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Electrophoresis of the non-structural proteins from normal and atrophic muscles of the rabbit and of man
258
BIOCHIMICA ET BIOPHYSICA ACTA
VOL. 11
(1953)
E L E C T R O P H O R E S I S OF T H E NON-STRUCTURAL P R O T E I N S FROM NORMAL AND A T R O P H I C MUSCLES OF T H E R A B B I T AND OF MAN* by A. M. F. H. H A A N
Laboratory [or Physiological Chemistry, The University, Utrecht (Netherla~ds)
INTRODUCTION
The experiments described in this paper aimed at ascertaining the influence of atrophy on the muscle proteins (globulin X, myogen, myoalbumin, myoglobin), soluble in salt solutions of low ionic strength and pH about 7, in which myosin and actomyosin are said to be insoluble. The proteins studied will further be called "easily soluble proteins" of muscle. They were studied by quantitative eleetrophoresis. According to the results of experiments, carried out in this laboratory by Bosc~f 1, the easily soluble proteins can be extracted completely by grinding the muscle with sand and a buffer solution of pH 7.15 and ionic strength o.13. This was confirmed in the course of the work presented here. The numerical evaluation of the rather complicated diagrams by fitting in Gauss curves did not give fully satisfactory results in the hands of BOSCH. He believes that the differences between results calculated from various diagrams that appear to be identical upon superimposed projection must be attributed to the difficulty in drawing a pencil line exactly through the middle of the projected curves obtained by the Svensson-Philpot technique and to the arbitrariness inherent in the choice of the Gauss curves. Therefore he advises always to make the electrophoresis diagrams under exactly identical conditions and to decide upon visual inspection of superimposed projections whether differences exist or not. This procedure has the great disadvantage that one is not able to express these differences quantitatively. We believe, however, that we have demonstrated that a quantitative analysis of the diagrams of extracts of skeletal muscles is possible. Our procedure will be described in detail below. Experiments were carried out with rabbit muscles and with human muscles. EXPERIMENTAL PART
~1ethods I n general the procedure of lBoscI~ 1 was followed. Therefore the same buffer solution was e m p l o y e d for extraction, dialysis and electrophoresis. BOSCH has tried several solutions. We have * This w o r k forms p a r t of the investigations on the b i o c h e m i s t r y of muscle diseases b y H. G. K. \VESTENBRINK a n d co-workers, s u p p o r t e d b y a g r a n t from the N e t h e r l a n d s Organisation for Pure Research (Z.W.O.).
Re/erences p. 269.
VOL.
11 ( 1 9 5 3 )
ELECTROPHORESIS OF MUS(:LE PROTEINS
25~ )
used a s o l u t i o n he r e c o m m e n d s , c o n t a i n i n g 0.05 M KC1, 0.023 h i N a 2 H P O 4 a n d O.Ol M t(H21'Oa, of p H 7.15 and ionic s t r e n g t h o.13, w h i c h gives tile m o s t d e t a i l e d d i a g r a m s of all s o l u t i o n s t e s t e d . Some m i n o r a l t e r a t i o n s d e s c r i b e d below were i n t r o d u c e d .
Extractiou o/the proteins T h e r a b b i t s were a n a e s t h e t i z e d w i t h N u m a l - R o c h e ( d i e t h y l a m m o n i u m a l l y i i s o p r o p y l b a r b i t u r a t e ) a n d bled b y t h e aorta. A f t e r excision t h e m u s c l e was s t o r e d for i h o u r a t l ~' C. I m m e d i a t e l y a f t e r g r i n d i n g a g mu scle w i t h a g s a n d a n d a ml e x t r a c t i o n fluid t h e nauscle d e b r i s were s p u n d o w n a t 1 ° C b y IO m i n u t e s ' c e n t r i f u g i n g a t I4,ooo g ( c e n t r i f u g i n g m a y also be clone a f t e r i or 2 h). C ( m t r a r y to 13OSCH'S e x p e r i e n c e viscous e x t r a c t s were n e v e r o b t a i n e d . No e x p l a n a t i o n of thi~ difference c a n be offered. I n t h e e x p e r i m e n t s on muscles, a t r o p h i e d as a c o n s e q u e n c e of i n a n i t i o n or v i t a m i n - E - f r e e feeding, it w a s n e c e s s a r y to excise t h e m u s c l e of one leg before s u b j e c t i n g t h e r a b b i t t o f a s t i n g or a v i t a m i n o s i s . Therefore t h i s m u s c l e c e r t a i n l y c o n t a i n e d more blood t h a n a m u s c l e e xc i s e d a f t e r t h e a n i m a l h a d been bled. As t h e e x p e r i m e n t s , referred to in T a b l e II, show, t h i s difference of blood c o n t e n t did not affect t h e results. The h u m a n m u s c l e s were also stored, as soon as possi bl e a f t e r t h e o p e r a t i o n , for i hour a t i ~' C before e x t r a c t s were made. N o r m a l h u m a n m u s c l e s were e x c i s e d d u r i n g l u n g o p e r a t i o n s .
Dialysis The d i a l y s i s was c a r r i e d o u t a t 5 ° C for a t l e a s t 12 h, u s u a l l y 18 t o 24 h. The p r e c i p i t a t e f o r m e d d u r i n g d i a l y s i s w a s s p u n d o w n b y c e n t r i f u g i n g a t o ° C a t 14,ooo g. F or r a b b i t m u s c l e e x t r a c t s 3 ° m i n u t e s ' c e n t r i f u g i n g was sufficient; in t h e case of h u n l a n m u s c l e e x t r a c t s t h e c e n t r i f u g i n g h a d t o be c o n t i n u e d for a t l e a s t 5 ° m i n u t e s in order to p r e v e n t fl oc c ul a t i on of s ome p r o t e i n (myosin) in t h e e l e c t r o p h o r e s i s cell.
Determination o[ protein content o/the extracts This was done b y m e a n s of an A b b e r e f r a c t o m e t e r . I n t h e case of n o r m a l m u s c l e s t h e p r o t e i n c o n t e n t was a l i t t l e h i g h e r t h a n 3 %. I n p r e l i m i n a r y e x p e r i m e n t s t h e r e s u l t s of t h e r e f r a c t o m e t r i c d e t e r m i n a t i o n s were c o m p a r e d w i t h t h e r e s u l t s of K j e l d a h l d e t e r m i n a t i o n s . The a g r e e m e n t w a s excellent.
Electrophoresis The d i a l y z e d p r o t e i n s o l u t i o n w a s d i l u t e d w i t h t h e buffer s o l u t i o n to a b o u t 2 %. E l e c t r o p h o r e s i s was carried o u t in a T i s e l i u s a p p a r a t u s (Striibin, Basle) a c c o r d i n g t o t h e S v e n s s o n - P h i l p o t m e t h o d . Ilford P a n F film, 35 ram, 25 ° S, w a s used. No difficulties w e re e n c o u n t e r e d w i t h t h e e x t r a c t s of w h i t e mu scles of r a b b i t s . I n t h e case of e x t r a c t s of red m u s c l e s of b o t h h u m a n a n d r a b b i t , i t a p p e a r e d to be n e c e s s a r y to c o v e r t h e u n c o l o u r e d p o r t i o n of t h e a s c e n d i n g b o u n d a r y in t i l e e l e c t r o p h o r e s i s cell d u r i n g p a r t of t h e r a t h e r long e x p o s u r e time, a n d to p r e v e n t t h e e n t r a n c e of s t r a y l i g h t from t h e r o o m i n t o the o p t i c a l system• Some m y o s i n s o l u t i o n s were e x a m i n e d , As t h e y were v e r y o p a l e s c e n t v e r y l ong e x p o s u r e t i m e s were req uired . D i a g r a m s of r e a s o n a b l e q u a l i t y were o b t a i n e d . Y e t we b e l i e v e t h a t cells as ns e d b y DUBUISSON et al. 2 are to be p r e f e r r e d to our cells of t h e c o m m o n t y p e for t h e i n v e s t i g a t i o n of m y o s i n solutions.
Numerical evaluation o/the diagrams
VIII
Th e g e n e r a l t y p e of d i a g r a m to be a n a l y z e d is s h o w n in Fig. i. I n p r i n c i p l e t h e a n a l y s i s was c a r r i e d o u t a c c o r d i n g to WIEDEMANN 3. A Gauss c u r v e was p l a c e d in p e a k V I I I * . T h e n o t h e r G a u s s c u r v e s were p l a c e d in the d i a g r a m to tile left a n d to t h e r i g h t , ,:' " ~:' ", V t a k i n g g r e a t care e a c h t i m e t h a t t h e • : ! i", ",, ~ t/ a r e a o v e r l a p p e d b y t w o Gauss c u r v e s was as n e a r l y e q u a l as p o s s i b l e to t h e a r e a of t h e d i a g r a m left u n c o v e r e d b e t w e e n b o t h curves. The t o p s of t h e Fig. 1. D i a g r a m of e x t r a c t of fresh n o r m a l r a b b i t m u s c l e Gauss c u r v e s need n o t coincide a t all w i t h Gauss c u r v e s fi t t e d in. w i t h t h e p e a k s w h i c h can be d i s t i n g u i s h e d in t h e d i a g r a m . The p a r t of t h e d i a g r a m b e t w e e n p e a k s I I a n d I I I is too fiat to be r e s o l v e d i n t o G a us s curves. The s urfa c e of * T h e c o m p o n e n t s are n u m b e r e d from I I to I X . A v e r y s m a l l c o m p o n e n t I, m o v i n g v e r y rapidly~ w a s neglected.
Re/ere~ces p. 269. i7
260
A . i . V . H . HAhN
VOL. 11 (1953)
this part was measured with a planimeter. The sum of the surfaces of the Gauss curves and the unresolvable intermediate part between II and III should equal the total surface determined by planimeter and at every point of the abscissa the sum of the ordinates of points of the Gauss curves should equal the ordinate of the diagram at that point (see Fig. i). Rabbit muscles The data assembled in Table I show that the preparation of the extracts as well as the numerical evaluation of the diagrams can be carried out in a highly reproducible way. In each of these experiments homologous muscles of the hind legs of a rabbit were compared. These muscles were excised immediately one after another. The differences between homologous muscles were scarcely greater when the muscles were excised with an interval of 21 to 28 days (see Table II). This statement is important in view of later experiments, in which a muscle of one leg had to be excised before the rabbit was subjected to fasting or vitamin-E deficiency in order to provoke atrophy of the homologous muscle of the other leg. Somewhat greater, though always still small, differences were found between various skeletal muscles of one rabbit (Table III), quite distinct differences, however, between the skeletal muscles o5 various rabbits (Table IV). The mobilities at pH 7.15 of the eight components which can be derived from the diagrams were determined in 17 cases. The means and the standard deviations are given in Table V. To determine the iso-electric points of the components portions of an extract were dialyzed against solutions of ionic strength o.13, which contained 0.05 M KC1 and various ratios of primary and secondary phosphate, so that they had the following pH's : 8.00, 7.4 ° , 7.00, 6.60, 6.20, 5.75, 5.30, 4.90* • With the protein solutions thus obtained pH-mobility curves of the various components were determined. The iso-electric points derived from these curves are given by Table VI. Similar determinations have been carried out by JACOBa. His results were: comp. VI : 5.7, comp. V I I : 6.0, comp. v n I : 6.2, comp. I X : 6.8. The iso-electric points of components II, I I I and IV are situated below 4.9. Component I X was split into 3 (ascending boundaries) or 2 (descending boundaries) components at pH values between 6. 4 and 6.0 respectively. Each mobility determination was carried out in triplicate. All determinations were carried out as rapidly as possible one after another without interruption during the night in order to prevent undesired alterations in the protein solutions. Rigor mortis appeared to entail a quite VIII distinct increase of the percentage of component V and possibly a slight increase of the intermediate part of the diagram between components I I and I I I , no matter V > whether the rigor mortis had developed in the course of 8 h at I ° C in the isolated muscle or in the muscle in situ (see Table V I I and Fig. 2). Muscular atrophy was induced by variFig. 2. Diagram of extract of normal rabbit muscle in rigor morris. ous means, viz. severance of a motoric nerve, * We did not prepare protein solutions of lower pH, as it is not possible to compose more acid phosphate buffer solutions, and buffers of other composition would give quite different diagrams (see BoscH1). References p. 269.
VOL. 11 (1953)
ELECTROPHORESIS OF MUSCLE PROTEINS
26I
TABLE I IIOMOLOGOUS MUSCLES
OF THE
ttlND
LEGS
OF
RABBITS
Muscles excised immediately one after another. Area of the Gauss curves I I to I X and of tile intermediate p a r t between I I and I I I expressed in percentage of total area m i n u s 6- (resp. e-) gradient. Rabbi! No.
Muscle
IX
VIII
VII
V1
1,"
I1"
III
lnterm, part
1I
Ascending boundaries 4
V a s t u s lateralis, left V a s t u s lateralis, right
14-7 14.6
3° . ° 29.9
24-3 24.1
3.7 3.7
11.8 11.6
4.8 4.8
2.5 2. 4
2.5 2. 7
5.8 5.8
IO
Gastrocnemius, left Gastrocnemius, right
17.2 17.1
25.2 24.9
24.9 25.0
12.3 12.3
5.6 5.7
4.0 4 .2
3.8 3.9
3.0 3 .0
3.8 3.9
12
Serratus, Serratus,
15.3 15. 7
30.0 30.6
25.3 25. 3
6.1 6. 3
6.7 6.8
3.8 3.8
4.1 4.1
3.4 3.2
4 .1 4.1
left right
Descending boundaries 4
V a s t u s lateralis, left V a s t u s lateralis, right
14. 5 14. 7
28.o 27.1
22. 7 22.8
3.2 3.3
7.4 7.6
4 .1 4.2
2.8 2.9
7.5 7.0
5.7 5,8
IO
Gastroenemius, left Gastrocnemius, right
17.2 17. 7
19.7 20.0
29.3 29.1
lO.3 io.i
7.3 7.3
6.7 6.6
1.8 1.7
5.2 5.5
3.6 3-5
12
Serratus, Serratus,
13. 4 13. 4
31.o 31.1
25.1 25.1
2.9 2.9
7.5 7 .2
4.4 4.4
2.9 2.9
2.5 2.9
6.5 6.5
left right
TABLE II I-IOMOLOGOUS MUSCLES (VASTUS LATERALIS) OF THE H I N D LEGS OF RABBITS, E X C I S E D W I T H AN I N T E R V A L OF 3 A N D 4 W E E K S
Area of the Gauss curves I I to I X and of the intermediate p a r t between I I and I I I expressed in percentage of total area m i n u s 6- (resp. e-) gradient. R a b b i t 3, muscle from right leg excised 21 days after left leg, r a b b i t s 8 and i i, 28 days. Rabbit No.
Leg
IX
VIII
VII
VI
V
1V
III
Interm, part
1I
Ascending boundaries 3
left right
9.6 9.0
28.9 28.5
26.4 27.0
6.0 6.2
IO.O 9-5
4.2 4 .6
4.6 4 .2
3.0 3.2
4.8 5.0
8
left right
21.6 20. 4
24.1 24. 3
27.2 26.8
3.9 3.1
9.7 9.2
2.2 3.o
2.2 2.6
4.2 4.0
4.2 4.0
ii
left right
14.6 14.1
32.1 32.9
22.2 21. 7
6.7 6.o
lO.2 9.7
7.2 8.0
3-7 3.5
1.5 1.7
3 .I 3-4
Descending boundaries 3
left right
8. 3 8.8
30.9 31.2
26. 5 25.8
4.8 4.6
io.o 9-7
5.1 5-5
2.4 2.4
5.3 5-5
4 .0 4 .2
8
left right
11. 7 11. 9
32.5 32.7
25.6 26.2
6.8 7.0
8.3 8.7
2.8 3.1
2.9 3 .1
4.5 4.3
4 .~
left right
13.6 13.2
27 .6 27.0
24.6 25.2
7.3 7.6
6.2 6.5
7.2 6.6
4.0 4.0
5.0 5-3
3.6 3 .8
II
Re]erences p. 269.
4 .(~
262
A.M.F.
tI. HAAN
VOL. 1 1
(1953)
TABLE III VARIOUS
MUSCLES
OF
ONE
RABBIT
A r e a of th e Gau ss c u r v e s I I to I X a n d of t h e i n t e r m e d i a t e p a r t b e t w e e n I I a n d I I I e x p r e s s e d in p e r c e n t a g e of t o t a l a r e a m i n u s ~5- (resp. e-) g r a d i e n t . 31usclc
IX
['1II
I'I1
VI
I |"
V
11I
Dilcrm. parl
11
4.t 3.9 4 .1 3.5
3.2 2.6 3.9 4. t
4.1 4.5 4-2 4.3
2.9 2.9 2.9 2-9
2.9 3.4 2.4 3 .6
6.5 6.5 6-9 6.5
Ascending boundaries Serratus Gastrocnemius Vastus lateralis Vastus iutermedius
I5.7 15. 4 15.6 i5.6
3o.6 3o. I 29.5 29.4
25.3 25.o 25.I 25.0
6.3 5.8 5.5 5.6
6.8 6.9 6.7 6.8
3.8 4.6 5.3 3.9
Descending botmdaries Serratus Gastrocnemius Vastus lateralis Vastus intermedius
13.4 13.2 13.2 12. 5
31.1 31.2 31.4 31. 7
25.1 25.o 28.8 26.1
2-9 2,9 3.0 2, 9
7 .2 7.2 6.9 6. 9
4.4 4.5 4.7 4.5
T A B L E IV MUSCLES
OF VARIOUS
RABBITS
Area of t h e Gauss c u r v e s I I to I X a n d of t h e i n t e r m e d i a t e p a r t b e t w e e n I [ a n d I I I e x p r e s s e d in p e r c e n t a g e of t o t a l a r e a m i n u s 3- (resp. e-) g r a d i e n t . Means a n d s t a n d a r d d e v i a t i o n s . Gauss curve
Number of detcrm,
IX VIII VII VI V IV III Interm. part II
Ascending boundaries
33 33 33 33 33 33 27 27 33
I6.4 33.4 24.2 6.0 8. 3
5 .0 3.3 4.7 4 .0
Descending boundaries
~ 4 .8 4_- 6. 7 !-2.7 _q: 1. 7 ~ 1.6 q- 1.7 + I.O ± 2.2 i 1.3
I5.5 ± 3.5 28. 7 ~ 3.6 23.9:]-3.2 5-7 £ 2.o 7.9 :k 1.7 4 - 7 / - 1.6 2.8 :,~ I.O 4.7 • 1.9 4.5 • t.I
TABLE V MUSCLES
OF VARIOUS
RABBITS
Mobilities of c o m p o n e n t s I I to I X e x p r e s s e d in lO -5 cm"- v o l t -1 sec -1. Means a n d s t a n d a r d d e v i a t i o n s of 17 d e t e r m i n a t i o n s . Component
IX VIII VII VI V IV III iI R,:'.h'r~'nces p. 169.
Descending boundaries 0.8 I. 3 1.7 2.2 2.7 3.3 4.o 6.4
::t- 0.02 +j- 0.05 :k o.o2 -~ 0.03 :L 0-03 ~: 0.05 ± o.o 5 ± o.o6
A scending boundaries 0. 7 1.2 1.6 1. 9 2.6 3.2 3.8 6.2
~: 0.02 _~ 0.03 +_ o.oI ~ o.o 3 ~: 0.02 :[= o.o6 ,- o.o6 ~ o.o6
VOl.. I I
(19.53)
ELECTROPHORESIS OF MUSCLE PROTEINS
2!J~
immobilization of a leg in a plaster cast, prolonged fasting and vitamin-l~2 dcticit~nc\,. ,5everance o/ a lnotoric ~zerve. P a r t of t h e n e r v u s ischiadicus of one leg was excised aml the skin s u t u r e d . T h e operation, w h i c h was t e r m i n a t e d in 5 m i n u t e s , was carried o u t u n d e r N u m a l - R o c h e narcosis. A p a r t from t h e p a r a l y s i s of t h e leg tile a n i m a l s were in p e r f e c t health, l I o m o l o g o n s muscle:~ of tile hind legs, one n o r m a l a n d one atrophied, were excised after 2 to 5 weeks, in one ease after 9 weeks. Immobility in a plaster cast. In order to p r e v e n t d a m a g e b y g n a w i n g it a p p e a r e d to be n e c e s s a r y to place a wooden collar a r o u n d t h e neck of t h e rabbit. A f t e r t h r e e weeks t h e v a s t u s lateralis muscles were r e m o v e d from b o t h hind legs a n d e x a m i n e d . Inanition. T w o r a b b i t s were used for this e x p e r i m e n t . T h e v a s t u s lateralis was r e m o v e d from one leg before t h e f a s t i n g period. (The r e s u l t s in Table II s h o w t h a t no c h a n g e occurs in n o r m a l muscle in t h e course of s o m e weeks.) T h e a n i m a l s died a f t e r f a s t i n g for 6 a n d 9 d a y s respectively. I n this t i m e t h e b o d y weight h a d decreased b y a b o u t 4 ° %. As b o t h a n i m a l s died d u r i n g t h e n i g h t t h e m u s c l e s were in rigor w h e n t h e e x t r a c t s were m a d e . As will a p p e a r f r o m T a b l e V I I I this did n o t affect t h e results. Vitamin-E deficiency. A r a b b i t was fed on tile GOETTSCH-PAPPENHEIMI~:R diet '5. T h e v a s t u s lateralis of one leg h a d been r e m o v e d before a d m i n i s t r a t i o n of t h e v i t a m i n - E - f r e e diet. T h e excretion of creatine a n d creatinine was d e t e r m i n e d e v e r y day. A f t e r a b o u t i8 d a y s t h e creatine excretion increased from a b o u t io m g to 2o to 40 m g p e r 24 h. T h e 29th d a y s h o w e d a s h a r p rise of t h e creatine excretion to a b o u t 26o mg. O n t h e n e x t d a y s it was s o m e w h a t lower again. T h e r a b b i t died on t h e 33rd day.
All atrophied muscles, which have been examined, with the exception of the muscle excized 9 weeks after severance of the nerve, had lost about 5o % of their original weight. No matter which method was used to cause the atrophy, a 5o% decrease in weight was accompanied by a 2- to 3-fold increase of the percentage of component II. This can be seen in Table VIII and Fig. 3. (The increase of the percentage of this component has alIX v i i i H ready been observed by CR~PAX8, though only in the case of denervated muscles). The diagram obtained from the muscle which had atrophied during 9 weeks after Fig. 3. D i a g r a m of e x t r a c t of a t r o p h i e d r a b b i t severance of the nervus ischiadicus is shown muscle ( 2 ½ to 5 weeks a f t e r s e v e r a n c e of n e r v e or i m m o b i l i s a t i o n in p l a s t e r c a s t ; or in Fig. 4. All peaks had disappeared with the after d e a t h b y i n a n i t i o n or v i t a m i n - E deexception of component II, which accounted ficiency). for about 5o % of the total area. As can be seen from Fig. 3 the unresolvable part of the diagram between components II and III also increases considerably, but this increase cannot be expressed numerically, as component I I I - - a n d sometimes component I V - - c a n n o t be distinguished anymore in tile diagrams of atrophied muscles, so that
the flat unresolvable part extends from comI V o r V (see T a b l e V I I I ) .
ponent
II to component
¢--,_
Human
muscles
Fig. 4- D i a g r a m of e x t r a c t of atrop h i e d r a b b i t muscle, 9 weeks after s e v e r a n c e of nerve.
Fifteen normal muscles (m. serratus anterior) were examined. Fig. 5 shows the prototype of the diagrams. It bears a strong resemblance to the diagrams of normal rabbit muscles. The mobilities of the
components very closely approached those of the components of the rabbit muscle diagrams. Therefore the same numbering could be used. Table IX shows the results Re/emnees p. 269.
264
VOL. 11 (I953)
A. M. F. H. HAAN T A B L E VI ISO-ELECTRIC
Ascending boundaries Descending boundaries
POINTS
V
VI
VII
VIII
IX
4.9 4.9
5.o 5 .o
5.3 5.3
5.8 6.o
6.2 6. 4
T A B L E VII COMPARISON
OF F R E S H
MUSCLES
AND
HOMOLOGOUS
MUSCLES
IN
RIGOR
Area of the Gauss curves II to I X and of the intermediate part between II and III expressed in percentage of total area minus d- (resp. e-) gradient. Ascending boundaries. Rigor developed in situ. Each pair of homologous muscles taken from a different rabbit. Muscle
Rigor
IX
VIII
VII
VI
V
IV
111
Intern*. part
11
Serratus
-+
15.7 12.4
3 °.6 25.4
25-3 25.1
6.3 6.3
6.8 14.8
3.8 3.5
4.1 3 .2
3.2 5 .o
4.1 4 .I
Gastrocnemius
-+
15.4 13.6
3o.I 24.5
25.0 24.8
5.8 5.8
6.9 13.2
4.6 4.2
4.0 3.3
2.6 3 .1
4.5 4.5
+
15.6 13.3
29.5 23.3
25.1 24.6
5-5 5.5
6.7 11.7
5.3 4.9
4 .l 3.5
3-9 5 .0
4 .2 4 .2
-+
15.6 13.°
29.4 22.9
25.0 25.2
5.6 5.7
6.9 15.9
3.9 3.7
3 .6 2.8
4 .1 6. 3
4.3 4.3
Vastus lateralis Vastus intermedius
of t h e n u m e r i c a l e v a l u a t i o n of 13 d i a g r a m s . T h e m o s t p r o n o u n c e d d i f f e r e n c e as c o m p a r e d t o r a b b i t m u s c l e is t h e c o m p a r a t i v e l y h i g h p e r c e n t a g e of c o m p o n e n t s I I a n d VII, a n d t h e c o m p a r a t i v e l y l o w p e r c e n t a g e of c o m p o n e n t V I I I . I n m o s t c a s e s c o m ponent IX was absent.
V///
vlff
F i g . 5. D i a g r a m
>
of e x t r a c t muscle.
of n o r m a l
- - >
human
Fig. 6. Diagram of extract of h u m a n muscle atrophied since several decades.
Atrophic muscles were excised from patients suffering from prolonged muscular i n a c t i v i t y , o f t e n e x t e n d i n g o v e r s e v e r a l d e c a d e s . T a b l e X s h o w s t h e r e s u l t s of t h e n u m e r i c a l e v a l u a t i o n of t h e d i a g r a m s o b t a i n e d f r o m 7 p a t i e n t s . A s c o m p a r e d t o n o r m a l m u s c l e s i t a p p e a r s t h a t i n g e n e r a l 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 I I a n d V I I I h a v e i n c r e a s e d a n d 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 V I a n d V I I h a v e d i m i n i s h e d , w h i l e c o m Re#rences p. 269.
AND
VIII
ATROPHIED
TABLE HOMOLOGOUS
by inanition, E =
MUSCLES
atrophy
RABBIT
b y i m m o b i l i z a t i o n in p l a s t e r c a s t , I =
NORMAL
atrophy
OF
atrophy by vitamin-E
Cause of atrophy
2
-SN
-SN
-SN
-SN
-SN
-SN
-SN
-P
-I
-I
-E
Rabbit No.
2
9
13
14
15
16
19
33
20
18
24
22
-33 d a y s
-6 days
-9 days
-3 weeks
-5 weeks
-4 weeks
-4 weeks
-4 weeks
-3 weeks
-21~ w e e k s
-2 weeks
3
Time of development of atrophy
I 12 39
480 162
57 ° 134
55 ° 230
290 12o
338 14o
290 12o
460 19o
4°0 137
75 ° 3o0
31o 154
4
mg total protein extracted from whole muscle
18.6 8. 4
16 9 . 9.6
26.8 12"5
13.o 9.2
12-3 7.7
I 1.7 12.2
9.5 8.2
12. 5 7-7
14.4 12.7
17.4 12.5
18.o 12.8
5
IX
3o.1 18.2
23.4 18.o
21.2 12.5
29.6 18. 4
24.8 lO.8
27-4 17.o
25.1 17.3
26.8 27-2
27-4 17.1
29.8 19.8
30.0 19.2
6
VIII
26.6 16. 5
29.8 14. 5
21.o 17.6
24. 4 17.6
20.9 9.9
25.o 15. 3
2o.2 12.2
25.2 18.o
30.4 15.1
21. 4 19.2
21.2 18. 9
7
VII
5.4 5.7
6.3 5.8
4.5 7 .6
7.9 7. I
9.1 8.1
6.8 5-7
8. 7 4.8
7.1 6. 4
5.6 5.7
7.2 5.7
6.7 5.9
8
V[
6.3 6.2
lO.7 18.2
8.2 11.4
7-8 6.8
7.9 5.7
7.3 7-0
7.7 7.8
6.9 12.o
6.1 7.0
9.7 7-9
9.5 9.5
9
V
6.3
4.1
5.2
6.0 5.6
6"4 5.1
6.2 4.9
6.0
4.0 5.0
6.6 6.2
6.8 7.5
zo
IV
4 .2
Percentage of total area
4.0
4.6
2.4
3.2
8.1
4 .0
4.2
4.5
2.2
2.2
II
IlI
32.8
14"8
26.8
3 o. I
24.2
20.2
14.2 25.6
I4.2
20.0
19.6
15.8
5.0
8.2
5.0
5.6
5.6
4.1
4.5
9.5
3.6
3.0
12
Interm. part
4. i Ii.i
6. 7 23.3
6.1 26.o
4.2 i o.o
5-3 13.6
6.8 17.i
5.2 13.2
6.4 15.2
4.0 i2.r
4.7 12. 5
4-9 lO. 4
13
II
4.6 4.3
32 37
35 35
23 23
15 16
23 24
15 16
29 29
16 17
3,5 36
15 16
24
mg comp. I I extracted from zchole muscle
I f c o m p o n e n t I I I or c o m p o n e n t s I I I a n d I V c o u l d n o t b e d i s t i n g u i s h e d in d i a g r a m s of a t r o p h i e d m u s c l e s , o n e f i g u r e is g i v e n f o r t h e u n r e s o l v a b l e p a r t b e t w e e n c o m p o n e n t s I I a n d I V o r I I a n d V. Ascending boundaries.
S N = a t r o p h y b y s e v e r a n c e of n e r v e , P = deficiency.
COMPARISON
t.dt
t~
©
N
©
0
0
t~ c~ H
~'~
~-~ ~-"
< © t"
20(;
A.M.F.H.
HAAN
TABLE NORMAL
VOL. 1 1
(1953)
IX
HUMAN
MUSCLES
A r e a of G a u s s c u r v e s I I t o V I I I a n d of i n t e r n l e d i a t e p a r t b e t w e e n I I a n d I I I e x p r e s s e d in p e r c e n t a g e of t o t a l a r e a n l i n u s 6- (resp. e-) g r a d i e n t . M e a n s a n d s t a n d a r d d e v i a t i o n s . Ascending b~mndaries Ga4¢:,x curve
Number of determination,s
VIII VII VI V IV llI Interm. part ii
Descending boundaries
Percentage
13 13 13 13 IO 4 4 13
27. 3 33.7 7.7 7.7 5'5 4.1 4.3 6.6
~_ 6.8 ±6.4 A_ 2.1 ~ 1.9 ~ I'5 ~: 1.6 b 1. 3 :~ 1.2
TABLE ATROPHIED
A r e a of G a u s s c u r v e s I I t o V I I I
Age of atrophy in ycars
Fatty degcnerations
VIII
Number of determinations
t'ercentage
I2 12 12 12 I2 4 ,t I2
26.0 36.2 7.3 6. 4 0. 7 5"3 5.3 7.2
4:5.7 ~6.o ~[ 2.0 ~_ 2.0 4 1. 4 ~ 1"3 ± 2.3 :}- 1.4
X
HUMAN
MUSCLES
a n d of i n t e r m e d i a t e p a r t e x p r e s s e d in p e r c e n t a g e of t o t a l m i n u s d- (resp. e-) g r a d i e n t . vI
VII
v
Iv
area
lll
lnterm. part
II
o o o o o o o
I7. 4 15.o 14. 4 i2.1 o o o
9.0 [ t .7 II. 3 it. 5 16.9 18.6 18.o
o o o O o o o
18.8 I3.[ 26.0 9.1 o o o
11.6 I2.8 8. 3 1I. 4 13.2 14.2 18.2
Ascending boundaries 2 3 4 1o 20 23 46
---q + + +
44.0 48.8 41.3 56.4 55.2 50.6 49.2
28.8
3.2
o o o o o o o
24. 3 28.8
4.3 20.0 27.2 30.6 32.8
o o o o o o o
Descending boundaries 2 3 4 lo 20 23 40
---@ + + +
40.0 46.2 36.2 52.2 51.8 52.4 50.2
24.4
5.2
29.4
9.0 26.6 35.8 33.2 31.6
TABLE AMOUNTS
o o o o o o o
28.8
OF
PROTEIN
o o o O o o o
XI
EXTRACTED
PER
G
MUSCLE
Easily soluble proteins Muscles
Normal rabbit muscles Atrophic rabbit muscles R a b b i t m u s c l e s in r i g o r m o r r i s Normal human muscles
Myosin
Number off determinations
mg per g
Number of determinations
44 12 12 12
24 ± 3.5 19±3.1 24 ~ 4.2 24 q- 4.1
16 8 Ii 12
mg per g
16 4 3 17
i 2.5 }2 2.6 ± 1.5 ~ 3.2
voL. 11 (1953)
ELECTROPHORESIS OF MUSCLE PROTEINS
2(~7
ponents I I I , IV and V have completely disappeared, in those cases in which thc~ atrophy had only existed for a few years the unresolvable intermediate part was present in the diagrams. If the atrophy had been manifest for m a n y years (~:.g. 4 o years) this intermediate part had completely disappeared. Fig. 6 shows the prototype of the diagrams of the latter muscles.
Myosin, 6xtracted /rom muscle by salt solution o~ pH 7.z5 and ionic strength o.I3 As described above, a precipitate forms upon dialysis of the muscle extracts against the KCl-phosphate solution of p H 7.15 and ionic strength o.13. This precipitate was spun down, repeatedly washed with the same solution and then dissolved in GreensteinEdsall solution (0.5 M KC1, 0.03 M NaHCO~; p H 8.1o; ionic strength 0.53 ). After dialysis against this solution the electrophoresis diagram obtained appeared to be practically identical with that oi a myosin solution prepared irom rabbit muscles according to STEYN-PARVt~ AND GERRITSEN7. Hence it appears that myosin m a y 1)e extracted from the muscle by a practically neutral solution of low ionic strength.
Amounts o/proteins extracted/rom normal and atrophied muscles Table XI shows the means of the amounts of the easily soluble proteins and of myosin, extracted per g muscle by KCl-phosphate solution of p H 7.15 and ionic strength o.13. Regarding the easily soluble proteins no difference exists between fresh normal rabbit muscles, fresh normal human muscles and rabbit muscles in rigor, but the amount extracted per g of atrophic rabbit muscles is significantly lower (P < o.ooi). The amount of myosin extracted per g tissue from fresh normal rabbit muscles and from fresh normal human muscles did not differ, the amounts extracted per g tissue from the atrophied rabbit muscles and from normal rabbit muscles in rigor were very much lower, however. Notwithstanding the fact that the total amount of easily extracted protein per g of atrophic rabbit muscle was lower than the amount extracted per g of normal muscle, and that the weight of the atrophic muscles was much less than the weight of the normal homologous muscles, the amount of the protein(s) giving rise to component I I of the diagram, extracted from the whole atrophied muscle, had not diminished as compared with the amount of this protein extracted from the whole homologous normal muscle. This can be seen from column 14 of Table V I I I , the figures of which have been calculated from the total amounts of easily soluble protein per whole muscle (column 4) and the percentage of component I I (column 13). Though the diagrams of extracts of atrophied muscles had also shown an increase of the flat unresolvable part between peaks I I and III, observed in the case of normal muscles, a similar calculation could not be carried out here, for this part of the diagram now extended from component I I to component IV or from component I I to component V. DISCUSSION I t should be realized that the high reproducibility of the numerical evaluation of the diagrams could only be attained if it was carried out by one and the same person, always following the same procedure. We have placed Gauss curves in the diagrams, starting with the rather arbitrarily chosen peak No. V I I I (the highest one in the case of extracts of rabbit muscle) and then trying to fill up the whole diagram with the
Re/erences p. 269.
268
A.M.F.H.
HAAN
VOL. 1 1
(1953)
smallest possible number of Gauss curves. The figures obtained for the various Gauss areas as percentages of the total area minus ~- (resp. e-) gradient may obviously only be regarded as characteristics of the muscle extracts serving for mutual comparison of these extracts. It will be necessary to employ other methods, e.g. salting out according to DERRIEN and enzyme determinations, in order to obtain a fuller understanding of what happens to the proteins in the course of the development of atrophy. The most remarkable fact established in this work seems to be the practically identical changes that are observed irrespective of whether the atrophy is caused by immobilization (as a consequence of severance of the motoric nerve or of application of a plaster cast), by inanition or by vitamin-E deficiency. In all cases the proteins that give rise to component II (and probably also those composing the flat part of the diagram between components II and III) are maintained, while the other proteins disappear in the course of the development of the atrophy. Special attention should furthermore be given to the difference between the diagrams of rabbit muscles, rendered atrophic by experiment and human atrophied muscles. C-ERRITSENs, working in this laboratory on the glucokinase content of normal and atrophied muscles, also found a difference between these two kinds of atrophy. The enzyme appeared to have been fully maintained in the atrophied rabbit muscles, but to have decreased considerably in the atrophied human muscles. In both cases we believe that the differences between rabbit and human muscles are caused by the different times during which the atrophies have existed. The intermediate unresolvable part of the electrophoresis diagrams of extracts of atrophied rabbit muscles and the glucokinase of these muscles would probably also have disappeared if it would have been possible to incite a very slowly developing muscular atrophy in rabbits.
SUMMARY T h e p r o t e i n s of n o r m a l a n d a t r o p h i e d skeletal m u s c l e s of t h e r a b b i t a n d of m a n , soluble in salt solutions of low ionic s t r e n g t h a n d p H a b o u t 7, were s t u d i e d b y m e a n s of electrophoresis. A t r o p h y of r a b b i t leg m u s c l e s w a s c a u s e d b y s e v e r a n c e of t h e m o t o r i c nerve, b y i m m o b i l i s a t i o n of a leg in a p l a s t e r cast, b y i n a n i t i o n a n d b y v i t a m i n - E deficiency. No m a t t e r w h i c h of t h e s e m e t h o d s was u s e d to i n d u c e m u s c u l a r a t r o p h y , t h e electrophoresis d i a g r a m s o b t a i n e d a l w a y s s h o w e d t h e s a m e a l t e r a t i o n s as c o m p a r e d w i t h t h e d i a g r a m s o b t a i n e d for n o r m a l muscles. T h e m o s t r e m a r k a b l e f e a t u r e w a s t h e increase of t h e p e r c e n t a g e s of t h e m o r e r a p i d l y m o v i n g c o m p o n e n t s a n d t h e decrease of t h e p e r c e n t a g e s of t h e slower c o m p o n e n t s . W h i l e t h e t o t a l a m o u n t of protein, e x t r a c t a b l e b y a salt solution of ionic s t r e n g t h o.13 a n d p H 7.15 f r o m t h e whole muscle, h a d c o n s i d e r a b l y decreased, t h e a m o u n t of t h e m o s t r a p i d l y m i g r a t i n g c o m p o n e n t (II), d i s t i n g u i s h a b l e in t h e d i a g r a m s , h a d remained constant. N o r m a l h u m a n m u s c l e s differed little f r o m n o r m a l r a b b i t muscles. T h e flat i n t e r m e d i a t e p a r t of t h e d i a g r a m s , w h i c h h a d b e e n m a i n t a i n e d in t h e case of t h e a t r o p h i e d r a b b i t muscles, h a d , however, c o m p l e t e l y d i s a p p e a r e d in t h e case of chronic h u m a n m u s c u l a r a t r o p h y caused b y i m m o b i l i z a t i o n , e x i s t i n g d u r i n g several decades. T h e t i m e of e x i s t e n c e of t h e a t r o p h y m a y be t h e c a u s e of this difference, as t h e a t r o p h y of t h e r a b b i t m u s c l e s h a d developed in t h e course of a few weeks. RI~SUMt~ L e s p r o t 6 i n e s des m u s c l e s s q u e l e t t i q u e s n o r m a u x et a t r o p h i e s de l ' h o m m e et du lapin, solubles d a n s des s o l u t i o n s salines de force ionique faible et de p H e n v i r o n 7, o n t dt6 dtudi6es p a r 61cctrophor6se. L ' a t r o p h i e des m u s c l e s de la p a t t e du lapin est p r o v o q u d e p a r ldsion du nerf m o t e u r , par i m m o b i l i s a t i o n de la p a t t e d a n s u n m o u l e de pl~tre, p a r i n a n i t i o n ou p a r carence en v i t a m i n e E. Quelle q u e soit la m 6 t h o d e utilis6e p o u r p r o v o q u e r l ' a t r o p h i e m u s c u l a i r e , les d i a g r a m m e s d'6lectrophor6se o b t e n u s p r d s e n t e n t t o u j o u r s les m ~ m e s modifications p a r r a p p o r t a u x d i a g r a m m e s o b t e n u s a v e c des m u s c l e s n o r m a u x . L a p l u s r e m a r q u a b l e de ces modifications est l ' a u g m e n t a t i o n des pour-
Re/erences p. 269.
voL. 11 (I953)
ELECTROPHORESIS OF MUSCLE PROTEINS
2~)9
centages des c o n s t i t u a n t s so ddpla~ant le plus rapidement, et la diminution des pourcentages des constituants los plus lents. Alors que le contenu total en prot6ines extractibles par une solution saline de forco ioniquo o.13 et de p H 7.15 i~ partir du muscle entier, diminue considdrablement, la teneur du consfituant qui poss~de la plus grande vitesse de migration (II), qu'on p e u t distinguer sur le diagramme, reste constante. Les muscles h u m a i n s n o r m a u x diffSren~c peu des muscles de lapins n o r m a u x . La partie intermddiaire plate du diagramme, qui est Inaintenue dans le cas des muscles de lapin atrophi6s, disparait cependant compl~tement dans le cas d ' u n e atrophie musculaire hunlaine chroniqne provoqude par une immobilisation de plusieurs dizaines d'anndes. Cette diffdrence est peut-6tre due /z la dur6e de l'atrophie, car l'atrophie des muscles du lapin est obtenue en quelqnes semaines.
ZUSAMMENFASSUNG Die Proteine normaler und atrophierter Skelettmuskeln von K a n i n c h e n und Menschen, die in Salzl6sungen m i t niedriger Ionenst~rke und einem p H von ungefS.hr 7 16slich sind, wurden m i t Hilfe der Elektrophorese untersucht. Die Atrophie der Beinmuskeln yon K a n i n c h e n wurde hervorgerufen durch T r e n n u n g des motorischen Nervs, durch Immobilisation des Beines im Gipsverband, durch Entkr~tftigung u n d durch Vitamin E-Mangel. Verglich m a n die erhaltenen elektrophoretischen D i a g r a m m e mit denen normaler Muskeln, so zeigten sie immer die gleiche Ver~nderung, ganz gleichgiiltig welche dieser Methoden zur Herbeifiihrung der Muskelatrophie b e n u t z t wurde. Das Ansteigen des Prozentgehalts der schneller beweglichen K o m p o n e n t e n und das Absinken des Prozentgehalts langsamer K o m p o n e n t e n war das meist bemerkenswerte Merkmal. WAhrend der Gesamtanteil des mit Salzl6sung der Ionenst~rke o.13 und p H 7.15 aus dem ganzen Muskel extrahierbaren Proteins betr~tchtlich a b n a h m , blieb der Anteil der a m schnellsten wandernden K o m p o n e n t e (II) welche m a n in den D i a g r a m m e n unterscheidet, k o n s t a n t . Normale nlenschliche Muskeln untersehieden sich wenig yon normalen K a n i n c h e n m u s k e l n . Der ttache Zwischenteil der Diagramme, der im Fall des atrophierten K a n i n c h e n m u s k e l s erhalten blieb, verschwand jedoch vollst~ndig im Fall der fiber mehrere J a h r z e h n t e bestehenden, durch Immobilisation verursachten menschlichen chronischen Muskelatrophie. Die Ursache dieses Unterschiedes k6nnte in der Dauer des Bestehens der Atrophie liegen, da sich die Atrophie des K a n i n c h e n m u s k e l s im Laufe einiger Wochen entwickelte. REFERENCES M. W. BOSCH, Biochim. Biophys. Acta, 7 (1951) 61; Thesis, U t r e c h t 1951. 2 M. DUBOISSON, A. DIST~CI4E AND A. DEBOT, Biochim. Biophys. Acta, 6 (I951/52) 97. 3 E. WIEDEMANN,Helv. Chim. Acta, 3 ° (1947) 892. 4 j. JACOB, Biochem. J., 42 (1948) 71. 5 M. GOETTSCH AND A. M. PAPPENHEIMER, J. Exp. Med., 54 (1931) 145. P. CR£PAX, Experientia, 5 (1949) 167. E. P. STEYN-PARV£ AND T. GERRITSEN, Biochim. Biophys. Acta, 8 (1952) lO 4. 8 T. GERRITSEN, Biochim. Biophys. Acta, 8 (1952) 466. Received January
I o t h , 1953
BIOCHIMICA ET BIOPHYSICA ACTA
VOL. 11
(1953)
E L E C T R O P H O R E S I S OF T H E NON-STRUCTURAL P R O T E I N S FROM NORMAL AND A T R O P H I C MUSCLES OF T H E R A B B I T AND OF MAN* by A. M. F. H. H A A N
Laboratory [or Physiological Chemistry, The University, Utrecht (Netherla~ds)
INTRODUCTION
The experiments described in this paper aimed at ascertaining the influence of atrophy on the muscle proteins (globulin X, myogen, myoalbumin, myoglobin), soluble in salt solutions of low ionic strength and pH about 7, in which myosin and actomyosin are said to be insoluble. The proteins studied will further be called "easily soluble proteins" of muscle. They were studied by quantitative eleetrophoresis. According to the results of experiments, carried out in this laboratory by Bosc~f 1, the easily soluble proteins can be extracted completely by grinding the muscle with sand and a buffer solution of pH 7.15 and ionic strength o.13. This was confirmed in the course of the work presented here. The numerical evaluation of the rather complicated diagrams by fitting in Gauss curves did not give fully satisfactory results in the hands of BOSCH. He believes that the differences between results calculated from various diagrams that appear to be identical upon superimposed projection must be attributed to the difficulty in drawing a pencil line exactly through the middle of the projected curves obtained by the Svensson-Philpot technique and to the arbitrariness inherent in the choice of the Gauss curves. Therefore he advises always to make the electrophoresis diagrams under exactly identical conditions and to decide upon visual inspection of superimposed projections whether differences exist or not. This procedure has the great disadvantage that one is not able to express these differences quantitatively. We believe, however, that we have demonstrated that a quantitative analysis of the diagrams of extracts of skeletal muscles is possible. Our procedure will be described in detail below. Experiments were carried out with rabbit muscles and with human muscles. EXPERIMENTAL PART
~1ethods I n general the procedure of lBoscI~ 1 was followed. Therefore the same buffer solution was e m p l o y e d for extraction, dialysis and electrophoresis. BOSCH has tried several solutions. We have * This w o r k forms p a r t of the investigations on the b i o c h e m i s t r y of muscle diseases b y H. G. K. \VESTENBRINK a n d co-workers, s u p p o r t e d b y a g r a n t from the N e t h e r l a n d s Organisation for Pure Research (Z.W.O.).
Re/erences p. 269.
VOL.
11 ( 1 9 5 3 )
ELECTROPHORESIS OF MUS(:LE PROTEINS
25~ )
used a s o l u t i o n he r e c o m m e n d s , c o n t a i n i n g 0.05 M KC1, 0.023 h i N a 2 H P O 4 a n d O.Ol M t(H21'Oa, of p H 7.15 and ionic s t r e n g t h o.13, w h i c h gives tile m o s t d e t a i l e d d i a g r a m s of all s o l u t i o n s t e s t e d . Some m i n o r a l t e r a t i o n s d e s c r i b e d below were i n t r o d u c e d .
Extractiou o/the proteins T h e r a b b i t s were a n a e s t h e t i z e d w i t h N u m a l - R o c h e ( d i e t h y l a m m o n i u m a l l y i i s o p r o p y l b a r b i t u r a t e ) a n d bled b y t h e aorta. A f t e r excision t h e m u s c l e was s t o r e d for i h o u r a t l ~' C. I m m e d i a t e l y a f t e r g r i n d i n g a g mu scle w i t h a g s a n d a n d a ml e x t r a c t i o n fluid t h e nauscle d e b r i s were s p u n d o w n a t 1 ° C b y IO m i n u t e s ' c e n t r i f u g i n g a t I4,ooo g ( c e n t r i f u g i n g m a y also be clone a f t e r i or 2 h). C ( m t r a r y to 13OSCH'S e x p e r i e n c e viscous e x t r a c t s were n e v e r o b t a i n e d . No e x p l a n a t i o n of thi~ difference c a n be offered. I n t h e e x p e r i m e n t s on muscles, a t r o p h i e d as a c o n s e q u e n c e of i n a n i t i o n or v i t a m i n - E - f r e e feeding, it w a s n e c e s s a r y to excise t h e m u s c l e of one leg before s u b j e c t i n g t h e r a b b i t t o f a s t i n g or a v i t a m i n o s i s . Therefore t h i s m u s c l e c e r t a i n l y c o n t a i n e d more blood t h a n a m u s c l e e xc i s e d a f t e r t h e a n i m a l h a d been bled. As t h e e x p e r i m e n t s , referred to in T a b l e II, show, t h i s difference of blood c o n t e n t did not affect t h e results. The h u m a n m u s c l e s were also stored, as soon as possi bl e a f t e r t h e o p e r a t i o n , for i hour a t i ~' C before e x t r a c t s were made. N o r m a l h u m a n m u s c l e s were e x c i s e d d u r i n g l u n g o p e r a t i o n s .
Dialysis The d i a l y s i s was c a r r i e d o u t a t 5 ° C for a t l e a s t 12 h, u s u a l l y 18 t o 24 h. The p r e c i p i t a t e f o r m e d d u r i n g d i a l y s i s w a s s p u n d o w n b y c e n t r i f u g i n g a t o ° C a t 14,ooo g. F or r a b b i t m u s c l e e x t r a c t s 3 ° m i n u t e s ' c e n t r i f u g i n g was sufficient; in t h e case of h u n l a n m u s c l e e x t r a c t s t h e c e n t r i f u g i n g h a d t o be c o n t i n u e d for a t l e a s t 5 ° m i n u t e s in order to p r e v e n t fl oc c ul a t i on of s ome p r o t e i n (myosin) in t h e e l e c t r o p h o r e s i s cell.
Determination o[ protein content o/the extracts This was done b y m e a n s of an A b b e r e f r a c t o m e t e r . I n t h e case of n o r m a l m u s c l e s t h e p r o t e i n c o n t e n t was a l i t t l e h i g h e r t h a n 3 %. I n p r e l i m i n a r y e x p e r i m e n t s t h e r e s u l t s of t h e r e f r a c t o m e t r i c d e t e r m i n a t i o n s were c o m p a r e d w i t h t h e r e s u l t s of K j e l d a h l d e t e r m i n a t i o n s . The a g r e e m e n t w a s excellent.
Electrophoresis The d i a l y z e d p r o t e i n s o l u t i o n w a s d i l u t e d w i t h t h e buffer s o l u t i o n to a b o u t 2 %. E l e c t r o p h o r e s i s was carried o u t in a T i s e l i u s a p p a r a t u s (Striibin, Basle) a c c o r d i n g t o t h e S v e n s s o n - P h i l p o t m e t h o d . Ilford P a n F film, 35 ram, 25 ° S, w a s used. No difficulties w e re e n c o u n t e r e d w i t h t h e e x t r a c t s of w h i t e mu scles of r a b b i t s . I n t h e case of e x t r a c t s of red m u s c l e s of b o t h h u m a n a n d r a b b i t , i t a p p e a r e d to be n e c e s s a r y to c o v e r t h e u n c o l o u r e d p o r t i o n of t h e a s c e n d i n g b o u n d a r y in t i l e e l e c t r o p h o r e s i s cell d u r i n g p a r t of t h e r a t h e r long e x p o s u r e time, a n d to p r e v e n t t h e e n t r a n c e of s t r a y l i g h t from t h e r o o m i n t o the o p t i c a l system• Some m y o s i n s o l u t i o n s were e x a m i n e d , As t h e y were v e r y o p a l e s c e n t v e r y l ong e x p o s u r e t i m e s were req uired . D i a g r a m s of r e a s o n a b l e q u a l i t y were o b t a i n e d . Y e t we b e l i e v e t h a t cells as ns e d b y DUBUISSON et al. 2 are to be p r e f e r r e d to our cells of t h e c o m m o n t y p e for t h e i n v e s t i g a t i o n of m y o s i n solutions.
Numerical evaluation o/the diagrams
VIII
Th e g e n e r a l t y p e of d i a g r a m to be a n a l y z e d is s h o w n in Fig. i. I n p r i n c i p l e t h e a n a l y s i s was c a r r i e d o u t a c c o r d i n g to WIEDEMANN 3. A Gauss c u r v e was p l a c e d in p e a k V I I I * . T h e n o t h e r G a u s s c u r v e s were p l a c e d in the d i a g r a m to tile left a n d to t h e r i g h t , ,:' " ~:' ", V t a k i n g g r e a t care e a c h t i m e t h a t t h e • : ! i", ",, ~ t/ a r e a o v e r l a p p e d b y t w o Gauss c u r v e s was as n e a r l y e q u a l as p o s s i b l e to t h e a r e a of t h e d i a g r a m left u n c o v e r e d b e t w e e n b o t h curves. The t o p s of t h e Fig. 1. D i a g r a m of e x t r a c t of fresh n o r m a l r a b b i t m u s c l e Gauss c u r v e s need n o t coincide a t all w i t h Gauss c u r v e s fi t t e d in. w i t h t h e p e a k s w h i c h can be d i s t i n g u i s h e d in t h e d i a g r a m . The p a r t of t h e d i a g r a m b e t w e e n p e a k s I I a n d I I I is too fiat to be r e s o l v e d i n t o G a us s curves. The s urfa c e of * T h e c o m p o n e n t s are n u m b e r e d from I I to I X . A v e r y s m a l l c o m p o n e n t I, m o v i n g v e r y rapidly~ w a s neglected.
Re/ere~ces p. 269. i7
260
A . i . V . H . HAhN
VOL. 11 (1953)
this part was measured with a planimeter. The sum of the surfaces of the Gauss curves and the unresolvable intermediate part between II and III should equal the total surface determined by planimeter and at every point of the abscissa the sum of the ordinates of points of the Gauss curves should equal the ordinate of the diagram at that point (see Fig. i). Rabbit muscles The data assembled in Table I show that the preparation of the extracts as well as the numerical evaluation of the diagrams can be carried out in a highly reproducible way. In each of these experiments homologous muscles of the hind legs of a rabbit were compared. These muscles were excised immediately one after another. The differences between homologous muscles were scarcely greater when the muscles were excised with an interval of 21 to 28 days (see Table II). This statement is important in view of later experiments, in which a muscle of one leg had to be excised before the rabbit was subjected to fasting or vitamin-E deficiency in order to provoke atrophy of the homologous muscle of the other leg. Somewhat greater, though always still small, differences were found between various skeletal muscles of one rabbit (Table III), quite distinct differences, however, between the skeletal muscles o5 various rabbits (Table IV). The mobilities at pH 7.15 of the eight components which can be derived from the diagrams were determined in 17 cases. The means and the standard deviations are given in Table V. To determine the iso-electric points of the components portions of an extract were dialyzed against solutions of ionic strength o.13, which contained 0.05 M KC1 and various ratios of primary and secondary phosphate, so that they had the following pH's : 8.00, 7.4 ° , 7.00, 6.60, 6.20, 5.75, 5.30, 4.90* • With the protein solutions thus obtained pH-mobility curves of the various components were determined. The iso-electric points derived from these curves are given by Table VI. Similar determinations have been carried out by JACOBa. His results were: comp. VI : 5.7, comp. V I I : 6.0, comp. v n I : 6.2, comp. I X : 6.8. The iso-electric points of components II, I I I and IV are situated below 4.9. Component I X was split into 3 (ascending boundaries) or 2 (descending boundaries) components at pH values between 6. 4 and 6.0 respectively. Each mobility determination was carried out in triplicate. All determinations were carried out as rapidly as possible one after another without interruption during the night in order to prevent undesired alterations in the protein solutions. Rigor mortis appeared to entail a quite VIII distinct increase of the percentage of component V and possibly a slight increase of the intermediate part of the diagram between components I I and I I I , no matter V > whether the rigor mortis had developed in the course of 8 h at I ° C in the isolated muscle or in the muscle in situ (see Table V I I and Fig. 2). Muscular atrophy was induced by variFig. 2. Diagram of extract of normal rabbit muscle in rigor morris. ous means, viz. severance of a motoric nerve, * We did not prepare protein solutions of lower pH, as it is not possible to compose more acid phosphate buffer solutions, and buffers of other composition would give quite different diagrams (see BoscH1). References p. 269.
VOL. 11 (1953)
ELECTROPHORESIS OF MUSCLE PROTEINS
26I
TABLE I IIOMOLOGOUS MUSCLES
OF THE
ttlND
LEGS
OF
RABBITS
Muscles excised immediately one after another. Area of the Gauss curves I I to I X and of tile intermediate p a r t between I I and I I I expressed in percentage of total area m i n u s 6- (resp. e-) gradient. Rabbi! No.
Muscle
IX
VIII
VII
V1
1,"
I1"
III
lnterm, part
1I
Ascending boundaries 4
V a s t u s lateralis, left V a s t u s lateralis, right
14-7 14.6
3° . ° 29.9
24-3 24.1
3.7 3.7
11.8 11.6
4.8 4.8
2.5 2. 4
2.5 2. 7
5.8 5.8
IO
Gastrocnemius, left Gastrocnemius, right
17.2 17.1
25.2 24.9
24.9 25.0
12.3 12.3
5.6 5.7
4.0 4 .2
3.8 3.9
3.0 3 .0
3.8 3.9
12
Serratus, Serratus,
15.3 15. 7
30.0 30.6
25.3 25. 3
6.1 6. 3
6.7 6.8
3.8 3.8
4.1 4.1
3.4 3.2
4 .1 4.1
left right
Descending boundaries 4
V a s t u s lateralis, left V a s t u s lateralis, right
14. 5 14. 7
28.o 27.1
22. 7 22.8
3.2 3.3
7.4 7.6
4 .1 4.2
2.8 2.9
7.5 7.0
5.7 5,8
IO
Gastroenemius, left Gastrocnemius, right
17.2 17. 7
19.7 20.0
29.3 29.1
lO.3 io.i
7.3 7.3
6.7 6.6
1.8 1.7
5.2 5.5
3.6 3-5
12
Serratus, Serratus,
13. 4 13. 4
31.o 31.1
25.1 25.1
2.9 2.9
7.5 7 .2
4.4 4.4
2.9 2.9
2.5 2.9
6.5 6.5
left right
TABLE II I-IOMOLOGOUS MUSCLES (VASTUS LATERALIS) OF THE H I N D LEGS OF RABBITS, E X C I S E D W I T H AN I N T E R V A L OF 3 A N D 4 W E E K S
Area of the Gauss curves I I to I X and of the intermediate p a r t between I I and I I I expressed in percentage of total area m i n u s 6- (resp. e-) gradient. R a b b i t 3, muscle from right leg excised 21 days after left leg, r a b b i t s 8 and i i, 28 days. Rabbit No.
Leg
IX
VIII
VII
VI
V
1V
III
Interm, part
1I
Ascending boundaries 3
left right
9.6 9.0
28.9 28.5
26.4 27.0
6.0 6.2
IO.O 9-5
4.2 4 .6
4.6 4 .2
3.0 3.2
4.8 5.0
8
left right
21.6 20. 4
24.1 24. 3
27.2 26.8
3.9 3.1
9.7 9.2
2.2 3.o
2.2 2.6
4.2 4.0
4.2 4.0
ii
left right
14.6 14.1
32.1 32.9
22.2 21. 7
6.7 6.o
lO.2 9.7
7.2 8.0
3-7 3.5
1.5 1.7
3 .I 3-4
Descending boundaries 3
left right
8. 3 8.8
30.9 31.2
26. 5 25.8
4.8 4.6
io.o 9-7
5.1 5-5
2.4 2.4
5.3 5-5
4 .0 4 .2
8
left right
11. 7 11. 9
32.5 32.7
25.6 26.2
6.8 7.0
8.3 8.7
2.8 3.1
2.9 3 .1
4.5 4.3
4 .~
left right
13.6 13.2
27 .6 27.0
24.6 25.2
7.3 7.6
6.2 6.5
7.2 6.6
4.0 4.0
5.0 5-3
3.6 3 .8
II
Re]erences p. 269.
4 .(~
262
A.M.F.
tI. HAAN
VOL. 1 1
(1953)
TABLE III VARIOUS
MUSCLES
OF
ONE
RABBIT
A r e a of th e Gau ss c u r v e s I I to I X a n d of t h e i n t e r m e d i a t e p a r t b e t w e e n I I a n d I I I e x p r e s s e d in p e r c e n t a g e of t o t a l a r e a m i n u s ~5- (resp. e-) g r a d i e n t . 31usclc
IX
['1II
I'I1
VI
I |"
V
11I
Dilcrm. parl
11
4.t 3.9 4 .1 3.5
3.2 2.6 3.9 4. t
4.1 4.5 4-2 4.3
2.9 2.9 2.9 2-9
2.9 3.4 2.4 3 .6
6.5 6.5 6-9 6.5
Ascending boundaries Serratus Gastrocnemius Vastus lateralis Vastus iutermedius
I5.7 15. 4 15.6 i5.6
3o.6 3o. I 29.5 29.4
25.3 25.o 25.I 25.0
6.3 5.8 5.5 5.6
6.8 6.9 6.7 6.8
3.8 4.6 5.3 3.9
Descending botmdaries Serratus Gastrocnemius Vastus lateralis Vastus intermedius
13.4 13.2 13.2 12. 5
31.1 31.2 31.4 31. 7
25.1 25.o 28.8 26.1
2-9 2,9 3.0 2, 9
7 .2 7.2 6.9 6. 9
4.4 4.5 4.7 4.5
T A B L E IV MUSCLES
OF VARIOUS
RABBITS
Area of t h e Gauss c u r v e s I I to I X a n d of t h e i n t e r m e d i a t e p a r t b e t w e e n I [ a n d I I I e x p r e s s e d in p e r c e n t a g e of t o t a l a r e a m i n u s 3- (resp. e-) g r a d i e n t . Means a n d s t a n d a r d d e v i a t i o n s . Gauss curve
Number of detcrm,
IX VIII VII VI V IV III Interm. part II
Ascending boundaries
33 33 33 33 33 33 27 27 33
I6.4 33.4 24.2 6.0 8. 3
5 .0 3.3 4.7 4 .0
Descending boundaries
~ 4 .8 4_- 6. 7 !-2.7 _q: 1. 7 ~ 1.6 q- 1.7 + I.O ± 2.2 i 1.3
I5.5 ± 3.5 28. 7 ~ 3.6 23.9:]-3.2 5-7 £ 2.o 7.9 :k 1.7 4 - 7 / - 1.6 2.8 :,~ I.O 4.7 • 1.9 4.5 • t.I
TABLE V MUSCLES
OF VARIOUS
RABBITS
Mobilities of c o m p o n e n t s I I to I X e x p r e s s e d in lO -5 cm"- v o l t -1 sec -1. Means a n d s t a n d a r d d e v i a t i o n s of 17 d e t e r m i n a t i o n s . Component
IX VIII VII VI V IV III iI R,:'.h'r~'nces p. 169.
Descending boundaries 0.8 I. 3 1.7 2.2 2.7 3.3 4.o 6.4
::t- 0.02 +j- 0.05 :k o.o2 -~ 0.03 :L 0-03 ~: 0.05 ± o.o 5 ± o.o6
A scending boundaries 0. 7 1.2 1.6 1. 9 2.6 3.2 3.8 6.2
~: 0.02 _~ 0.03 +_ o.oI ~ o.o 3 ~: 0.02 :[= o.o6 ,- o.o6 ~ o.o6
VOl.. I I
(19.53)
ELECTROPHORESIS OF MUSCLE PROTEINS
2!J~
immobilization of a leg in a plaster cast, prolonged fasting and vitamin-l~2 dcticit~nc\,. ,5everance o/ a lnotoric ~zerve. P a r t of t h e n e r v u s ischiadicus of one leg was excised aml the skin s u t u r e d . T h e operation, w h i c h was t e r m i n a t e d in 5 m i n u t e s , was carried o u t u n d e r N u m a l - R o c h e narcosis. A p a r t from t h e p a r a l y s i s of t h e leg tile a n i m a l s were in p e r f e c t health, l I o m o l o g o n s muscle:~ of tile hind legs, one n o r m a l a n d one atrophied, were excised after 2 to 5 weeks, in one ease after 9 weeks. Immobility in a plaster cast. In order to p r e v e n t d a m a g e b y g n a w i n g it a p p e a r e d to be n e c e s s a r y to place a wooden collar a r o u n d t h e neck of t h e rabbit. A f t e r t h r e e weeks t h e v a s t u s lateralis muscles were r e m o v e d from b o t h hind legs a n d e x a m i n e d . Inanition. T w o r a b b i t s were used for this e x p e r i m e n t . T h e v a s t u s lateralis was r e m o v e d from one leg before t h e f a s t i n g period. (The r e s u l t s in Table II s h o w t h a t no c h a n g e occurs in n o r m a l muscle in t h e course of s o m e weeks.) T h e a n i m a l s died a f t e r f a s t i n g for 6 a n d 9 d a y s respectively. I n this t i m e t h e b o d y weight h a d decreased b y a b o u t 4 ° %. As b o t h a n i m a l s died d u r i n g t h e n i g h t t h e m u s c l e s were in rigor w h e n t h e e x t r a c t s were m a d e . As will a p p e a r f r o m T a b l e V I I I this did n o t affect t h e results. Vitamin-E deficiency. A r a b b i t was fed on tile GOETTSCH-PAPPENHEIMI~:R diet '5. T h e v a s t u s lateralis of one leg h a d been r e m o v e d before a d m i n i s t r a t i o n of t h e v i t a m i n - E - f r e e diet. T h e excretion of creatine a n d creatinine was d e t e r m i n e d e v e r y day. A f t e r a b o u t i8 d a y s t h e creatine excretion increased from a b o u t io m g to 2o to 40 m g p e r 24 h. T h e 29th d a y s h o w e d a s h a r p rise of t h e creatine excretion to a b o u t 26o mg. O n t h e n e x t d a y s it was s o m e w h a t lower again. T h e r a b b i t died on t h e 33rd day.
All atrophied muscles, which have been examined, with the exception of the muscle excized 9 weeks after severance of the nerve, had lost about 5o % of their original weight. No matter which method was used to cause the atrophy, a 5o% decrease in weight was accompanied by a 2- to 3-fold increase of the percentage of component II. This can be seen in Table VIII and Fig. 3. (The increase of the percentage of this component has alIX v i i i H ready been observed by CR~PAX8, though only in the case of denervated muscles). The diagram obtained from the muscle which had atrophied during 9 weeks after Fig. 3. D i a g r a m of e x t r a c t of a t r o p h i e d r a b b i t severance of the nervus ischiadicus is shown muscle ( 2 ½ to 5 weeks a f t e r s e v e r a n c e of n e r v e or i m m o b i l i s a t i o n in p l a s t e r c a s t ; or in Fig. 4. All peaks had disappeared with the after d e a t h b y i n a n i t i o n or v i t a m i n - E deexception of component II, which accounted ficiency). for about 5o % of the total area. As can be seen from Fig. 3 the unresolvable part of the diagram between components II and III also increases considerably, but this increase cannot be expressed numerically, as component I I I - - a n d sometimes component I V - - c a n n o t be distinguished anymore in tile diagrams of atrophied muscles, so that
the flat unresolvable part extends from comI V o r V (see T a b l e V I I I ) .
ponent
II to component
¢--,_
Human
muscles
Fig. 4- D i a g r a m of e x t r a c t of atrop h i e d r a b b i t muscle, 9 weeks after s e v e r a n c e of nerve.
Fifteen normal muscles (m. serratus anterior) were examined. Fig. 5 shows the prototype of the diagrams. It bears a strong resemblance to the diagrams of normal rabbit muscles. The mobilities of the
components very closely approached those of the components of the rabbit muscle diagrams. Therefore the same numbering could be used. Table IX shows the results Re/emnees p. 269.
264
VOL. 11 (I953)
A. M. F. H. HAAN T A B L E VI ISO-ELECTRIC
Ascending boundaries Descending boundaries
POINTS
V
VI
VII
VIII
IX
4.9 4.9
5.o 5 .o
5.3 5.3
5.8 6.o
6.2 6. 4
T A B L E VII COMPARISON
OF F R E S H
MUSCLES
AND
HOMOLOGOUS
MUSCLES
IN
RIGOR
Area of the Gauss curves II to I X and of the intermediate part between II and III expressed in percentage of total area minus d- (resp. e-) gradient. Ascending boundaries. Rigor developed in situ. Each pair of homologous muscles taken from a different rabbit. Muscle
Rigor
IX
VIII
VII
VI
V
IV
111
Intern*. part
11
Serratus
-+
15.7 12.4
3 °.6 25.4
25-3 25.1
6.3 6.3
6.8 14.8
3.8 3.5
4.1 3 .2
3.2 5 .o
4.1 4 .I
Gastrocnemius
-+
15.4 13.6
3o.I 24.5
25.0 24.8
5.8 5.8
6.9 13.2
4.6 4.2
4.0 3.3
2.6 3 .1
4.5 4.5
+
15.6 13.3
29.5 23.3
25.1 24.6
5-5 5.5
6.7 11.7
5.3 4.9
4 .l 3.5
3-9 5 .0
4 .2 4 .2
-+
15.6 13.°
29.4 22.9
25.0 25.2
5.6 5.7
6.9 15.9
3.9 3.7
3 .6 2.8
4 .1 6. 3
4.3 4.3
Vastus lateralis Vastus intermedius
of t h e n u m e r i c a l e v a l u a t i o n of 13 d i a g r a m s . T h e m o s t p r o n o u n c e d d i f f e r e n c e as c o m p a r e d t o r a b b i t m u s c l e is t h e c o m p a r a t i v e l y h i g h p e r c e n t a g e of c o m p o n e n t s I I a n d VII, a n d t h e c o m p a r a t i v e l y l o w p e r c e n t a g e of c o m p o n e n t V I I I . I n m o s t c a s e s c o m ponent IX was absent.
V///
vlff
F i g . 5. D i a g r a m
>
of e x t r a c t muscle.
of n o r m a l
- - >
human
Fig. 6. Diagram of extract of h u m a n muscle atrophied since several decades.
Atrophic muscles were excised from patients suffering from prolonged muscular i n a c t i v i t y , o f t e n e x t e n d i n g o v e r s e v e r a l d e c a d e s . T a b l e X s h o w s t h e r e s u l t s of t h e n u m e r i c a l e v a l u a t i o n of t h e d i a g r a m s o b t a i n e d f r o m 7 p a t i e n t s . A s c o m p a r e d t o n o r m a l m u s c l e s i t a p p e a r s t h a t i n g e n e r a l 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 I I a n d V I I I h a v e i n c r e a s e d a n d 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 V I a n d V I I h a v e d i m i n i s h e d , w h i l e c o m Re#rences p. 269.
AND
VIII
ATROPHIED
TABLE HOMOLOGOUS
by inanition, E =
MUSCLES
atrophy
RABBIT
b y i m m o b i l i z a t i o n in p l a s t e r c a s t , I =
NORMAL
atrophy
OF
atrophy by vitamin-E
Cause of atrophy
2
-SN
-SN
-SN
-SN
-SN
-SN
-SN
-P
-I
-I
-E
Rabbit No.
2
9
13
14
15
16
19
33
20
18
24
22
-33 d a y s
-6 days
-9 days
-3 weeks
-5 weeks
-4 weeks
-4 weeks
-4 weeks
-3 weeks
-21~ w e e k s
-2 weeks
3
Time of development of atrophy
I 12 39
480 162
57 ° 134
55 ° 230
290 12o
338 14o
290 12o
460 19o
4°0 137
75 ° 3o0
31o 154
4
mg total protein extracted from whole muscle
18.6 8. 4
16 9 . 9.6
26.8 12"5
13.o 9.2
12-3 7.7
I 1.7 12.2
9.5 8.2
12. 5 7-7
14.4 12.7
17.4 12.5
18.o 12.8
5
IX
3o.1 18.2
23.4 18.o
21.2 12.5
29.6 18. 4
24.8 lO.8
27-4 17.o
25.1 17.3
26.8 27-2
27-4 17.1
29.8 19.8
30.0 19.2
6
VIII
26.6 16. 5
29.8 14. 5
21.o 17.6
24. 4 17.6
20.9 9.9
25.o 15. 3
2o.2 12.2
25.2 18.o
30.4 15.1
21. 4 19.2
21.2 18. 9
7
VII
5.4 5.7
6.3 5.8
4.5 7 .6
7.9 7. I
9.1 8.1
6.8 5-7
8. 7 4.8
7.1 6. 4
5.6 5.7
7.2 5.7
6.7 5.9
8
V[
6.3 6.2
lO.7 18.2
8.2 11.4
7-8 6.8
7.9 5.7
7.3 7-0
7.7 7.8
6.9 12.o
6.1 7.0
9.7 7-9
9.5 9.5
9
V
6.3
4.1
5.2
6.0 5.6
6"4 5.1
6.2 4.9
6.0
4.0 5.0
6.6 6.2
6.8 7.5
zo
IV
4 .2
Percentage of total area
4.0
4.6
2.4
3.2
8.1
4 .0
4.2
4.5
2.2
2.2
II
IlI
32.8
14"8
26.8
3 o. I
24.2
20.2
14.2 25.6
I4.2
20.0
19.6
15.8
5.0
8.2
5.0
5.6
5.6
4.1
4.5
9.5
3.6
3.0
12
Interm. part
4. i Ii.i
6. 7 23.3
6.1 26.o
4.2 i o.o
5-3 13.6
6.8 17.i
5.2 13.2
6.4 15.2
4.0 i2.r
4.7 12. 5
4-9 lO. 4
13
II
4.6 4.3
32 37
35 35
23 23
15 16
23 24
15 16
29 29
16 17
3,5 36
15 16
24
mg comp. I I extracted from zchole muscle
I f c o m p o n e n t I I I or c o m p o n e n t s I I I a n d I V c o u l d n o t b e d i s t i n g u i s h e d in d i a g r a m s of a t r o p h i e d m u s c l e s , o n e f i g u r e is g i v e n f o r t h e u n r e s o l v a b l e p a r t b e t w e e n c o m p o n e n t s I I a n d I V o r I I a n d V. Ascending boundaries.
S N = a t r o p h y b y s e v e r a n c e of n e r v e , P = deficiency.
COMPARISON
t.dt
t~
©
N
©
0
0
t~ c~ H
~'~
~-~ ~-"
< © t"
20(;
A.M.F.H.
HAAN
TABLE NORMAL
VOL. 1 1
(1953)
IX
HUMAN
MUSCLES
A r e a of G a u s s c u r v e s I I t o V I I I a n d of i n t e r n l e d i a t e p a r t b e t w e e n I I a n d I I I e x p r e s s e d in p e r c e n t a g e of t o t a l a r e a n l i n u s 6- (resp. e-) g r a d i e n t . M e a n s a n d s t a n d a r d d e v i a t i o n s . Ascending b~mndaries Ga4¢:,x curve
Number of determination,s
VIII VII VI V IV llI Interm. part ii
Descending boundaries
Percentage
13 13 13 13 IO 4 4 13
27. 3 33.7 7.7 7.7 5'5 4.1 4.3 6.6
~_ 6.8 ±6.4 A_ 2.1 ~ 1.9 ~ I'5 ~: 1.6 b 1. 3 :~ 1.2
TABLE ATROPHIED
A r e a of G a u s s c u r v e s I I t o V I I I
Age of atrophy in ycars
Fatty degcnerations
VIII
Number of determinations
t'ercentage
I2 12 12 12 I2 4 ,t I2
26.0 36.2 7.3 6. 4 0. 7 5"3 5.3 7.2
4:5.7 ~6.o ~[ 2.0 ~_ 2.0 4 1. 4 ~ 1"3 ± 2.3 :}- 1.4
X
HUMAN
MUSCLES
a n d of i n t e r m e d i a t e p a r t e x p r e s s e d in p e r c e n t a g e of t o t a l m i n u s d- (resp. e-) g r a d i e n t . vI
VII
v
Iv
area
lll
lnterm. part
II
o o o o o o o
I7. 4 15.o 14. 4 i2.1 o o o
9.0 [ t .7 II. 3 it. 5 16.9 18.6 18.o
o o o O o o o
18.8 I3.[ 26.0 9.1 o o o
11.6 I2.8 8. 3 1I. 4 13.2 14.2 18.2
Ascending boundaries 2 3 4 1o 20 23 46
---q + + +
44.0 48.8 41.3 56.4 55.2 50.6 49.2
28.8
3.2
o o o o o o o
24. 3 28.8
4.3 20.0 27.2 30.6 32.8
o o o o o o o
Descending boundaries 2 3 4 lo 20 23 40
---@ + + +
40.0 46.2 36.2 52.2 51.8 52.4 50.2
24.4
5.2
29.4
9.0 26.6 35.8 33.2 31.6
TABLE AMOUNTS
o o o o o o o
28.8
OF
PROTEIN
o o o O o o o
XI
EXTRACTED
PER
G
MUSCLE
Easily soluble proteins Muscles
Normal rabbit muscles Atrophic rabbit muscles R a b b i t m u s c l e s in r i g o r m o r r i s Normal human muscles
Myosin
Number off determinations
mg per g
Number of determinations
44 12 12 12
24 ± 3.5 19±3.1 24 ~ 4.2 24 q- 4.1
16 8 Ii 12
mg per g
16 4 3 17
i 2.5 }2 2.6 ± 1.5 ~ 3.2
voL. 11 (1953)
ELECTROPHORESIS OF MUSCLE PROTEINS
2(~7
ponents I I I , IV and V have completely disappeared, in those cases in which thc~ atrophy had only existed for a few years the unresolvable intermediate part was present in the diagrams. If the atrophy had been manifest for m a n y years (~:.g. 4 o years) this intermediate part had completely disappeared. Fig. 6 shows the prototype of the diagrams of the latter muscles.
Myosin, 6xtracted /rom muscle by salt solution o~ pH 7.z5 and ionic strength o.I3 As described above, a precipitate forms upon dialysis of the muscle extracts against the KCl-phosphate solution of p H 7.15 and ionic strength o.13. This precipitate was spun down, repeatedly washed with the same solution and then dissolved in GreensteinEdsall solution (0.5 M KC1, 0.03 M NaHCO~; p H 8.1o; ionic strength 0.53 ). After dialysis against this solution the electrophoresis diagram obtained appeared to be practically identical with that oi a myosin solution prepared irom rabbit muscles according to STEYN-PARVt~ AND GERRITSEN7. Hence it appears that myosin m a y 1)e extracted from the muscle by a practically neutral solution of low ionic strength.
Amounts o/proteins extracted/rom normal and atrophied muscles Table XI shows the means of the amounts of the easily soluble proteins and of myosin, extracted per g muscle by KCl-phosphate solution of p H 7.15 and ionic strength o.13. Regarding the easily soluble proteins no difference exists between fresh normal rabbit muscles, fresh normal human muscles and rabbit muscles in rigor, but the amount extracted per g of atrophic rabbit muscles is significantly lower (P < o.ooi). The amount of myosin extracted per g tissue from fresh normal rabbit muscles and from fresh normal human muscles did not differ, the amounts extracted per g tissue from the atrophied rabbit muscles and from normal rabbit muscles in rigor were very much lower, however. Notwithstanding the fact that the total amount of easily extracted protein per g of atrophic rabbit muscle was lower than the amount extracted per g of normal muscle, and that the weight of the atrophic muscles was much less than the weight of the normal homologous muscles, the amount of the protein(s) giving rise to component I I of the diagram, extracted from the whole atrophied muscle, had not diminished as compared with the amount of this protein extracted from the whole homologous normal muscle. This can be seen from column 14 of Table V I I I , the figures of which have been calculated from the total amounts of easily soluble protein per whole muscle (column 4) and the percentage of component I I (column 13). Though the diagrams of extracts of atrophied muscles had also shown an increase of the flat unresolvable part between peaks I I and III, observed in the case of normal muscles, a similar calculation could not be carried out here, for this part of the diagram now extended from component I I to component IV or from component I I to component V. DISCUSSION I t should be realized that the high reproducibility of the numerical evaluation of the diagrams could only be attained if it was carried out by one and the same person, always following the same procedure. We have placed Gauss curves in the diagrams, starting with the rather arbitrarily chosen peak No. V I I I (the highest one in the case of extracts of rabbit muscle) and then trying to fill up the whole diagram with the
Re/erences p. 269.
268
A.M.F.H.
HAAN
VOL. 1 1
(1953)
smallest possible number of Gauss curves. The figures obtained for the various Gauss areas as percentages of the total area minus ~- (resp. e-) gradient may obviously only be regarded as characteristics of the muscle extracts serving for mutual comparison of these extracts. It will be necessary to employ other methods, e.g. salting out according to DERRIEN and enzyme determinations, in order to obtain a fuller understanding of what happens to the proteins in the course of the development of atrophy. The most remarkable fact established in this work seems to be the practically identical changes that are observed irrespective of whether the atrophy is caused by immobilization (as a consequence of severance of the motoric nerve or of application of a plaster cast), by inanition or by vitamin-E deficiency. In all cases the proteins that give rise to component II (and probably also those composing the flat part of the diagram between components II and III) are maintained, while the other proteins disappear in the course of the development of the atrophy. Special attention should furthermore be given to the difference between the diagrams of rabbit muscles, rendered atrophic by experiment and human atrophied muscles. C-ERRITSENs, working in this laboratory on the glucokinase content of normal and atrophied muscles, also found a difference between these two kinds of atrophy. The enzyme appeared to have been fully maintained in the atrophied rabbit muscles, but to have decreased considerably in the atrophied human muscles. In both cases we believe that the differences between rabbit and human muscles are caused by the different times during which the atrophies have existed. The intermediate unresolvable part of the electrophoresis diagrams of extracts of atrophied rabbit muscles and the glucokinase of these muscles would probably also have disappeared if it would have been possible to incite a very slowly developing muscular atrophy in rabbits.
SUMMARY T h e p r o t e i n s of n o r m a l a n d a t r o p h i e d skeletal m u s c l e s of t h e r a b b i t a n d of m a n , soluble in salt solutions of low ionic s t r e n g t h a n d p H a b o u t 7, were s t u d i e d b y m e a n s of electrophoresis. A t r o p h y of r a b b i t leg m u s c l e s w a s c a u s e d b y s e v e r a n c e of t h e m o t o r i c nerve, b y i m m o b i l i s a t i o n of a leg in a p l a s t e r cast, b y i n a n i t i o n a n d b y v i t a m i n - E deficiency. No m a t t e r w h i c h of t h e s e m e t h o d s was u s e d to i n d u c e m u s c u l a r a t r o p h y , t h e electrophoresis d i a g r a m s o b t a i n e d a l w a y s s h o w e d t h e s a m e a l t e r a t i o n s as c o m p a r e d w i t h t h e d i a g r a m s o b t a i n e d for n o r m a l muscles. T h e m o s t r e m a r k a b l e f e a t u r e w a s t h e increase of t h e p e r c e n t a g e s of t h e m o r e r a p i d l y m o v i n g c o m p o n e n t s a n d t h e decrease of t h e p e r c e n t a g e s of t h e slower c o m p o n e n t s . W h i l e t h e t o t a l a m o u n t of protein, e x t r a c t a b l e b y a salt solution of ionic s t r e n g t h o.13 a n d p H 7.15 f r o m t h e whole muscle, h a d c o n s i d e r a b l y decreased, t h e a m o u n t of t h e m o s t r a p i d l y m i g r a t i n g c o m p o n e n t (II), d i s t i n g u i s h a b l e in t h e d i a g r a m s , h a d remained constant. N o r m a l h u m a n m u s c l e s differed little f r o m n o r m a l r a b b i t muscles. T h e flat i n t e r m e d i a t e p a r t of t h e d i a g r a m s , w h i c h h a d b e e n m a i n t a i n e d in t h e case of t h e a t r o p h i e d r a b b i t muscles, h a d , however, c o m p l e t e l y d i s a p p e a r e d in t h e case of chronic h u m a n m u s c u l a r a t r o p h y caused b y i m m o b i l i z a t i o n , e x i s t i n g d u r i n g several decades. T h e t i m e of e x i s t e n c e of t h e a t r o p h y m a y be t h e c a u s e of this difference, as t h e a t r o p h y of t h e r a b b i t m u s c l e s h a d developed in t h e course of a few weeks. RI~SUMt~ L e s p r o t 6 i n e s des m u s c l e s s q u e l e t t i q u e s n o r m a u x et a t r o p h i e s de l ' h o m m e et du lapin, solubles d a n s des s o l u t i o n s salines de force ionique faible et de p H e n v i r o n 7, o n t dt6 dtudi6es p a r 61cctrophor6se. L ' a t r o p h i e des m u s c l e s de la p a t t e du lapin est p r o v o q u d e p a r ldsion du nerf m o t e u r , par i m m o b i l i s a t i o n de la p a t t e d a n s u n m o u l e de pl~tre, p a r i n a n i t i o n ou p a r carence en v i t a m i n e E. Quelle q u e soit la m 6 t h o d e utilis6e p o u r p r o v o q u e r l ' a t r o p h i e m u s c u l a i r e , les d i a g r a m m e s d'6lectrophor6se o b t e n u s p r d s e n t e n t t o u j o u r s les m ~ m e s modifications p a r r a p p o r t a u x d i a g r a m m e s o b t e n u s a v e c des m u s c l e s n o r m a u x . L a p l u s r e m a r q u a b l e de ces modifications est l ' a u g m e n t a t i o n des pour-
Re/erences p. 269.
voL. 11 (I953)
ELECTROPHORESIS OF MUSCLE PROTEINS
2~)9
centages des c o n s t i t u a n t s so ddpla~ant le plus rapidement, et la diminution des pourcentages des constituants los plus lents. Alors que le contenu total en prot6ines extractibles par une solution saline de forco ioniquo o.13 et de p H 7.15 i~ partir du muscle entier, diminue considdrablement, la teneur du consfituant qui poss~de la plus grande vitesse de migration (II), qu'on p e u t distinguer sur le diagramme, reste constante. Les muscles h u m a i n s n o r m a u x diffSren~c peu des muscles de lapins n o r m a u x . La partie intermddiaire plate du diagramme, qui est Inaintenue dans le cas des muscles de lapin atrophi6s, disparait cependant compl~tement dans le cas d ' u n e atrophie musculaire hunlaine chroniqne provoqude par une immobilisation de plusieurs dizaines d'anndes. Cette diffdrence est peut-6tre due /z la dur6e de l'atrophie, car l'atrophie des muscles du lapin est obtenue en quelqnes semaines.
ZUSAMMENFASSUNG Die Proteine normaler und atrophierter Skelettmuskeln von K a n i n c h e n und Menschen, die in Salzl6sungen m i t niedriger Ionenst~rke und einem p H von ungefS.hr 7 16slich sind, wurden m i t Hilfe der Elektrophorese untersucht. Die Atrophie der Beinmuskeln yon K a n i n c h e n wurde hervorgerufen durch T r e n n u n g des motorischen Nervs, durch Immobilisation des Beines im Gipsverband, durch Entkr~tftigung u n d durch Vitamin E-Mangel. Verglich m a n die erhaltenen elektrophoretischen D i a g r a m m e mit denen normaler Muskeln, so zeigten sie immer die gleiche Ver~nderung, ganz gleichgiiltig welche dieser Methoden zur Herbeifiihrung der Muskelatrophie b e n u t z t wurde. Das Ansteigen des Prozentgehalts der schneller beweglichen K o m p o n e n t e n und das Absinken des Prozentgehalts langsamer K o m p o n e n t e n war das meist bemerkenswerte Merkmal. WAhrend der Gesamtanteil des mit Salzl6sung der Ionenst~rke o.13 und p H 7.15 aus dem ganzen Muskel extrahierbaren Proteins betr~tchtlich a b n a h m , blieb der Anteil der a m schnellsten wandernden K o m p o n e n t e (II) welche m a n in den D i a g r a m m e n unterscheidet, k o n s t a n t . Normale nlenschliche Muskeln untersehieden sich wenig yon normalen K a n i n c h e n m u s k e l n . Der ttache Zwischenteil der Diagramme, der im Fall des atrophierten K a n i n c h e n m u s k e l s erhalten blieb, verschwand jedoch vollst~ndig im Fall der fiber mehrere J a h r z e h n t e bestehenden, durch Immobilisation verursachten menschlichen chronischen Muskelatrophie. Die Ursache dieses Unterschiedes k6nnte in der Dauer des Bestehens der Atrophie liegen, da sich die Atrophie des K a n i n c h e n m u s k e l s im Laufe einiger Wochen entwickelte. REFERENCES M. W. BOSCH, Biochim. Biophys. Acta, 7 (1951) 61; Thesis, U t r e c h t 1951. 2 M. DUBOISSON, A. DIST~CI4E AND A. DEBOT, Biochim. Biophys. Acta, 6 (I951/52) 97. 3 E. WIEDEMANN,Helv. Chim. Acta, 3 ° (1947) 892. 4 j. JACOB, Biochem. J., 42 (1948) 71. 5 M. GOETTSCH AND A. M. PAPPENHEIMER, J. Exp. Med., 54 (1931) 145. P. CR£PAX, Experientia, 5 (1949) 167. E. P. STEYN-PARV£ AND T. GERRITSEN, Biochim. Biophys. Acta, 8 (1952) lO 4. 8 T. GERRITSEN, Biochim. Biophys. Acta, 8 (1952) 466. Received January
I o t h , 1953