- Email: [email protected]
End-to-end aggregation of myosin
598
SHORT COMMUNICATIONS
together with cystine, norvaline and isovaline with methionine, diaminopimelic acid with allo-isoleucine and citrulline with proline. The use of an internal standard in another role has been recently suggested by SIEGEL AND ROACH 11, who use fl-2-thienyl-DL-alanine as a means of monitoring the daily reliability of analyses on the i5o-cm column. We have been using L-2-amino guanido propionic acid (available from California Corporation for Biochemical Research) for this purpose on the I5-cm column (Fig. I). An ideal internal standardization might well result from using a mixture of known concentrations of both compounds. This should not only provide a measure of the daily reliability of the instrument but also permit a daily correction for any differences in analytical sensitivity between long and short columns (resulting from errors in pipetting, pump rates, ninhydrin stability, etc.). In summary, norleucine has been found to be a reliable internal standard for amino acid analysis by the chromatographic technique of SPACKMAN, STEIN AND MOORE. The internal standardization permits a correction for mechanical errors during steps leading to analysis of protein hydrolysates, and places these analyses in the forefront as highly precise tools for determining protein concentrations. The authors are indebted to Professor H. NEURATH for helpful discussions and to Miss K. LADUE for her technical assistance. This work has been supported in part by American Cancer Society (Grants P-45 and P-79), the National Institutes of Health, U.S. Public Health Service (RG-4617) and by funds from the State of Washington, Initiative 171.
Department of Biochemistry, University of Washington, Seattle, Wash. (U.S.A.)
K . A . WALSH J . R . BROWN
1 E. LAYNE, in S. P. COLOWlCK AND O. KAPLAN, Methods in Emymology, Vol. 3, Academic Press, Inc., New York, 1957, p. 447. 2 M, P. TOMBS, F. SOUTER AND N. F. MACLAGAN, Biochem. J., 73 (1959) 167. s O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (195 I) 265. 4 D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. 5 C. S. HANES, C. K. HARRIS, M. A. MOSCARELLO AND E. TIGANE, Can.J. Biochem. Physiol., 39 (I961) 163. e E. TIGANE, E. H. M. WADE, J. T. WONG AND C. S. HANES, Can. J. Biochem. Physiol., 39 (1961) 427. K. A. PIEZ AND L. MORRIS, Anal. Biochem., I (196o) 187. 8 T. W. GOODWlN AND R. A. MORTON, Bioehem. J., 4 ° (1946) 628. 9 E. L. SMITH AND A. STOCI
Received November 27th, 1961 Biochim. Biophys. Acta, 58 (1962) 596-598
End-to-end aggregation of myosin* NODA2 has recently reported the presence of a component sedimenting with a sedimentation coefficient of about 7 S in natural actomyosin under conditions when clearing 3 can be observed. From preliminary measurements he concluded that the 7-S peak was due to highly asymmetrical particles--their length being of the order of about * A preliminary
report of
this w o r k has been presented 1.
Biochim. Biophys. Acta, 58 (1962) 598-6Ol
599
SHORT COMMUNICATIONS
I/~--but was unable to identify them with any of the known myofibrillar proteins of muscle. The present report deals with further investigations on this problem. Natural actomyosin in a solution containing 2 m M ATP and 2 m M Mg at 1 o.18 was centrifuged at IOOOOO × g for 2 h. Some of the protein remained in the supernatant, and on examination of the analytical centrifuge a single peak with an extrapolated sedimentation coefficient of roughly 7 S was found. It should be noted that this is nearly equal to the sedimentation rate of the myosin monomer at higher ionic strength.
8.0
I00
5.0
I00
90
L" 4.0
90
8 0 ~:
7o ~
8o ~
0
70 ~
IR
3.0
~o ~
•
~
60 ~
~4,0 2.0
- -40
~o ~
~2.0 I'0 I 0
~
--20 ~
w
--I0 ~
I _ 0.15
I 0.20
KCI CONCENTRATION (M)
to 0.,25
0 __1
0
I
___] _ __.1__~ 2 3 4
[_
5
6
A TP CONCENTRATION(raM)
Fig. I. The effect of (a) KC1 concentration a n d (b) A T P concentration on the a m o u n t of protein recovered in t h e I o o o o o × g s u p e r n a t a n t ( 0 - - 0 ) and on the birefringence ( O - - O ) of the i oo ooo × g s u p e r n a t a n t . Conditions: (a) Actomyosin, I.O mg/ml, o.o2 M p h o s p h a t e buffer (pH 7.o), 2 m M ATP, a n d 2. 5 m M MgCI=; KCI as indicated on the abscissa. (b) Actomyosin, 1.6 m g / m l , o.o2 M p h o s p h a t e buffer (pH 7.o), o.z 5 M KC1 and 2.5 m M MgCI=; A T P as indicated on the abscissa. P r o t e i n d e t e r m i n a t i o n s were m a d e w i t h the use of a biuret m e t h o d ; An was determined a t a velocity gradient of 13oo sec -1.
When the concentration of KC1 or ATP was increased the amount of the 7-S component increased monotonically, but the total birefringence went through a maximum and then decreased (Fig. I). The birefringence of flow, An, of the supernatant was almost equal to, or slightly higher than, that of the uncentrifuged solution. The extinction angle of the supernatant was lower than that of the uncentrifuged system at all velocity gradients (Fig. 2). The supernatant containing the 7-S component showed ATPase activity having all the characteristics of myosin. It was activated by Ca 2+ but not by Mg2+--unless F-actin was added (Table I). In the presence of F-actin in 0.05 M KCI typical superprecipitation could be observed upon addition of I m M ATP. Thus, in conjunction with the results described above, it appears that myosin forms well-defined aggregates in the clear actomyosin system. The results obtained at higher KCI or ATP concentrations (Fig. I) could be interpreted in terms of there being, first, an increase in the 7-S component at the expense of the more rapidly Biochim. Biophys. Acta, 58 (1962) 598--6ox
600
SHORT COMMUNICATIONS
sedimenting ones 1, followed by the dissociation of the 7-S component to monomers-reflected in the decrease of flow birefringence. In view of the differences between the flow-birefringence properties before and after centrifugation, it also appears that the aggregation of myosin was enhanced by the removal of the components sedimenting at a higher rate. Similar aggregates could be observed with myosin alone under the conditions under which these experiments were carried out although the results were more erratic. Clearly, to decide 50~--
50
40
4O
tt t~
Lu
50
30
~-
t~
0
L
20 L
20
Lo~
-41o
L
0 !,
I
0
I0
~
0
20
30
VELOCITY GRADIENT (sec-O
Fig. 2. E x t i n c t i o n angle a n d birefringence of cleared a c t o m y o s i n before a n d a f t e r c e n t r i f u g a t i o n a t 10o0oo × g for 2 h. Conditions: A c t o m y o s i n , 2.2 m g ] m l , o.02 N / ! h i s t i d i n e buffer (pH 6.8), 0.17 M KC1, 2 m M A T P a n d 2. 5 m M MgC1 v Birefringence m e a s u r e m e n t s were carried o u t a t t h e velocity g r a d i e n t s i n d i c a t e d on t h e abscissa. O - - O , e x t i n c t i o n a n g l e before c e n t r i f u g a t i o n ; Q - I , e x t i n c t i o n angle a f t e r c e n t r i f u g a t i o n ; & - A, birefringence before c e n t r i f u g a t i o n ; A - - A , birefringence a f t e r c e n t r i f u g a t i o n . TABLE I ATPAsE A C T I V I T Y O F 7-S C O M P O N E N T T h e p r o t e i n in t h e s u p e r n a t a n t of clear a c t o m y o s i n (see text) w a s p r e c i p i t a t e d b y dialysis a g a i n s t water, collected b y c e n t r i f u g a t i o n a n d dissolved in o.6 M KC1. A T P a s e a c t i v i t y w a s a s s a y e d in t h e following s y s t e m : 0.0 5 M KC1, o.o2 M h i s t i d i n e buffer (pH 7.0), 1.2 m M A T P . I rnl c o n t a i n i n g O,I 4 m g p r o t e i n w a s i n c u b a t e d a t 2o ° for 30 m i n . T h e Pt liberated w a s d e t e r m i n e d w i t h t h e F i s k e - S u b b a r o w procedure. Additions
~molesof P~
None 4 m M Mg ~+ 4 m M Mg ~+ + o.14 m g F - a c t i n 4 rnM C# +
o.o3 0.03 o.9I o.96
Biochim. Biophys. Acta, 58 (1962) 598-6Ol
SHORT COMMUNICATIONS
601
whether actin plays a role in the formation of the myosin aggregates requires further study. The flow birefringence of myosin at low ionic strength in the absence of ATP 4 may be partly due to the same aggregates. A tentative conclusion about the shape of these myosin aggregates can be arrived at on the basis of the sedimentation and birefringence measurements. By combining the SVEDBERGequation 5 with PERRIN'S tWO relations between the axial ratio and the translationaN and rotary 7 frictional coefficients, respectively, it is possible to calculate the axial ratio and the length of the particle from the sedimentation coefficient and the rotary-diffusion coefficient, provided one assumes that the partial specific volume of the monomers may be used for the aggregates and that ~ = NV/M, where N is Avogadro's number, M the molecular weight, and V the hydrated volume of the particles. The particle length for the 7-S component, with 8 ---- 2", is thus 24ooo A, and the axial ratio p = IIOO. It can be shown with the use of the above equations that for a given sedimentation coefficient, at high axial ratios, the length is proportional to the axial ratio. Thus it appears that the myosin aggregate having essentially the same sedimentation coefficient, at high axial ratios, the length is proportional to the axial ratio, and that the myosin aggregate having essentially the same sedimentation coefficient as the monomer must be due to end-to-end aggregation. The finding of typical myosin ATPase activity after the removal of rapidly sedimenting components constitutes another piece of evidence in favor of the view, elaborated elsewhere9, that. clearing of actomyosin is accompanied by dissociation. Further studies on these elongated myosin aggregates formed under conditions closely resembling those prevailing in muscle may contribute to a better understanding of the fine structure of the myosin filaments observable under the electron microscope. This research has been supported by grants from the National Heart Institute, the Muscular Dystrophy Association of America, Inc., and the Life Insurance Medical Research Fund.
Cardiac Biochemistry Res earth Laboratory, Department of Medicine, H . N O D A * * Massachusetts General Hospital and Harvard Medical School, K. MARUYAMA*** Boston, Mass. (U.S.A.) J. GERGELY 1 H. NODA, K. MARU~rAMA AND J. GERGXLY, TC xo, Abstr. 5th Ann. Meeting, Biophys. Soc., St. Louis, Mo., 1961. 2 H. NODA, J. Biochem., 48 (196o) 31o. s S. S. SPlCER, J. Biol. Chem., 199 (1952) 289. 4 H. NODA AND S. EBASttI, Biochim. Biophys. Acta, 41 (I96O} 386. 5 T. S'¢XDBERG AND K . O . PEDERSEN, The Ultracentrifuge, O x f o r d U n i v e r s i t y Press, L o n d o n , 194 o. 6 F. PERRIN, J. phys. radium, 7 (1936) I. F. PERRIN, J. phys. radium, 5 (1934) 497. 8 H. SCtIERAGA,J. T. EDSALL AND J. O. GADD, J. Chem. Phys., 19 (1951) IiOI. 9 K. MARUYAMA AND J, GERGELY,TC I I , Abstr. 5th Ann. Meeting, Biophys. Soc., St. Louis, Mo., 1961.
Received November 28th, 1961 * T h i s v a l u e is c a l c u l a t e d f r o m Fig. 2 w i t h t h e u s e of t h e t a b l e g i v e n b y SCHERAGA et al. 8. measurements w e r e m a d e in a c o n c e n t r a t i o n r a n g e w h e r e ~ w a s f o u n d to be essentially i n d e p e n d e n t of t h e c o n c e n t r a t i o n . ** P r e s e n t a d d r e s s : D e p a r t m e n t of B i o p h y s i c s a n d B i o c h e m i s t r y , F a c u l t y of Science, U n i v e r s i t y of T o k y o , T o k y o (Japan}. *** R e s e a r c h Fellow of t h e H e l e n H a y W h i t n e y F o u n d a t i o n , while o n l e a v e f r o m t h e Biologica I n s t i t u t e , College of E d u c a t i o n , U n i v e r s i t y of T o k y o , T o k y o (Japan).
The
Biochim. Biophys. Acta, 58 (I962) 598-6Ol
SHORT COMMUNICATIONS
together with cystine, norvaline and isovaline with methionine, diaminopimelic acid with allo-isoleucine and citrulline with proline. The use of an internal standard in another role has been recently suggested by SIEGEL AND ROACH 11, who use fl-2-thienyl-DL-alanine as a means of monitoring the daily reliability of analyses on the i5o-cm column. We have been using L-2-amino guanido propionic acid (available from California Corporation for Biochemical Research) for this purpose on the I5-cm column (Fig. I). An ideal internal standardization might well result from using a mixture of known concentrations of both compounds. This should not only provide a measure of the daily reliability of the instrument but also permit a daily correction for any differences in analytical sensitivity between long and short columns (resulting from errors in pipetting, pump rates, ninhydrin stability, etc.). In summary, norleucine has been found to be a reliable internal standard for amino acid analysis by the chromatographic technique of SPACKMAN, STEIN AND MOORE. The internal standardization permits a correction for mechanical errors during steps leading to analysis of protein hydrolysates, and places these analyses in the forefront as highly precise tools for determining protein concentrations. The authors are indebted to Professor H. NEURATH for helpful discussions and to Miss K. LADUE for her technical assistance. This work has been supported in part by American Cancer Society (Grants P-45 and P-79), the National Institutes of Health, U.S. Public Health Service (RG-4617) and by funds from the State of Washington, Initiative 171.
Department of Biochemistry, University of Washington, Seattle, Wash. (U.S.A.)
K . A . WALSH J . R . BROWN
1 E. LAYNE, in S. P. COLOWlCK AND O. KAPLAN, Methods in Emymology, Vol. 3, Academic Press, Inc., New York, 1957, p. 447. 2 M, P. TOMBS, F. SOUTER AND N. F. MACLAGAN, Biochem. J., 73 (1959) 167. s O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (195 I) 265. 4 D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. 5 C. S. HANES, C. K. HARRIS, M. A. MOSCARELLO AND E. TIGANE, Can.J. Biochem. Physiol., 39 (I961) 163. e E. TIGANE, E. H. M. WADE, J. T. WONG AND C. S. HANES, Can. J. Biochem. Physiol., 39 (1961) 427. K. A. PIEZ AND L. MORRIS, Anal. Biochem., I (196o) 187. 8 T. W. GOODWlN AND R. A. MORTON, Bioehem. J., 4 ° (1946) 628. 9 E. L. SMITH AND A. STOCI
Received November 27th, 1961 Biochim. Biophys. Acta, 58 (1962) 596-598
End-to-end aggregation of myosin* NODA2 has recently reported the presence of a component sedimenting with a sedimentation coefficient of about 7 S in natural actomyosin under conditions when clearing 3 can be observed. From preliminary measurements he concluded that the 7-S peak was due to highly asymmetrical particles--their length being of the order of about * A preliminary
report of
this w o r k has been presented 1.
Biochim. Biophys. Acta, 58 (1962) 598-6Ol
599
SHORT COMMUNICATIONS
I/~--but was unable to identify them with any of the known myofibrillar proteins of muscle. The present report deals with further investigations on this problem. Natural actomyosin in a solution containing 2 m M ATP and 2 m M Mg at 1 o.18 was centrifuged at IOOOOO × g for 2 h. Some of the protein remained in the supernatant, and on examination of the analytical centrifuge a single peak with an extrapolated sedimentation coefficient of roughly 7 S was found. It should be noted that this is nearly equal to the sedimentation rate of the myosin monomer at higher ionic strength.
8.0
I00
5.0
I00
90
L" 4.0
90
8 0 ~:
7o ~
8o ~
0
70 ~
IR
3.0
~o ~
•
~
60 ~
~4,0 2.0
- -40
~o ~
~2.0 I'0 I 0
~
--20 ~
w
--I0 ~
I _ 0.15
I 0.20
KCI CONCENTRATION (M)
to 0.,25
0 __1
0
I
___] _ __.1__~ 2 3 4
[_
5
6
A TP CONCENTRATION(raM)
Fig. I. The effect of (a) KC1 concentration a n d (b) A T P concentration on the a m o u n t of protein recovered in t h e I o o o o o × g s u p e r n a t a n t ( 0 - - 0 ) and on the birefringence ( O - - O ) of the i oo ooo × g s u p e r n a t a n t . Conditions: (a) Actomyosin, I.O mg/ml, o.o2 M p h o s p h a t e buffer (pH 7.o), 2 m M ATP, a n d 2. 5 m M MgCI=; KCI as indicated on the abscissa. (b) Actomyosin, 1.6 m g / m l , o.o2 M p h o s p h a t e buffer (pH 7.o), o.z 5 M KC1 and 2.5 m M MgCI=; A T P as indicated on the abscissa. P r o t e i n d e t e r m i n a t i o n s were m a d e w i t h the use of a biuret m e t h o d ; An was determined a t a velocity gradient of 13oo sec -1.
When the concentration of KC1 or ATP was increased the amount of the 7-S component increased monotonically, but the total birefringence went through a maximum and then decreased (Fig. I). The birefringence of flow, An, of the supernatant was almost equal to, or slightly higher than, that of the uncentrifuged solution. The extinction angle of the supernatant was lower than that of the uncentrifuged system at all velocity gradients (Fig. 2). The supernatant containing the 7-S component showed ATPase activity having all the characteristics of myosin. It was activated by Ca 2+ but not by Mg2+--unless F-actin was added (Table I). In the presence of F-actin in 0.05 M KCI typical superprecipitation could be observed upon addition of I m M ATP. Thus, in conjunction with the results described above, it appears that myosin forms well-defined aggregates in the clear actomyosin system. The results obtained at higher KCI or ATP concentrations (Fig. I) could be interpreted in terms of there being, first, an increase in the 7-S component at the expense of the more rapidly Biochim. Biophys. Acta, 58 (1962) 598--6ox
600
SHORT COMMUNICATIONS
sedimenting ones 1, followed by the dissociation of the 7-S component to monomers-reflected in the decrease of flow birefringence. In view of the differences between the flow-birefringence properties before and after centrifugation, it also appears that the aggregation of myosin was enhanced by the removal of the components sedimenting at a higher rate. Similar aggregates could be observed with myosin alone under the conditions under which these experiments were carried out although the results were more erratic. Clearly, to decide 50~--
50
40
4O
tt t~
Lu
50
30
~-
t~
0
L
20 L
20
Lo~
-41o
L
0 !,
I
0
I0
~
0
20
30
VELOCITY GRADIENT (sec-O
Fig. 2. E x t i n c t i o n angle a n d birefringence of cleared a c t o m y o s i n before a n d a f t e r c e n t r i f u g a t i o n a t 10o0oo × g for 2 h. Conditions: A c t o m y o s i n , 2.2 m g ] m l , o.02 N / ! h i s t i d i n e buffer (pH 6.8), 0.17 M KC1, 2 m M A T P a n d 2. 5 m M MgC1 v Birefringence m e a s u r e m e n t s were carried o u t a t t h e velocity g r a d i e n t s i n d i c a t e d on t h e abscissa. O - - O , e x t i n c t i o n a n g l e before c e n t r i f u g a t i o n ; Q - I , e x t i n c t i o n angle a f t e r c e n t r i f u g a t i o n ; & - A, birefringence before c e n t r i f u g a t i o n ; A - - A , birefringence a f t e r c e n t r i f u g a t i o n . TABLE I ATPAsE A C T I V I T Y O F 7-S C O M P O N E N T T h e p r o t e i n in t h e s u p e r n a t a n t of clear a c t o m y o s i n (see text) w a s p r e c i p i t a t e d b y dialysis a g a i n s t water, collected b y c e n t r i f u g a t i o n a n d dissolved in o.6 M KC1. A T P a s e a c t i v i t y w a s a s s a y e d in t h e following s y s t e m : 0.0 5 M KC1, o.o2 M h i s t i d i n e buffer (pH 7.0), 1.2 m M A T P . I rnl c o n t a i n i n g O,I 4 m g p r o t e i n w a s i n c u b a t e d a t 2o ° for 30 m i n . T h e Pt liberated w a s d e t e r m i n e d w i t h t h e F i s k e - S u b b a r o w procedure. Additions
~molesof P~
None 4 m M Mg ~+ 4 m M Mg ~+ + o.14 m g F - a c t i n 4 rnM C# +
o.o3 0.03 o.9I o.96
Biochim. Biophys. Acta, 58 (1962) 598-6Ol
SHORT COMMUNICATIONS
601
whether actin plays a role in the formation of the myosin aggregates requires further study. The flow birefringence of myosin at low ionic strength in the absence of ATP 4 may be partly due to the same aggregates. A tentative conclusion about the shape of these myosin aggregates can be arrived at on the basis of the sedimentation and birefringence measurements. By combining the SVEDBERGequation 5 with PERRIN'S tWO relations between the axial ratio and the translationaN and rotary 7 frictional coefficients, respectively, it is possible to calculate the axial ratio and the length of the particle from the sedimentation coefficient and the rotary-diffusion coefficient, provided one assumes that the partial specific volume of the monomers may be used for the aggregates and that ~ = NV/M, where N is Avogadro's number, M the molecular weight, and V the hydrated volume of the particles. The particle length for the 7-S component, with 8 ---- 2", is thus 24ooo A, and the axial ratio p = IIOO. It can be shown with the use of the above equations that for a given sedimentation coefficient, at high axial ratios, the length is proportional to the axial ratio. Thus it appears that the myosin aggregate having essentially the same sedimentation coefficient, at high axial ratios, the length is proportional to the axial ratio, and that the myosin aggregate having essentially the same sedimentation coefficient as the monomer must be due to end-to-end aggregation. The finding of typical myosin ATPase activity after the removal of rapidly sedimenting components constitutes another piece of evidence in favor of the view, elaborated elsewhere9, that. clearing of actomyosin is accompanied by dissociation. Further studies on these elongated myosin aggregates formed under conditions closely resembling those prevailing in muscle may contribute to a better understanding of the fine structure of the myosin filaments observable under the electron microscope. This research has been supported by grants from the National Heart Institute, the Muscular Dystrophy Association of America, Inc., and the Life Insurance Medical Research Fund.
Cardiac Biochemistry Res earth Laboratory, Department of Medicine, H . N O D A * * Massachusetts General Hospital and Harvard Medical School, K. MARUYAMA*** Boston, Mass. (U.S.A.) J. GERGELY 1 H. NODA, K. MARU~rAMA AND J. GERGXLY, TC xo, Abstr. 5th Ann. Meeting, Biophys. Soc., St. Louis, Mo., 1961. 2 H. NODA, J. Biochem., 48 (196o) 31o. s S. S. SPlCER, J. Biol. Chem., 199 (1952) 289. 4 H. NODA AND S. EBASttI, Biochim. Biophys. Acta, 41 (I96O} 386. 5 T. S'¢XDBERG AND K . O . PEDERSEN, The Ultracentrifuge, O x f o r d U n i v e r s i t y Press, L o n d o n , 194 o. 6 F. PERRIN, J. phys. radium, 7 (1936) I. F. PERRIN, J. phys. radium, 5 (1934) 497. 8 H. SCtIERAGA,J. T. EDSALL AND J. O. GADD, J. Chem. Phys., 19 (1951) IiOI. 9 K. MARUYAMA AND J, GERGELY,TC I I , Abstr. 5th Ann. Meeting, Biophys. Soc., St. Louis, Mo., 1961.
Received November 28th, 1961 * T h i s v a l u e is c a l c u l a t e d f r o m Fig. 2 w i t h t h e u s e of t h e t a b l e g i v e n b y SCHERAGA et al. 8. measurements w e r e m a d e in a c o n c e n t r a t i o n r a n g e w h e r e ~ w a s f o u n d to be essentially i n d e p e n d e n t of t h e c o n c e n t r a t i o n . ** P r e s e n t a d d r e s s : D e p a r t m e n t of B i o p h y s i c s a n d B i o c h e m i s t r y , F a c u l t y of Science, U n i v e r s i t y of T o k y o , T o k y o (Japan}. *** R e s e a r c h Fellow of t h e H e l e n H a y W h i t n e y F o u n d a t i o n , while o n l e a v e f r o m t h e Biologica I n s t i t u t e , College of E d u c a t i o n , U n i v e r s i t y of T o k y o , T o k y o (Japan).
The
Biochim. Biophys. Acta, 58 (I962) 598-6Ol