 153 (1968) 3974 H. IWASAKI AND S. SHIDARA, Plant Cell Physiol., IO (1969) 291. 5 M. A. CUSANOVICH, S. M. TEDRO AND M. D. KAMEN, Arch. Biochem. Biophys., 141 (197 o) 557. 6 Q. H. GIBSON AND M. P. KAMEN, J. Biol. Chem., 241 (1966) 1969. 7 Y. IMAI, K. IMAI, R. SATO AND T. HORIO, J. Biochem. Tokyo, 65 (1969) 225. 8 M. A. CUSANOVICH AND Q. H. GIBSON, in preparation. 9 M. D. KAMEN, K. M. D u s , T. FLATMARK AND H. DEKLERK, in T. E. KING AND M. KLINGENBERG, Treatise on Electron Transport and Coupled Energy Transfer in Biological Systems,
Marcel Dekker, New York, in the press. K. M. Du s, R. G. BARTSCH AND M. D. KAMEN, ,[. Biol. Chem., 237 (1962) 3083 . D. A. YPHANTIS, Ann. N . Y . Acad. Sci., 88 (196o) 586. A. 13. ROBINSON, P h . D . Thesis, University of California at San Diego, 1968. K. E. VAN HOLDE AND R. L. BALDWIN, J. Phys. Chem., 62 (1958) 734. M. A. CUSANOVICH, P h . D . Thesis, University of California at San Diego, 1967. T. MEYER, P h . D . Thesis, University of California at San Diego, 197 ° K. D u s , H. DEKLERK, R. G. }{ARTSCH, T. FIORIO AND M. D. KAMEN, Proc. Nat. Acad. Sci. U.S., 57 (1967) 367 . 17 E. REISLER AND H. EISENBER(L Biochemistry, 8 (1969) 4572. 18 A. ULLMAN, M. E. GOLDBERG, P. PERRIN AND J, MONOD, Biochemistry, 7 (1968) 261. io II 12 13 14 15 16
Biochim. Biophys. Acta, 236 (1971) 238-241
BIOCHIMICAET BIOPHYSICAACTA
BBA 35794 MOLECULAR W E I G H T S OY SOME CYTOCHROMES cc'
MICHAEL A. CUSANOVICH Department of Chemistry, University of Arizona, Tuscon, Ariz. 85721 (U.S.A.) (Received November 9th, 197o)
SUMMARY The molecular weights of seven different cytochromes cc' have been determined by sedimentation equilibrium centrifugation. Studies were conducted in a variety of solvents and it was found that 6 M guanidine promoted the dissociation of three of the cytochromes cc' into subunits. The apparent size of Chromatium cytochrome cc' was unaffected by 6 M guanidine. However, the Chromatium cytochrome was completely dissociated into subunits of approx. I2 ooo molecular weight in the presence of zoo mM NaOH. This last result suggests that the existence of a diheme peptide as previously reported should be re-evaluated.
The cytochromes cc', long implicated in bacterial photosynthesis 1-a and recently isolated from the denitrifying bacteria Pseudomonas denitrificans 4,~, have been the subject of detailed physicochemical studies. 5-s. In a number of these studies, the possibility of subunits was suggested. Available evidence (compiled in ref. 9) has indicated most of the cytochromes cc' are diheme proteins having a molecular weight of approx. 28 ooo. Further, tile amino acid sequence of a diheme peptide from Chromatium cytochrome cc' has been reported 1°. To clarify the question of subunits, the molecular weights of seven different cytochromes cc' were determined. Molecular weights were studied by the short column sedimentation equilibrium method described by YPHANTIS11 and calculated by a modification a2 of the method of VAN HOLDE AND BALDWIN 13, in which the synthetic boundary run was omitted. The cytochromes cc' used in this work were isolated as previously reportedS, 14-17. The purified proteins were diluted to the appropriate concentration (x-5 mg/mi) and dialyzed overnight against 0.02 M Tris-o.5 M NaC1, pH 7.3 (Tris NaCI), o.I M potassium phosphate-6 M guanidine HC1, pH 7-4 (6 M guanidine), or the buffer indicated. Protein concentrations were estimated assuming a molecular weight of x4 ooo per heine and the heine concentrations determined from pyridine hemochromogens5. In all eases, a 5 of o.725 was assumed and the temperature maintained at 2o °. Molecular weights were calculated with a computer program written by D. Hopkins (Department of Biology, University of California at San Diego). This proBiochim. Biophys. Acta, 236 (I97I) 238-241
239
MOLECULAR WEIGHTS OF CYI'OCHROMES CC' TABLE I MOLECULAR W E I G H T S OF CYTOCHROME
ce'
Cytochrome ~
Molecular weight × lO -3 Tris-NaCl
6 31 guanidine
R. spheroides c y t o c h r o m e cc" R. rubrum cytochrome cc" (28 840 ) P. denitrificans c y t o c h r e m e ce' (26 400) R. gelatinosa cytochrou;.~ cc' (15 ooo) R. palustris cytochrome c' (14 820) R. capsulatus cytochronle ec' (27 720) Chromatium cytochrome cc' (27 900)
23-4 25.2 27. 4 27.0 13.2 22. 5 27.6
i ± ± ± ± ± ~
19.6 1.5.1 17.3 20. 3 18.o 21. 3 29.5
± £ ± ± ± ± ±
Horse h e a r t cytochronle c
II.I
Z~ 1 , 2
II.8
~Z I . I
0.8 3.6 1.9 2.5 1. 5 3.0 2.0
4.1 1.9 1.9 1.2 2. 5 1.2 1.2
Other
27.2 ± 1.6 (A) 19.3 ± 3.0 (B) 30.0 26. 4 25.3 29.4 I2.O 12.1
± ± ± ± ~ i
0.3 2.6 0.2 1.8 I. 4 1.3
(C) (D) (E) (F) (G) (A)
All d e t e r m i n a t i o n s were made under conditions described in the text. The particular c y t o c h r o m e was in the ferri state unless noted otherwise. A density for 6 M guanidine of 1.14 was used. Protein concentrations varied between I and 5 mg/ml, in all cases determinations were made at least at two different protein concentrations. (A) Solvent o.oi M Tris, p H 7.3. (B) Solvent 6 M guanidine, R. palustris c y t o c h r o m e cc' was reduced with an excess of Na2S204. (C) Solvent Tris-NaC1, Chromatium cytochrome cc' was reduced with an excess of Na2S204. (D) Same as (C) except the protein was s a t u r a t e d with CO. (E) Solvent o.i M Tris-2 M NaC1, p H 7.3. (F) Solvent 0.025 M p o t a s s i u m p h o s p h a t e -0.025 M sodium acetate p H 5.1. (G) Solvent o.I M N a O H . * I n parentheses is the molecular weight determined from amino acid analysis (see ref. 9).
gram calculated the average molecular weight for a run and the molecular weight at various distances across the centrifuge cell. Thus, an indication of the homogenity of the sample was obtained. Table I summarizes the results obtained. Horse heart cytochrome c (Sigma Type IV) was used as a standard and gave essentially identical molecular weights in Tris-NaC1 and 6 M guanidine. Rhodospirillum rubrum, Pseudomonas denitrificans and Rhodopseudomonas spheroides cytochrome cc' are substantially dissociated in the presence of 6 M guanidine. Dissociation was indicated by the lower average molecular weight and the fact that the samples show a distribution of molecular sizes across the centrifuge cell. The distribution of molecular sizes across the centrifuge cell can be interpreted as resulting from incomplete dissociation into subunits. Thus, the presence of a small amount of dimers would cause the calculated molecular weights to increase as the distance from the center of rotation decreased. This distribution is shown in more detail in Table II where results obtained with P. denitrificans cytochrome cc' are given as an example. Rhodopseudomonas gelatinosa eytochrome cc', although dissociated as judged from the decreased molecular weight in 6 M guanidine, (as compared to Tris-NaC1), was found to be homogeneous. The molecular weight determined in 6 M guanidine was not one-half of the Tris-NaC1 molecular weight, suggesting that the hydrodynamic properties of R. gelatinosa eytochrome cc' were substantially modified. This makes it impossible at this time to determine if R. gelatinosa cytoehrome cc' is dissociated into subunits. Biochim. Biophys. Acta, 236 (1971) 238-241
240
M. A. CUSANOVICH
TABLE [ l SEDIMENTATION
l'ris-NaCl
EQUILIBRIUM
O F P . denitrificans C Y T O C H R O M E
CO"
6 M guanidine
Distance ~
M o l . wt. × ro -a D i s t a n c e
M o l . wt. × l o 3
28.o 33.6 39.2 44.8 5o.4 56.o 61.6 67.2 72.8 78.4 84.0
31.1 29.7 24.5 25.2 27.0 27.8 25. 9 27. 5 28.1 27.7 27.5
t3.2 t6-5 16.2 15. 4 15.o 16.9 r8. i 18.o 17. 4 17.8 19.3
20. 4 31.6 36.8 42.0 47.2 52.4 57.6 62.8 68.0 73.2 78.4
* The distance s h o w n is the percentage from the top of the cell to where the m e a s u r e m e u t .was made. Because of the limitations of the Schlieren optics used in this work, the top 2O°'o and b o t t o m IO'~, of the cell could not be used in the calculations presented.
Rhodopseudomonas palustris cytochrome c' has been shown to be a monoheme protein 16 with a molecular weight of approx. 14 ooo, a result confirmed in this work (Tris-NaC1). However, in the presence of 6 M guanidine the apparent molecular weight increases. This observation would suggest that in the presence of 6 M guanidine there is an increased partial specific volume. However it is not possible to make a definitive statement with the data available. The molecular weights of Chromatium cytochrome cc' and Rhodopseudomonas capsulatus cytochrome cc' were not affected by the presence of 6 M guanidine. Chromatium cytochrome cc' was studied more extensively in order to correlate molecular weight with kinetic studies 8. As shown in Table I, incubation in the solvents listed, except o.I M NaOH, have no effect on the apparent molecular weight, whereas kinetic studies of CO binding in the same solvents suggest the presence of two molecular species 8. However, o.I M NaOH completely dissociates Chromatium cytochrome cc' into subunits having a molecular weight approximately one-half of the Tris NaC1 value. The effect of o.I M NaOH on tile partial specific volume of Chromatium cytochrome cc' is unknown, but it is unlikely that the variation of~ would be large enough to account for the apparent decrease in molecular weight (see discussion below). Thus, it can be concluded that Chromatium cytochrorne co' is made up of at least two subunits. Summarizing the results reported, R. spheroides, R. rubrum, P. denitrificans and Chromatium cytochromes cc' consist of subunits which can be dissociated. The effect of guanidine on R. gelatinosa cytochrome cc' is unclear at this time, but neither R. capsulatus cytochrome cc' nor Chromatium cytochromes cc' can be dissociated in this solvent. It should be pointed out the number of heroes per molecule is not known in the case of R. capsulatus cytochrome cc' since the reported molecular weight based on amino acid analysis (13 86o per heme) is not consistent with the results obtained in this study (II 25o per heme or 22 5oo per 2 hemes). The results obtained in Tris-NaC1 for the various cytochromes, other than R. capsulatus and R. sflheroides B i o c h i m . B i o p h y s . A c t a , 236 (I97 I) 238-241
MOLECULAR WEIGHTS OF CYTOCHROMES CC'
241
cytoehromes cc' which have not been previously reported are in good agreement with estimates from other sources 9. The major problem in the interpretation presented is the effect of guanidine on the partial specific volmne of the proteins studied. A number of studies have been made on a variety of proteins and ~ was only slightly, if at all, affected by 6 M guanidine17, is. Further, as shown in Table I, 6 M guanidine has no apparent effect on the hydrodynamic properties of horse heart cytochrome c. Nothing can be said at this point in regard to the role of dimerization in the function of the cytochromes cc'; indeed, tile existence of a naturally occurring monoheine cytochrome cc' (R. palustris cytochrome c') suggests that dimerization is not important in determining the physico-chemical properties of purified cytochromes cc'. The results reported here with Chromatium cytochromes cc' suggest that further investigation into the subunit structure is important as the results presented here are contradictory to the existence of a diheme peptide as previously reported 1°. I would like to thank Dr. M. D. Kamen, in whose laboratory a portion of this work was done. REFERENCES i J. M. OLSON AND B. CHANCE, Arch. Biochem. Biophys., 88 (196o) 28. 2 S. MORITA, M. EDWARDS AND J. GIBSON, Biochim. Biophys. Acta, lO8 (1965) 45. 3 M. A. CUSANOVICH, R. G. BARTSCH AND M. D. KAMEN, Biochim. Biophys. Acta, 153 (1968) 3974 H. IWASAKI AND S. SHIDARA, Plant Cell Physiol., IO (1969) 291. 5 M. A. CUSANOVICH, S. M. TEDRO AND M. D. KAMEN, Arch. Biochem. Biophys., 141 (197 o) 557. 6 Q. H. GIBSON AND M. P. KAMEN, J. Biol. Chem., 241 (1966) 1969. 7 Y. IMAI, K. IMAI, R. SATO AND T. HORIO, J. Biochem. Tokyo, 65 (1969) 225. 8 M. A. CUSANOVICH AND Q. H. GIBSON, in preparation. 9 M. D. KAMEN, K. M. D u s , T. FLATMARK AND H. DEKLERK, in T. E. KING AND M. KLINGENBERG, Treatise on Electron Transport and Coupled Energy Transfer in Biological Systems,
Marcel Dekker, New York, in the press. K. M. Du s, R. G. BARTSCH AND M. D. KAMEN, ,[. Biol. Chem., 237 (1962) 3083 . D. A. YPHANTIS, Ann. N . Y . Acad. Sci., 88 (196o) 586. A. 13. ROBINSON, P h . D . Thesis, University of California at San Diego, 1968. K. E. VAN HOLDE AND R. L. BALDWIN, J. Phys. Chem., 62 (1958) 734. M. A. CUSANOVICH, P h . D . Thesis, University of California at San Diego, 1967. T. MEYER, P h . D . Thesis, University of California at San Diego, 197 ° K. D u s , H. DEKLERK, R. G. }{ARTSCH, T. FIORIO AND M. D. KAMEN, Proc. Nat. Acad. Sci. U.S., 57 (1967) 367 . 17 E. REISLER AND H. EISENBER(L Biochemistry, 8 (1969) 4572. 18 A. ULLMAN, M. E. GOLDBERG, P. PERRIN AND J, MONOD, Biochemistry, 7 (1968) 261. io II 12 13 14 15 16
Biochim. Biophys. Acta, 236 (1971) 238-241