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Heat stable protein from skeletal muscle
178
SHORT COMMUNICATIONS, PRELIMINARY NOTt,;S
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11956)
noted t h a t the strong reflections corresponding to 9.05 A and lO.8 A occur [is more extended arcs merging into the equatorial spots. The observation of this series of meridional reflections in such detail w a r r a n t s f u r t h e r investigation and it is proposed to continue with this work. I a m indebted to Mr. S. J. EDMONDS for the s u p p l y of the muscles and for his assistance in their preparation. I t is a pleasure to t h a n k Dr. S. G. TOMLIN for his encouragement and interest and Professor W. T. ASTBURY for a valuable discussion during his visit to this University.
Physics Department, University of Adelaide (South Australia)
C. R. WORTHINGTON
S. J. EDMONDS, Australian J. Marine and Freshwater Research (to be published). 2 R. S. BEAR, J. Am. Chem. Soe., 67 (1945) 1625. 3 W. T. ASTBURY, Exptl. Cell Research, 1 (1947) 234. 4 R. O. HERZOG AND W. JANCKE, Naturwissenschaflen, 14 (1926) 1223. 5 W. LOTMAR AND L. E. R. PICKEN, Helv. Chim. Acta, 25 (1942) 538. 6 R. S. BEAR AND C. M. M. CANNAN, Nature, 168 (I95 I) 684. H. E. HUXLEY AND J. C. I~ENDREW, Nature, 17o (1952) 882. 8 C. R. WORTHINGTON, dr. Sci. Instr. (in the press). 9 H. ]~. HUXLEY, Proc. Roy. Soc. B, 141 (1953) .59. Received October I5th, 1955 z
Heat stable protein from skeletal muscle A substance h a v i n g the general properties of a protein has been isolated from muscle b y the following m e t h o d : F o u r - h u n d r e d g r a m s of ground beef are stirred with 32o ml of 2 M MgC12 and 88o ml of w a t e r containing 12 ml of N HCI. The suspension, the p H of which should be 4.5, is heated to 9o ° and filtered hot. The filtrate is neutralized with N a O H and treated with i volume of methanol. The precipitate is sedimented and dissolved in 35o ml of o.4M MgC12 adjusted to p H 5.o. The solution is heated to 9 o°, the precipitate discarded. The s u p e r n a t a n t is neutralized, the precipitate again discarded. The s u p e r n a t a n t is dialyzed at 4 ° against 2o volumes of distilled water. The protein crystallizes in the form of thin needles or coarse sphenoids (Fig. 1). Crystallization is repeated by the same procedure. The yield is o. 4 g. The same m e t h o d has yielded material of the same crystalline appearance when applied to h u m a n muscle. The extraction of the protein was unsuccessful when NaCI or BaC12 was used instead of MgC12. Crystallization did not take place in the absence of Ca, Ba or Mg. MnCI 2 and CoC12 dissolved the protein b u t did not p r o m o t e crystallization. Zn and heavy metals did not dissolve it. I t s solubility was a p p r o x i m a t e l y as folh)ws: In the absence of salt, above p H 7 and below p H 4; in o . o 2 M MgC12, only below p H 4; in o . 4 M MgC12, only above p H 5The crystalline p r e p a r a t i o n s yielded a high ash (6.i % when BaC12 was used for crystallization). This decreased to o.25 % after dialysis against o.oi N HC1. Nitrogen content was J 4.0 %, carbon 42.6 % ; p h o s p h o r u s and sugar were not detected. The viscosity of solutions in o.41tl MgC12, p H 7.o, was measured at 3 ° ° in Ostwald type pipettes and yielded an intrinsic viscosity It/] = 0.38. Hence 1, Simha's factor v = [~/] loo/~ = 5~ (assuming o.74 for the partial specific volume ~); hence 2, an axial ratio of 24: 1. The protein was homogeneous in the ultracentrifuge in o.4:~¢ MgC12, p H 7.o, and yieMed s = 2. 7" IO-13 at infinite dilution. F r o m this and the axial ratio, with the help of the simplified form of Perrin's e q u a t i o n a, the following values were obtained : radius of an ellipsoid of revolution, b = 1.o 3 mt~; semi long axis, a = 25 m/~; molecular weight = 91,ooo. I n O . o l N HC1, p H 2.5, ultracentrifugation revealed two components, whose sedimentation rates at zero concentration appeared to be of the order of m a g n i t u d e of 2 and 3' lO 13 very approximately. The curve relating s to concentration was, however, so convex t o w a r d the origin t h a t no satisfactory extrapolation could be made. The substance shares with the other muscle proteins, m y o s i n 4, actin 4, and the peptonlyosins*,5, 6, the p r o p e r t y of being insoluble in high salt concentration at p H below 4- It resembles p e p t o m y o s i n B 6 in molecular shape, being slightly less elongated. However, since the sedimen" P e p t o m y o s i n A also is insoluble below p H 4 in 31 NaC1. This fact was not stated i n the original description.
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19 (1956)
s n O R T COMMUNICATIONS, PRELIMINARY NOTES
179
Fig. l. H e a t stable m u s c l e protein. (Magnification 850 × ) ration rate of v e r y elongated particles d e p e n d s p r i m a r i l y on w i d t h a n d v e r y little on length, t h e h o m o g e n e i t y s u g g e s t e d b y a s h a r p s e d i m e n t i n g b o u n d a r y m a y be illusory, a n d t h e results c o m p a t i b l e with t h e a s s m n p t i o n t h a t t h e m a t e r i a l consists of thread-like particles of u n i f o r m width, in v a r i o u s degrees of linear aggregation. O s m o t i c p r e s s u r e m e a s u r e m e n t s b y t h e senior a u t h o r ' s m e t h o d 7 yielded only erratic results, a fact u s u a l l y o b s e r v e d with m a t e r i a l t h a t can be s u s p e c t e d of p h y s i c a l instability. JAQUES BOURDILLON Division o/Laboratories and Research, New York Slate Department of Health, WINFIELD H. BAKER
Albany, N . Y . (U.S.A.) 1 j . T. EDSALL, in The Proteins, edited b y H. NEURATH AZqD K. BAILEY, A c a d e m i c Press, New York, 1953, Vol. I, P a r t B, p. 689 (Table). 2 T . SVEDBERG AND K. O. PEDERSEN, The Ultracentrifuge, C l a r e n d o n Press, Oxford, 194 o, (Table). a A. R. PEACOCKE AXD H. K. SCHACH,~IAN, Biochim. Biophys. Acta, 15 (1954) 198. 4 A. SZENT-GY6RGYI, Chemistry o/Muscular Co~ztraclion, A c a d e m i c Press, Inc., N e w York, 2nd ed. 5 j . BOURDILLON, Arch. Biochem., 16 (1948) 61. 6 j . BOURDILLON, J. Am. Chem. Soc. (in press). 7 j. BOURDILLON, J. Biol. Chem., 127 (1939) 617. Received N o v e m b e r 5th,
Inc., p. 41
1951,
1955
Synthesis of ribose by the rat* T h e b i o s y n t h e s i s of ribose I h a s been p o s t u l a t e d to occur (a) directly f r o m h e x o s e b y loss of an e n d c a r b o n a n d (b) f r o m s m a l l e r u n i t s b y c o n d e n s a t i o n , e.g. C 2 + C a. D a t a o b t a i n e d in p r e v i o u s e x p e r i m e n t s 2 h a v e been i n t e r p r e t e d to indicate t h a t in t h e chick, t h e s y n t h e s i s of this p e n t o s e p r o b a b l y occurs b y t h e l a t t e r m e c h a n i s m , possibly b y a c o n d e n s a t i o n of g l y c e r a l d e h y d e 3 - p h o s p h a t e a n d a n active C 2 u n i t (e.g. f r o m f r u c t o s e - 6 - p h o s p h a t e a) c a t a l y z e d b y t r a n s k e t o l a s e . T h e t e c h n i q u e e m p l o y e d was to isolate glycogen a n d ribose f r o m t h e c o m b i n e d i n t e r n a l o r g a n s * P r e s e n t e d a t t h e m e e t i n g of t h e A m e r i c a n C h e m i c a l Society, Cincinnati, Ohio, April, 1955. I2
SHORT COMMUNICATIONS, PRELIMINARY NOTt,;S
VOL. 1 9
11956)
noted t h a t the strong reflections corresponding to 9.05 A and lO.8 A occur [is more extended arcs merging into the equatorial spots. The observation of this series of meridional reflections in such detail w a r r a n t s f u r t h e r investigation and it is proposed to continue with this work. I a m indebted to Mr. S. J. EDMONDS for the s u p p l y of the muscles and for his assistance in their preparation. I t is a pleasure to t h a n k Dr. S. G. TOMLIN for his encouragement and interest and Professor W. T. ASTBURY for a valuable discussion during his visit to this University.
Physics Department, University of Adelaide (South Australia)
C. R. WORTHINGTON
S. J. EDMONDS, Australian J. Marine and Freshwater Research (to be published). 2 R. S. BEAR, J. Am. Chem. Soe., 67 (1945) 1625. 3 W. T. ASTBURY, Exptl. Cell Research, 1 (1947) 234. 4 R. O. HERZOG AND W. JANCKE, Naturwissenschaflen, 14 (1926) 1223. 5 W. LOTMAR AND L. E. R. PICKEN, Helv. Chim. Acta, 25 (1942) 538. 6 R. S. BEAR AND C. M. M. CANNAN, Nature, 168 (I95 I) 684. H. E. HUXLEY AND J. C. I~ENDREW, Nature, 17o (1952) 882. 8 C. R. WORTHINGTON, dr. Sci. Instr. (in the press). 9 H. ]~. HUXLEY, Proc. Roy. Soc. B, 141 (1953) .59. Received October I5th, 1955 z
Heat stable protein from skeletal muscle A substance h a v i n g the general properties of a protein has been isolated from muscle b y the following m e t h o d : F o u r - h u n d r e d g r a m s of ground beef are stirred with 32o ml of 2 M MgC12 and 88o ml of w a t e r containing 12 ml of N HCI. The suspension, the p H of which should be 4.5, is heated to 9o ° and filtered hot. The filtrate is neutralized with N a O H and treated with i volume of methanol. The precipitate is sedimented and dissolved in 35o ml of o.4M MgC12 adjusted to p H 5.o. The solution is heated to 9 o°, the precipitate discarded. The s u p e r n a t a n t is neutralized, the precipitate again discarded. The s u p e r n a t a n t is dialyzed at 4 ° against 2o volumes of distilled water. The protein crystallizes in the form of thin needles or coarse sphenoids (Fig. 1). Crystallization is repeated by the same procedure. The yield is o. 4 g. The same m e t h o d has yielded material of the same crystalline appearance when applied to h u m a n muscle. The extraction of the protein was unsuccessful when NaCI or BaC12 was used instead of MgC12. Crystallization did not take place in the absence of Ca, Ba or Mg. MnCI 2 and CoC12 dissolved the protein b u t did not p r o m o t e crystallization. Zn and heavy metals did not dissolve it. I t s solubility was a p p r o x i m a t e l y as folh)ws: In the absence of salt, above p H 7 and below p H 4; in o . o 2 M MgC12, only below p H 4; in o . 4 M MgC12, only above p H 5The crystalline p r e p a r a t i o n s yielded a high ash (6.i % when BaC12 was used for crystallization). This decreased to o.25 % after dialysis against o.oi N HC1. Nitrogen content was J 4.0 %, carbon 42.6 % ; p h o s p h o r u s and sugar were not detected. The viscosity of solutions in o.41tl MgC12, p H 7.o, was measured at 3 ° ° in Ostwald type pipettes and yielded an intrinsic viscosity It/] = 0.38. Hence 1, Simha's factor v = [~/] loo/~ = 5~ (assuming o.74 for the partial specific volume ~); hence 2, an axial ratio of 24: 1. The protein was homogeneous in the ultracentrifuge in o.4:~¢ MgC12, p H 7.o, and yieMed s = 2. 7" IO-13 at infinite dilution. F r o m this and the axial ratio, with the help of the simplified form of Perrin's e q u a t i o n a, the following values were obtained : radius of an ellipsoid of revolution, b = 1.o 3 mt~; semi long axis, a = 25 m/~; molecular weight = 91,ooo. I n O . o l N HC1, p H 2.5, ultracentrifugation revealed two components, whose sedimentation rates at zero concentration appeared to be of the order of m a g n i t u d e of 2 and 3' lO 13 very approximately. The curve relating s to concentration was, however, so convex t o w a r d the origin t h a t no satisfactory extrapolation could be made. The substance shares with the other muscle proteins, m y o s i n 4, actin 4, and the peptonlyosins*,5, 6, the p r o p e r t y of being insoluble in high salt concentration at p H below 4- It resembles p e p t o m y o s i n B 6 in molecular shape, being slightly less elongated. However, since the sedimen" P e p t o m y o s i n A also is insoluble below p H 4 in 31 NaC1. This fact was not stated i n the original description.
VOL.
19 (1956)
s n O R T COMMUNICATIONS, PRELIMINARY NOTES
179
Fig. l. H e a t stable m u s c l e protein. (Magnification 850 × ) ration rate of v e r y elongated particles d e p e n d s p r i m a r i l y on w i d t h a n d v e r y little on length, t h e h o m o g e n e i t y s u g g e s t e d b y a s h a r p s e d i m e n t i n g b o u n d a r y m a y be illusory, a n d t h e results c o m p a t i b l e with t h e a s s m n p t i o n t h a t t h e m a t e r i a l consists of thread-like particles of u n i f o r m width, in v a r i o u s degrees of linear aggregation. O s m o t i c p r e s s u r e m e a s u r e m e n t s b y t h e senior a u t h o r ' s m e t h o d 7 yielded only erratic results, a fact u s u a l l y o b s e r v e d with m a t e r i a l t h a t can be s u s p e c t e d of p h y s i c a l instability. JAQUES BOURDILLON Division o/Laboratories and Research, New York Slate Department of Health, WINFIELD H. BAKER
Albany, N . Y . (U.S.A.) 1 j . T. EDSALL, in The Proteins, edited b y H. NEURATH AZqD K. BAILEY, A c a d e m i c Press, New York, 1953, Vol. I, P a r t B, p. 689 (Table). 2 T . SVEDBERG AND K. O. PEDERSEN, The Ultracentrifuge, C l a r e n d o n Press, Oxford, 194 o, (Table). a A. R. PEACOCKE AXD H. K. SCHACH,~IAN, Biochim. Biophys. Acta, 15 (1954) 198. 4 A. SZENT-GY6RGYI, Chemistry o/Muscular Co~ztraclion, A c a d e m i c Press, Inc., N e w York, 2nd ed. 5 j . BOURDILLON, Arch. Biochem., 16 (1948) 61. 6 j . BOURDILLON, J. Am. Chem. Soc. (in press). 7 j. BOURDILLON, J. Biol. Chem., 127 (1939) 617. Received N o v e m b e r 5th,
Inc., p. 41
1951,
1955
Synthesis of ribose by the rat* T h e b i o s y n t h e s i s of ribose I h a s been p o s t u l a t e d to occur (a) directly f r o m h e x o s e b y loss of an e n d c a r b o n a n d (b) f r o m s m a l l e r u n i t s b y c o n d e n s a t i o n , e.g. C 2 + C a. D a t a o b t a i n e d in p r e v i o u s e x p e r i m e n t s 2 h a v e been i n t e r p r e t e d to indicate t h a t in t h e chick, t h e s y n t h e s i s of this p e n t o s e p r o b a b l y occurs b y t h e l a t t e r m e c h a n i s m , possibly b y a c o n d e n s a t i o n of g l y c e r a l d e h y d e 3 - p h o s p h a t e a n d a n active C 2 u n i t (e.g. f r o m f r u c t o s e - 6 - p h o s p h a t e a) c a t a l y z e d b y t r a n s k e t o l a s e . T h e t e c h n i q u e e m p l o y e d was to isolate glycogen a n d ribose f r o m t h e c o m b i n e d i n t e r n a l o r g a n s * P r e s e n t e d a t t h e m e e t i n g of t h e A m e r i c a n C h e m i c a l Society, Cincinnati, Ohio, April, 1955. I2