or a white muscle. G. SZEWT-GYORGYI

Transcription

1 131. P R O T E I N S OF THE M Y U F l B R I L A. G. SZEWT-GYORGYI Ladies and gentlemen, I would l i k e t o review here a number of questions which involve some properties of t h e fibrous muscle proteins, how these properties lead t o contractions, what t h e changes are which occur during contractions, and also t o expand on t h e paper presented by Dr. Cassens by discussing how some of these proteins are responsible for t h e specialized s t r u c t u r e of muscle and what reactions may control t h e format i o n of these s t r u c t u r e s and a c t i v i t i e s. The muscle proteins can be conveniently divided i n t o d i f f e r e n t f r a c t i o n s according t o s o l u b i l i t y and also according t o function. If you grind up muscle and add a solvent of low ionic strength you will f i n d that 35% of t h e p r o t e i n s w i l l be r e a d i l y solubilized without, as I think w i l l be shown on the f i r s t slide, really disrupting t h e specialized s t r u c t u r e of t h e muscle. The striated p a t t e r n t h a t apgeass in unextracted muscle, as shown by Dr. Cassens, w i l l remain. What i s solubilized i s t h e enzymes of t h e glycolytic cycle, t h e phosphate-producing enzymes l i k e myokinase and creetinekinase, and r e a l l y t h e residue, t h a t is, t h e insoluble portion remaining, w i l l show t h e s t r u c t u r a l regularity of t h e muscle even c l e a r e r than before the extraction, W e do not know exactly where these soluble enzymes are localized or i f they are localized, but we think they are somewhere i n t h e sarcoplasm and t h a t they are not associated with t h e filamentous s t r u c t u r e of muscle. I will not discuss these proteins any more. They do not concern us nor have anything t o do with t h e culinary aspects of meat. O f course t h e relative m u n t s of these soluble enzymes will g r e a t l y change depending on whether w e are dealing with a red muscle or a white muscle. Now i n muscle which w a s very w e l l ground up, you w i l l f i n d t h a t with t h e soluble proteins, a number of granules w i l l be extracted. These granules compose t h e mitochondria and also t h e s m o t h endoplasmic reticulum, which i s sometimes erroneously described as ribosomes, together with some a c t u a l ribosomes. Here, t h e r e w i l l be a great deal mre i n t e r e s t f o r a person who i s i n t e r e s t e d i n how contraction i s controlled, became the endoplasmic reticulum, which i s t h i s membranous system including t h e t r i a d system with longitudinal components i s closely connected with t h e problem of how stimulation t r a v e l s inward t o t h e muscles. One can, as Ebochet first d i d, i s o l a t e t h i s relaxing o r endoplasmic reticulum system and f i n d t h a t it a c t s as a relaxing system on t h e muscle. It indeed, even i n an underrated condition, can a c t as a calcium pwp, concentrating calcium, and t h i s a c t i v i t y w i l l regulate t h e occurrence o f c o c t r a c t i o n. Finally t h e r e are about f i f t y or f i f t y - f i v e p e r cent of t h e proteins which are not extractable with solvent of low ionic strength and which are responsible f o r t h e composition of myofibril and contraction. These proteins can be solubilized by using solvent of high ionic strength, e.g., 0.6 mlw m r e

2 salt, and include i n case of mst muscles with t h e exception of annelid and m l u s c a muscles, mainly t h r e e proteins, t h e properties of which I will t r y t o discuss i n somewhat mre d e t a i l. The proteins remaining a f t e r extraction with high ionic strength solutions are c a l l e d stroma proteins and consist mainly of collagen. The proportion of stoma protein i n a muscle again w i l l vary depending upon t h e type of muscle; I t h i n k it i s i n inverse r e l a t i o n ship with t h e s i z e of t h e muscle. W e use rabbit psoas muscle f o r many experiments because of i t s lack of connective t i s s u e, i t s softness, and t h e extreme ease of separating t h e fibers from each other. The next s l i d e will show some of t h e properties and the d i s t r i b u t i o n of t h e m a i n muscle groupings. L e t ' s start with myosin, which i s a p r o t e i n of about 500,000 o r 450,000 molecular weight, having a length of 1600 hgstroms. It i s important t o r e a l i z e t h a t t h e t h i c k filaments are made up of myosin and t h a t when discussing t h e banded s t r u c t u r e of muscle, i.e., t h e A-band, t h e Z-band, and t h e I-band, t h a t t h e length of t h i s A-band i s 1.6 microns. The individual myosin molecule i s one-tenth of t h i s length, or 0.16 microns, so t h e r e must be some type of aggregation of t h e qyosin molecules t o make up t h e A-bands. The lqyosin m l e c u l e has a number of properties. It has ATPase a c t i v i t y. It w i l l combine with a c t i n and t h e complex of actomyosin, which i s formed f r o m a c t i n and myosin, shows t h e simple proper type of contract i o n when ATP i s added, provided t h e conditions are correct -- t h a t i s t o say, at low ionic strength. Actomyosin has an unusual s o l u b i l i t y property since it i s p r e c i p i t a t e d at low ionic strength at which it contracts but it i s dissolved at higher ionic strength. It i s myosin which has t h e ATPase a c t i v i t y of t h e myofibrillar proteins, and what i s mst important, contraction, individual, o r f o r t h a t matter, contraction of t h e nyofibril, occurs only under conditions where myosin i s p r e c i p i t a t e d. T h i s makes a headache i f you hegpen t o be a biochemist, because you cannot measure changes i n length and shape during t h i s contraction by using t h e techniques which were designed t o study molecular solutions. Actually, perhaps t h e muscle proteins are one of those very few protein systems t h a t we don't see and we can't readily measure any length and shape changes, although they contract v i s i b l y somehow. I w i l l return a l i t t l e b i t later t o myosin, but I would l i k e t o discuss now t h e properties of a c t i n. Actin i s again an unusual protein. It can exist i n a monomeric form which i s c a l l e d globular actin, and which will polymerize under c e r t a i n i d e a l conditions and form fibrous actin. The globular a c t i n has a molecular weight of probably 60,000, and t h e F-actin has a molecular weight of several million. Actually, F-actin i s a very regular aggregation of mnomeric p a r t i c l e s, globular p a r t i c l e s. It i s an aggregation, but it forms a double h e l i c a l chain, of t h e form shown by Dr. Cassens, and it has an almost i n d e f i n i t e length. That i s why t h e molecular weight i s i n d e f i n i t e although it i s at l e a s t several millfon. What i s very i n t e r e s t i n g i s t h a t the globular a c t i n i s associated with ATP, but t h e fibrous a c t i n i s associated w i t h ADP. Curing t h e polymerization process and only during t h i s polymerization process, inorganic phosphate i s l i b e r a t e d. This chemical change i s closely assoc i a t e d with t h e s t r u c t u r e o r a l t e r a t i o n of t h i s molecule.

3 133. Now, there are some other interesting properties of actin. One of these is, as found by Gergely, t h a t the ATP associated with G-actin i s readily exchangeable with external ATP. Therefore, i f ATP labeled with radioactive carbon i s added t o a solution of globular actin, t h e label will appear i n t h e ATP bound t o t h e actin. I n t h e case of F-actin, the ADP does not exchange with ADP i n solution, and i f you add labeled ADP t o a solution of F-actin, it w i l l stay i n t h e solution and not appear i n the ADP bound t o the F-actin. This i s important and if I w i l l have some t i m e left, I w i l l expound on it because it gives us a t o o l t o study t h e state of the a c t i n i n muscle at rest o r i n contraction and t o study whether any kind of change from one state t o t h e other occurs during contraction. Now, l e t ' s discuss myosin further. m s i n i s a f a i r l y complex molecule, and under controlled conditions, treatment with protolytic enzymes will s p l i t it i n t o two types of components. The next slide w i l l show t h i s reaction. Trypsin, chymtripsin o r s u b t i l i s i n w i l l all give t h i s type of s p l i t t i n g. The miyosin m l e c u l e i s s p l i t e s s e n t i a l l y into two types of components, called heavy meromyosin o r l i g h t meromyosin. The next s l i d e w i l l show I think one of the e a r l y experiments involving the treatment of myosin with trypsin f o r varying lengths of time. The incubation periods i n t h i s experiment were two minutes, four minutes, s i x minutes, and twleve minutes with a one t o t w o hundred trypsin-to-myosin ratio. You can see t h a t myosin splits i n t o slower and faster sedimenting components. The slower sedimenting component which can be c r y s t a l l i z e d i s called l i g h t meromyosin and the faster sedhenting component i s called heavy meroqyosin. A t t h e time t h i s experiment was conis b u i l t up of at ducted, we thought these r e s u l t s indicated t h a t -sin least two kinds of components which are attached end-to-end t o each other. What i s of great i n t e r e s t i s that the heavier portion o r heavy meromyosin has t h e center which i s responsible f o r the ATPase a c t i v i t y of myosin, also possesses the center which combines with actin. The l i g h t e r component or l i g h t merorqyosin is responsible f o r the s o l u b i l i t y properties of qyosin, t h a t is, the precipitakion at low ionic strength and a l s o the solution at high ionic strength. Heavy meromyosin i s soluble regaxdless of t h e ionic strength. Also, l i g h t meromyosin i s capable of forming extremely regular structures. If you measure t h e alpha-helix content of l i g h t meromyosin, you will f i n d t h a t l i g h t meromyosin f r a c t i o n 1, which i s about 25/35 of the t o t a l l i g h t meromyosin i s almost lo@ alpha h e l i c a l. It i s one of t h e f e w proteins which behave as a f u l l y called alpha-helix. If a solution of light meromyosin i s precipitated by dilution and t h e precipitated protein examined d i r e c t l y under a microscope, a very regular 430x periodicity appears. This i s one of t h e characteristic periods present i n muscle and it i s now explained as representing the myosin periodicity. Therefore, qyosin may be considered as having a head and a t a i l, and these expectations have been borne out very nicely i n t h e electron microscope by a number of investigators s t a r t i n g from Rice and Hugh Huxley and Zobel and Carlson and so on. I would l i k e t o examine some of these electron micrographs now t o see how these molecules look before returning t o discuss some of the possible steps which may occur during contraction. The next s l i d e i s a summary of the properties and the molecular weights of heavy and l i g h t meromyosin. The molecular weight of heavy meromyosin i s about 300,000 and t h a t of l i g h t meromyosin i s about 150,000. The heavy meromyosin i s an AllPase and has all t h e ATPase a c t i v i t y of t h e

4 134. parent myosin, while the l i g h t meromyosin has t h e s o l u b i l i t y properties of myosin but has no A!llPase-activity and no combining a c t i v i t y with actin. The next slide shows t h e 430Aperiodic structure of l i g h t meromyosin. This structure can be seen with even unstained preparations. The next slide shows an electron micrograph of a preparation of myosin taken by Hugh Huxley. The head and tail portions of myosin can be e a s i l y seen. The average length of the molecules i s about 1600x o r so, which agrees with t h e values obtained i n solution. Actually t h e head portion p l u s a small p a r t of t h e t a i l represents the heavy meromyosin part, and the rest of the tail portion i s associated with t h e l i g h t meromyosin p a r t. Huxley also found t h a t during precipitation of myosin molecules t o form "crystals" a dimer forms. T h i s dimer has the heads of the molecule facing the heads away from each other. The t a i l portions of the molecule l i k e t o aggregate and form structures. These portions tend t o associate with each other s t a r t i n g t o form filaments, with the heads of the filaments facing away f r o m the center. There w i l l be a c e n t r a l s h a f t which has no roughness o r no head portions. That is, t h e filaments formed i n this process will not contain heavy meronlyosin p a r t i c l e s i n the center of t h e filaments but the rest of the crystal, o r filament, w i l l have these heavy meromyosin protuberances. This i s shown in the last slide where you see a growing myosin filament with the center portion of the filament smooth and f r e e of t h e hemy meromyosin projections. Even though t h e filament grows i n length, t h e center of the s h a f t will remain smooth, while the l a t e r a l portions away f r o m t h e center will have these i r r e g u l a r i t i e s o r these knobs, which probably correspond t o t h e heavy meroqyosin. Thus, appearance of t h i s filament i s very similar t o the t h i c k filament, which occurs naturally i n muscle. The present concept of Huxley i s that the c e n t r a l p a r t of t h e t h i c k filaments are made up of t h e l i g h t meromyosin portion of myosin which bind together i n some unknown but regular fashion, while p a s t of t h e heavy meromyosin portion i s sticking up fromthese filaments and i s responsible f o r the combination of the t h i c k filaments with a c t i n (or t h i n filaments). The next s l i d e shows some comparisons of filaments obtained from myosin with natural thick f i l a ments obtained from muscle. The two types of filaments look very similar. The next slide shows the structure of fibrous or F-actin 85 obtained by Haasen and bwry. Perhaps you can see the winding of t h e double strand and the further subdivision of each strand of t h e filament into subu n i t s o r particles which are associated with the mmomeric or globular actin Unit of 60,000. The next s l i d e i s an electron micrograph of a t h i n filament taken by Huxley. I t h i n k between the two arrows you can see how the two strands are winding around each other t o form the filament. I n addition t o the double h e l i c a l structure of actin, Huxley has shown that there i s some d i r e c t i o n a l i t y within t h e actin thread, and that, I think, can be quite c l e a r l y seen in these regions 360x apart, where you see t h e crossing over of the t w o strands. These F-actin strands also show t h i s d i r e c t i o n a l i t y when mixed with heavy meromyosin. You w i l l remember t h a t t h e heavy meromyosin i s the portion of myosin which i n t e r a c t s with actin. The next slide shows the head-like heavy meroilryosin structures attached t o one a c t i n filament. You notice t h a t t h e head-like structures all face one direction indicating some type of p o l a r i t y o r d i r e c t i o n a l i t y of the actin. It appears t h a t the actin filament, o r the t h i n filament, faces i n opposite directions on t h e two sides of the Z-band.

5 135. I think that is all forthe slides so now let's discuss some properties of the contraction system. I mentioned that the individual contraction system is the actomyosin system in the presence of the ATP, i.e., the complex of actin with qyosin, which in the presence of ATP will contract. During contraction, inorganic phosphate is liberated and this will be replaced by ATP once the stimulation has occurred and the act of contraction is over. The original state must be restored before a second cycle of contraction. Now, I would like to discuss a few aspects of the contraction cycle. First, how is muscle kept at rest? In muscle we have ATP present, we have actin, and we have myosin. At present, the concept is that the reason we don't have contraction in resting formations is due to the relaxing factor system, which pumps away all the calcium which is needed in trace accounts for contraction. The effect of stimulation is that calcium is liberated from the intermediazy vesicle of the sarcoplasmic reticulum. This calcium allows actin and myosin to combine and start the whole contraction cycle. The second part which I want to emphasize is that the evidence indicates that in the resting state actin and myosin are separated. The connections between the thick and thin filaments are not operating. That is the reason why muscle is so soft in the resting state, and you can stretch muscle in the resting state with very little resistance. You are able to pull out the thin filaments fromthe thick filaments. There is actually no continuity within the muscle except p&s of the sarcolemma and some membranous components. The first effect of excitation is to form actomyosin. This is responsible for the jincreased elastic mdulus, increased viscosity, and the increased hardness of the muscle over that of the resting state. It is possible to dissect muscle to yield a preparation which has the actin and myosin still intact. The mscle is punctional but the ATP has been extracted. Such a mscle is in rigor mortis, and actually, rigor mrtis may be defined as a combination of actin and Wosin because of the disappearance of ATP. It is partly the presence of ATP which keeps the actin and myosin filaments separated from each other. Once it diss.g,pea.rs you have actomyosin and your muscle will be inextensible and that I am told is quite an awkward state to be in. The third thing which I will mention rather briefly is the type of change which occurs during contraction. In essence,the lack of overall change in the X-ray diffraction pattern of the actomyosin system during contraction led Huxley to suggest that contraction is not a change in the intemlecular structure of the actomyosin system but is simply a sliding of the thin and thick filaments inward together. In a resting muscle or in a stretched muscle, the ends of the thin filaments do not meet but leave a space which forms the H-band of the myofibril. In contraction, the length of the A-band remains constant but the thick filaments slide inwmd. This is mediated by truss connections among the filaments in a manner which is yet uncleax. Quite recently, we have obtained evidence that there rnw be a change in fibrous actin which could be explained by something similar to local depolymerization of actin at the places where qyosin and actin

6 136. i n t e r a c t. That i s t o say, i n muscle i n r i g o r or i n muscle at rest, t h e nucleotide associated with t h e a e t i n i s unavailable f o r exchange w i t h outside ATP. During contraction or i n systems undergoing superprecipitation of t h e actoqyosin system, the nucleotide bound t o a c t i n w i l l be released and w i l l pass i n t o solution. W e now t h i n k t h a t there i s a possibility--and t h i s i s a bare figment of speculation--that somehow t h e movement during contracticn may be mediated at t h e places of i n t e r a c t i o n of myosin with actin. Thus a l o c a l change i n t h e s t r u c t u r e of actin causes the t h i n filament t o s t r e t c h a f t e r i n t e r a c t i o n with an active s i t e of myosin and allows a neighboring active s i t e t o get i n contact with myosin which can then also undergo t h i s cycle of change. I n rather general terms, I could describe contraction i n the following fashion: regardless of t h e actual function o r actual mechanism of contraction muscle i s r e a l l y not well-informed i n physics. It hasn't learned i t s lesson, and i f you keep a c h a i r or you t r y t o hold a c h a i r i n an outstretched position, you g e t t i r e d pretty fast, even i f you don't do any work. The c h a i r i s not rnoving since you j u s t hold it, but you s t i l l g e t very t i r e d i n a very short time. What it means i s t h a t even under isometric conditions when you don't do any work, t h e contraction process i s going on. That i s t o say, cycles of i n t e r a c t i o n s are occurring. The explanation of t h i s phenomenon i s unclear as i s t h a t of another even more surprising phenomenon, so-called fat effect, which says t h a t the t o t a l energy mobilization of the muscle depends on the work it w i l l have t o do or, i n o t h e r words, on the load which i s put at the end of t h e muscle. The muscle will perform work t o l i f t t h i s load. It mobilizes a g r e a t e r amount of energy than i s required i f it w e r e t o shorten i n an unloaded condition. T h i s has been shown physiologically i n recent experiizents on ATP l i b e r a t i o n and proton l i b e r a t i o n under such conditions. It i s a very surprising thing t h a t t h e molecules appear t o know what i s intended sometimes several lengths of molecules away. One possible explanation i s t h e following. L e t ' s assume that myosin filaments and a c t i n filaments have d i f f e r e n t periodicit i e s. It doesn't matter what t h e p e r i o d i c i t y i s j u s t so long as they are not t h e same. To accomplish contraction, cross links w i l l have t o be established at c e r t a h sites. Regadless of what t h e other conditions are, these cross links w i l l drive the two filaments apart. There i s a structural change i n t h e myosin o r i n the a c t i n which may be of short duration, but i n order t o have contraction, t h e s i t e from t h e myosin and t h e s i t e f r o m t h e a c t i n must come together. The number of sites established w i l l depend on the r a t e of the shortening. I f t h e r e i s more t i m e available f o r establishing t h e s i t e and t h e reaction, then t h e t w o systems move i n respect t o each other w i t h a slower speed. T h i s i n t u r n m e a n s t h a t more sites can be established. If you have an unloaded situation, the muscle w i l l shorten quickly. I n loaded condition it will shorten slower. So i f t h e muscle i s loaded, t h e r e w i l l be t i m e enough f o r more cross l i n k s o r active sites t o be established, and these active sites which will l i b e r a t e more energy by means of ATPase cleavage of ATP. I t h i n k I have exhausted my t i m e, and you will have t o forgive me i f I was a l i t t l e b i t unclear f o r when you t a l k of too m a n y things and you s a y too l i t t l e about each, t h i s i s often t h e result. However, I thought I would t r y t o bring you up t o date from t h e very beginning, indicating how we think about these problems and how t h e physical c h a r a c t e r i s t i c s and

7 137. chemical reactions will ultimately decide both t h e appearance and consistency of muscle, or i f you want t o c a l l it, meat. Thank you very much. DR. GOLL: Thank you, D r. Szent-Gyorgyi, f o r t h a t very excellent resume of t h e properties of t h e muscle lqyofibrillor proteins and some of the properties of muscle contraction. D r. Szent-Gyorgyi made reference briefly i n h i s t a l k t o some of the problems which are inherent i n t h e study of t h e muscle proteins. It appears t h a t i n many cases t h e methods available f o r the preparation and purification of t h e muscle proteins form t h e major obstacle t o t h e i r study. Fibrous proteins i n general present unique problems i n methods f o r separation, p u r i f i c a t i o n and study. We are happy t o have with us t h i s mrning Dr. Harry Snyder of Iowa State University who w i l l discuss some of the methods which are available f o r preparation, p u r i f i c a t i o n and i s o l a t i o n of fibrous proteins. Harry got h i s doctoral degree at t h e University of California at Daes and i s now interested i n t h e study of post-mrtern changes i n muscle. The t i t l e of Dr. Snyder's paper t h i s morning i s "Methods Available t o Separate, Isolate and Study Fibrous B o te i n s". DR. SNYIER: Thank you, Darrel. ############