- Email: [email protected]
Electrophoretic study of the “easily soluble” proteins of normal and atrophied rabbits' muscles and of normal human muscles
346
BIOCHIMICA ET BIOPHYSICA ACTA
VOL. 8
(1952)
Short Communications and Preliminary Notes
ELECTROPHORETIC S T U D Y OF THE "EASILY SOLUBLE" PROTEINS OF NORMAL A N D A T R O P H I E D RABBITS' MUSCLES AND OF NORMAL HUMAN MUSCLES by A. M. F. H. H A A N
Laboratory o~ Physiological Chemistry, The University, Utrecht (Netherlands)
T h e c o m p o s i t i o n of t h e m i x t u r e of t h e " e a s i l y soluble" p r o t e i n s of v a r i o u s r a b b i t s ' m u s c l e s a n d of h u m a n skeletal m u s c l e s - - v i z , t h e p r o t e i n s e x t r a c t e d b y K C l - p h o s p h a t e of ionic s t r e n g t h o.1B a n d p H 7 . x 5 - - w a s d e t e r m i n e d b y electrophoresis as described b y B o s c H x. T h e e x p e r i m e n t s on r a b b i t s were carried o u t w i t h t h e v a s t u s lateralis a n d t h e g a s t r o c n e m i u s . T h e differences b e t w e e n two n o r m a l c o n t r a - l a t e r a l m u s c l e s were a l w a y s scarcely perceptible, e v e n w h e n t h e y were r e m o v e d w i t h a week i n t e r v a l . F i f t e e n r a b b i t s were s u b j e c t e d to different t r e a t m e n t s c a u s i n g m u s c u l a r atrop h y . As a c o n t r o l t h e n o r m a l c o n t r a - l a t e r a l m u s c l e w a s a l w a y s ,4 used. All a t r o p h i c a l m u s c l e s i n v e s t i g a t e d s h o w e d t h e s a m e e l e c t r o p h o r e t i c p a t t e r n , i n d e p e n d e n t of t h e c a u s e of a t r o p h y , b u t differing v e r y m u c h f r o m t h e n o r m a l p a t t e r n . I n accorda n c e w i t h CREPAX2 we f o u n d t h a t in a t r o p h y , c a u s e d b y dissect i o n of t h e m o t o r i c nerve, t h e p e r c e n t a g e of c o m p o n e n t II (called m y o a l b u m i n b y t h e D u B u i s s o N school) h a d increased, while t h e p e r c e n t a g e s of c o m p o n e n t s VII, V I I I a n d I X h a d decreased (percentages of t h e t o t a l surface of t h e d i a g r a m ) . I t f u r t h e r a p p e a r e d t h a t also t h e p e r c e n t a g e of t h e level p a r t of the diagram between components II and V had distinctly increased. Similar r e s u l t s were o b t a i n e d a f t e r v i t a m i n E defic i e n t feeding, s t a r v a t i o n a n d i m m o b i l i s a t i o n . I n d e p e n d e n t of t h e c a u s e of t h e a t r o p h y a n a t r o p h i e d m u s c l e w h i c h h a d lost a b o u t h a l f its original w e i g h t c o n t a i n e d per g a b o u t twice t h e original a m o u n t of p r o t e i n II a n d of t h e p r o t e i n s i n d i c a t e d b y t h e level p a r t of t h e d i a g r a m , while 7r
Fig. i. E l e c t r o p h o r e t i c p a t t e r n s of e x t r a c t s of different m u s cles. Buffer, u s e d for e x t r a c t i o n , dialysis a n d electrophoresis: 0.0 5 M K C l + o.o2 3 M N a a H P O 4 + o.oi M K H ~ P O 4 (/~ = o.I3, p H = 7.15). A s c e n d i n g b o u n d a r i e s . A. n o r m a l r a b b i t ' s skeletal m u s c l e B. a t r o p h i e d r a b b i t ' s skeletal m u s c l e C. n o r m a l h u m a n skeletal m u s c l e D. r a b b i t ' s skeletal m u s c l e in rigor E. r a b b i t ' s h e a r t m u s c l e F. r a b b i t ' s d i a p h r a g m G. r a b b i t ' s s t o m a c h m u s c l e H. r a b b i t ' s u t e r u s
VOL. 8 (1952)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
347
the total amount of easily soluble proteins per g had decreased to some extent. Thus it appeared t h a t protein II and the components of the level part of the diagram had only increased in relative proportion to the other components, and t h a t their absolute amounts in the total muscle had remained constant. Rigor mortis also caused a distinct change in the diagram as compared to the fresh contralateral muscle. The percentage of component V had increased 2 to 3 fold, while the percentages of components VIII and I X had decreased. The protein composition of muscles in rigor mortis thus differs essentially from t h a t of normal as well as from t h a t of atrophic muscles. As an introduction to the study of patients suffering from muscle diseases, twelve normal human muscles were investigated. The electrophoretic diagrams closely resembled the diagrams obtained with rabbit's skeletal muscles. The latter diagrams, however, differed very much from the diagrams obtained from rabbit's heart, uterus, diaphragm and stomach, while the diagrams of these latter muscles were also mutually different. This work forms part of the investigations on the chemistry of muscle diseases by H. G. K. WESTENBRtHH and collaborators. I t has been communicated in the Meeting of the Netherlands Society for Biochemistry on December 1st, I951. Full details will be published. The support of the Netherlands Organisation for Pure Scientific Research (Z.W.O.) is gratefully acknowledged. REFERENCES 1 M. W. BOSCH, Biochim. Biopkys. Aeta, 7 (1951) 61. P. CREP.~X, Experientia, 5 (1949) 167. Received January 2ISt, 1952
QUATERNARY AMMONIUM SALTS AS INHIBITORS OF ACETYLCHOLINE ESTERASE II. pH DEPENDENCE OF T H E INHIBITORY EFFECTS OF
QUATERNARY AMMONIUM SALTS, AND T HE DISSOCIATION CONSTANT OF T H E ANIONIC SITE by F E L I X BERGMANN AND A R E L A SHIMONI
Department o~ Pharmacology, The Hebrew U~ivcrsity - Hadassah Medical School, Jerusalem (Israel)
Quaternary ammonium salts, in which one part or the other of the ester grouping R - O - C - R '
II
O is missing, were compared as inhibitors for ACh esterase. Whereas the inhibitory effect decreases in the series prostigmine > acetophenone trimethylammonium iodide > choline chloride > tetraethylammonium bromide, all members of the series show a very similar relative decrease of activity with decreasing pH. Near p H 5 the activity of all quaternary ammonium salts approaches zero. On the other hand, over the whole range of p H 7-1o.5 their inhibitory efficiency remains practically constant. The progressive inactivation with decreasing p H can not be ascribed to the inactivation of the nucleophilic group G 1 in the esteratic part of the active surface1, as claimed by WILSON~, since e.g. the tetraethylammonium ion has no "specific" chemical affinity to such a group (its alkyl chains are attached to the active surface by unspecific VAN DER WAALS forces). The dependence of inhibitory effects on p H changes must be due to the combination o/the anionic site wit]~ the hydrogen ion, which inactivates both G 1 in the esteratic site and the negative charge of the anionic site. The dissociation constant KEH~+, determined previously as characteristic for G1, thus appears now to be incorrect and requires redetermination.
BIOCHIMICA ET BIOPHYSICA ACTA
VOL. 8
(1952)
Short Communications and Preliminary Notes
ELECTROPHORETIC S T U D Y OF THE "EASILY SOLUBLE" PROTEINS OF NORMAL A N D A T R O P H I E D RABBITS' MUSCLES AND OF NORMAL HUMAN MUSCLES by A. M. F. H. H A A N
Laboratory o~ Physiological Chemistry, The University, Utrecht (Netherlands)
T h e c o m p o s i t i o n of t h e m i x t u r e of t h e " e a s i l y soluble" p r o t e i n s of v a r i o u s r a b b i t s ' m u s c l e s a n d of h u m a n skeletal m u s c l e s - - v i z , t h e p r o t e i n s e x t r a c t e d b y K C l - p h o s p h a t e of ionic s t r e n g t h o.1B a n d p H 7 . x 5 - - w a s d e t e r m i n e d b y electrophoresis as described b y B o s c H x. T h e e x p e r i m e n t s on r a b b i t s were carried o u t w i t h t h e v a s t u s lateralis a n d t h e g a s t r o c n e m i u s . T h e differences b e t w e e n two n o r m a l c o n t r a - l a t e r a l m u s c l e s were a l w a y s scarcely perceptible, e v e n w h e n t h e y were r e m o v e d w i t h a week i n t e r v a l . F i f t e e n r a b b i t s were s u b j e c t e d to different t r e a t m e n t s c a u s i n g m u s c u l a r atrop h y . As a c o n t r o l t h e n o r m a l c o n t r a - l a t e r a l m u s c l e w a s a l w a y s ,4 used. All a t r o p h i c a l m u s c l e s i n v e s t i g a t e d s h o w e d t h e s a m e e l e c t r o p h o r e t i c p a t t e r n , i n d e p e n d e n t of t h e c a u s e of a t r o p h y , b u t differing v e r y m u c h f r o m t h e n o r m a l p a t t e r n . I n accorda n c e w i t h CREPAX2 we f o u n d t h a t in a t r o p h y , c a u s e d b y dissect i o n of t h e m o t o r i c nerve, t h e p e r c e n t a g e of c o m p o n e n t II (called m y o a l b u m i n b y t h e D u B u i s s o N school) h a d increased, while t h e p e r c e n t a g e s of c o m p o n e n t s VII, V I I I a n d I X h a d decreased (percentages of t h e t o t a l surface of t h e d i a g r a m ) . I t f u r t h e r a p p e a r e d t h a t also t h e p e r c e n t a g e of t h e level p a r t of the diagram between components II and V had distinctly increased. Similar r e s u l t s were o b t a i n e d a f t e r v i t a m i n E defic i e n t feeding, s t a r v a t i o n a n d i m m o b i l i s a t i o n . I n d e p e n d e n t of t h e c a u s e of t h e a t r o p h y a n a t r o p h i e d m u s c l e w h i c h h a d lost a b o u t h a l f its original w e i g h t c o n t a i n e d per g a b o u t twice t h e original a m o u n t of p r o t e i n II a n d of t h e p r o t e i n s i n d i c a t e d b y t h e level p a r t of t h e d i a g r a m , while 7r
Fig. i. E l e c t r o p h o r e t i c p a t t e r n s of e x t r a c t s of different m u s cles. Buffer, u s e d for e x t r a c t i o n , dialysis a n d electrophoresis: 0.0 5 M K C l + o.o2 3 M N a a H P O 4 + o.oi M K H ~ P O 4 (/~ = o.I3, p H = 7.15). A s c e n d i n g b o u n d a r i e s . A. n o r m a l r a b b i t ' s skeletal m u s c l e B. a t r o p h i e d r a b b i t ' s skeletal m u s c l e C. n o r m a l h u m a n skeletal m u s c l e D. r a b b i t ' s skeletal m u s c l e in rigor E. r a b b i t ' s h e a r t m u s c l e F. r a b b i t ' s d i a p h r a g m G. r a b b i t ' s s t o m a c h m u s c l e H. r a b b i t ' s u t e r u s
VOL. 8 (1952)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
347
the total amount of easily soluble proteins per g had decreased to some extent. Thus it appeared t h a t protein II and the components of the level part of the diagram had only increased in relative proportion to the other components, and t h a t their absolute amounts in the total muscle had remained constant. Rigor mortis also caused a distinct change in the diagram as compared to the fresh contralateral muscle. The percentage of component V had increased 2 to 3 fold, while the percentages of components VIII and I X had decreased. The protein composition of muscles in rigor mortis thus differs essentially from t h a t of normal as well as from t h a t of atrophic muscles. As an introduction to the study of patients suffering from muscle diseases, twelve normal human muscles were investigated. The electrophoretic diagrams closely resembled the diagrams obtained with rabbit's skeletal muscles. The latter diagrams, however, differed very much from the diagrams obtained from rabbit's heart, uterus, diaphragm and stomach, while the diagrams of these latter muscles were also mutually different. This work forms part of the investigations on the chemistry of muscle diseases by H. G. K. WESTENBRtHH and collaborators. I t has been communicated in the Meeting of the Netherlands Society for Biochemistry on December 1st, I951. Full details will be published. The support of the Netherlands Organisation for Pure Scientific Research (Z.W.O.) is gratefully acknowledged. REFERENCES 1 M. W. BOSCH, Biochim. Biopkys. Aeta, 7 (1951) 61. P. CREP.~X, Experientia, 5 (1949) 167. Received January 2ISt, 1952
QUATERNARY AMMONIUM SALTS AS INHIBITORS OF ACETYLCHOLINE ESTERASE II. pH DEPENDENCE OF T H E INHIBITORY EFFECTS OF
QUATERNARY AMMONIUM SALTS, AND T HE DISSOCIATION CONSTANT OF T H E ANIONIC SITE by F E L I X BERGMANN AND A R E L A SHIMONI
Department o~ Pharmacology, The Hebrew U~ivcrsity - Hadassah Medical School, Jerusalem (Israel)
Quaternary ammonium salts, in which one part or the other of the ester grouping R - O - C - R '
II
O is missing, were compared as inhibitors for ACh esterase. Whereas the inhibitory effect decreases in the series prostigmine > acetophenone trimethylammonium iodide > choline chloride > tetraethylammonium bromide, all members of the series show a very similar relative decrease of activity with decreasing pH. Near p H 5 the activity of all quaternary ammonium salts approaches zero. On the other hand, over the whole range of p H 7-1o.5 their inhibitory efficiency remains practically constant. The progressive inactivation with decreasing p H can not be ascribed to the inactivation of the nucleophilic group G 1 in the esteratic part of the active surface1, as claimed by WILSON~, since e.g. the tetraethylammonium ion has no "specific" chemical affinity to such a group (its alkyl chains are attached to the active surface by unspecific VAN DER WAALS forces). The dependence of inhibitory effects on p H changes must be due to the combination o/the anionic site wit]~ the hydrogen ion, which inactivates both G 1 in the esteratic site and the negative charge of the anionic site. The dissociation constant KEH~+, determined previously as characteristic for G1, thus appears now to be incorrect and requires redetermination.