Proteins of the Myofibril

131.

PROTEINS OF THE MYUFlBRIL

A. G. SZEWT-GYORGYI

Ladies and gentlemen, I would like to review here a number of questions which involve some properties of the fibrous muscle proteins, how these properties lead to contractions, what the changes are which occur during contractions, and also to expand on the paper presented by Dr. Cassens by discussing how some of these proteins are responsible for the specialized structure of muscle and what reactions may control the forma- tion of these structures and activities.

The muscle proteins can be conveniently divided into different fractions according to solubility and also according to function. If you grind up muscle and add a solvent of low ionic strength you will find that 35% of the proteins will be readily solubilized without, as I think will be shown on the first slide, really disrupting the specialized structure of the muscle. The striated pattern that apgeass in unextracted muscle, as shown by Dr. Cassens, will remain. What is solubilized is the enzymes of the glycolytic cycle, the phosphate-producing enzymes like myokinase and creetinekinase, and really the residue, that is, the insoluble portion remaining, will show the structural regularity of the muscle even clearer than before the extraction, We do not know exactly where these soluble enzymes are localized or if they are localized, but we think they are somewhere in the sarcoplasm and that they are not associated with the filamentous structure of muscle. I will not discuss these proteins any more. They do not concern us nor have anything to do with the culinary aspects of meat. Of course the relative munts of these soluble enzymes will greatly change depending on whether we are dealing with a red muscle or a white muscle. Now in muscle which was very well ground up, you will find that with the soluble proteins, a number of granules will be extracted. These granules compose the mitochondria and also the smoth endoplasmic reticulum, which is sometimes erroneously described as ribosomes, together with some actual ribosomes. Here, there will be a great deal mre interest for a person who is interested in how contraction is controlled, became the endoplasmic reticulum, which is this membranous system including the triad system with longitudinal components is closely connected with the problem of how stimulation travels inward to the muscles. One can, as Ebochet first did, isolate this relaxing or endoplasmic reticulum system and find that it acts as a relaxing system on the muscle. It indeed, even in an underrated condition, can act as a calcium pwp, concentrating calcium, and this activity will regulate the occurrence of coctraction. Finally there are about fifty or fifty-five per cent of the proteins which are not extractable with solvent of low ionic strength and which are responsible for the composition of myofibril and contraction. These proteins can be solubilized by using solvent of high ionic strength, e.g., 0.6 mlw mre salt, and include in case of mst muscles with the exception of annelid and mlusca muscles, mainly three proteins, the properties of which I will try to discuss in somewhat mre detail. The proteins remaining after extraction with high ionic strength solutions are called stroma proteins and consist mainly of collagen. The proportion of stoma protein in a muscle again will vary depending upon the type of muscle; I think it is in inverse relation- ship with the size of the muscle. We use rabbit psoas muscle for many experiments because of its lack of connective tissue, its softness, and the extreme ease of separating the fibers from each other.

The next slide will show some of the properties and the distribu- tion of the main muscle groupings. Let's start with myosin, which is a protein of about 500,000 or 450,000 molecular weight, having a length of 1600 hgstroms. It is important to realize that the thick filaments are made up of myosin and that when discussing the banded structure of muscle, i.e., the A-band, the Z-band, and the I-band, that the length of this A-band is 1.6 microns. The individual myosin molecule is one-tenth of this length, or 0.16 microns, so there must be some type of aggregation of the qyosin molecules to make up the A-bands.

The lqyosin mlecule has a number of properties. It has ATPase activity. It will combine with actin and the complex of actomyosin, which is formed from actin and myosin, shows the simple proper type of contraction when ATP is added, provided the conditions are correct -- that is to say, at low ionic strength. Actomyosin has an unusual solubility property since it is precipitated at low ionic strength at which it contracts but it is dissolved at higher ionic strength. It is myosin which has the ATPase activity of the myofibrillar proteins, and what is mst important, contraction, individual, or for that matter, contraction of the nyofibril, occurs only under conditions where myosin is precipitated. This makes a headache if you hegpen to be a biochemist, because you cannot measure changes in length and shape during this contraction by using the tech- niques which were designed to study molecular solutions. Actually, perhaps the muscle proteins are one of those very few protein systems that we don't see and we can't readily measure any length and shape changes, although they contract visibly somehow.

I will return a little bit later to myosin, but I would like to discuss now the properties of actin. Actin is again an unusual protein. It can exist in a monomeric form which is called globular actin, and which will polymerize under certain ideal conditions and form fibrous actin. The globular actin has a molecular weight of probably 60,000, and the F-actin has a molecular weight of several million. Actually, F-actin is a very regular aggregation of mnomeric particles, globular particles. It is an aggregation, but it forms a double helical chain, of the form shown by Dr. Cassens, and it has an almost indefinite length. That is why the molecular weight is indefinite although it is at least several millfon.

What is very interesting is that the globular actin is associated with ATP, but the fibrous actin is associated with ADP. Curing the polymerization process and only during this polymerization process, inorganic phosphate is liberated. This chemical change is closely associated with the structure or alteration of this molecule. 133.

Now, there are some other interesting properties of actin. One of these is, as found by Gergely, that the ATP associated with G-actin is readily exchangeable with external ATP. Therefore, if ATP labeled with radioactive carbon is added to a solution of globular actin, the label will appear in the ATP bound to the actin. In the case of F-actin, the ADP does not exchange with ADP in solution, and if you add labeled ADP to a solution of F-actin, it will stay in the solution and not appear in the ADP bound to the F-actin. This is important and if I will have some time left, I will expound on it because it gives us a tool to study the state of the actin in muscle at rest or in contraction and to study whether any kind of change from one state to the other occurs during contraction.

Now, let's discuss myosin further. msin is a fairly complex molecule, and under controlled conditions, treatment with protolytic enzymes will split it into two types of components. The next slide will show this reaction. Trypsin, chymtripsin or subtilisin will all give this type of splitting. The miyosin mlecule is split essentially into two types of components, called heavy meromyosin or light meromyosin.

The next slide will show I think one of the early experiments involving the treatment of myosin with trypsin for varying lengths of time. The incubation periods in this experiment were two minutes, four minutes, six minutes, and twleve minutes with a one to two hundred trypsin-to-myosin ratio. You can see that myosin splits into slower and faster sedimenting components. The slower sedimenting component which can be crystallized is called light meromyosin and the faster sedhenting component is called heavy meroqyosin. At the time this experiment was con- ducted, we thought these results indicated that -sin is built up of at least two kinds of components which are attached end-to-end to each other. What is of great interest is that the heavier portion or heavy meromyosin has the center which is responsible for the ATPase activity of myosin, also possesses the center which combines with actin. The lighter component or light merorqyosin is responsible for the solubility properties of qyosin, that is, the precipitakion at low ionic strength and also the solution at high ionic strength. Heavy meromyosin is soluble regaxdless of the ionic strength. Also, light meromyosin is capable of forming extremely regular structures. If you measure the alpha-helix content of light meromyosin, you will find that light meromyosin fraction 1, which is about 25/35 of the total light meromyosin is almost lo@ alpha helical. It is one of the few proteins which behave as a fully called alpha-helix. If a solution of light meromyosin is precipitated by dilution and the precipitated protein examined directly under a microscope, a very regular 430x periodicity appears. This is one of the characteristic periods present in muscle and it is now explained as representing the myosin periodicity. Therefore, qyosin may be considered as having a head and a tail, and these expectations have been borne out very nicely in the electron microscope by a number of investigators starting from Rice and Hugh Huxley and Zobel and Carlson and so on. I would like to examine some of these electron micrographs now to see how these molecules look before returning to discuss some of the possible steps which may occur during contraction.

The next slide is a summary of the properties and the molecular weights of heavy and light meromyosin. The molecular weight of heavy meromyosin is about 300,000 and that of light meromyosin is about 150,000. The heavy meromyosin is an AllPase and has all the ATPase activity of the 134. parent myosin, while the light meromyosin has the solubility properties of myosin but has no A!llPase-activity and no combining activity with actin. The next slide shows the 430Aperiodic structure of light 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 easily seen. The average length of the molecules is about 1600x or so, which agrees with the values obtained in solution. Actually the head portion plus a small part of the tail represents the heavy meromyosin part, and the rest of the tail portion is associated with the light meromyosin part.

Huxley also found that during precipitation of myosin molecules to form "crystals" a dimer forms. This dimer has the heads of the molecule facing the heads away from each other. The tail portions of the molecule like to aggregate and form structures. These portions tend to associate with each other starting to form filaments, with the heads of the filaments facing away from the center. There will be a central shaft which has no roughness or no head portions. That is, the filaments formed in this process will not contain heavy meronlyosin particles in the center of the filaments but the rest of the crystal, or filament, will have these heavy meromyosin protuberances. This is shown in the last slide where you see a growing myosin filament with the center portion of the filament smooth and free of the hemy meromyosin projections. Even though the filament grows in length, the center of the shaft will remain smooth, while the lateral portions away fromthe center will have these irregularities or these knobs, which probably correspond to the heavy meroqyosin. Thus, appearance of this filament is very similar to the thick filament, which occurs naturally in muscle. The present concept of Huxley is that the central part of the thick filaments are made up of the light meromyosin portion of myosin which bind together in some unknown but regular fashion, while past of the heavy meromyosin portion is sticking up fromthese filaments and is responsible for the combination of the thick filaments with actin (or thin filaments). The next slide shows some comparisons of filaments obtained from myosin with natural thick filaments 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 the double strand and the further subdivision of each strand of the filament into sub- units or particles which are associated with the mmomeric or globular actin Unit of 60,000.

The next slide is an electron micrograph of a thin filament taken by Huxley. I think between the two arrows you can see how the two strands are winding around each other to form the filament. In addition to the double helical structure of actin, Huxley has shown that there is some directionality within the actin thread, and that, I think, can be quite clearly seen in these regions 360x apart, where you see the crossing over of the two strands. These F-actin strands also show this directionality when mixed with heavy meromyosin. You will remember that the heavy meromyosin is the portion of myosin which interacts with actin. The next slide shows the head-like heavy meroIlryosin structures attached to one actin filament. You notice that the head-like structures all face one direction indicating some type of polarity or directionality of the actin. It appears that the actin filament, or the thin filament, faces in opposite directions on the two sides of the Z-band. 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 re- placed 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 136.

interact. That is to say, in muscle in rigor or in muscle at rest, the nucleotide associated with the aetin is unavailable for exchange with out- side ATP. During contraction or in systems undergoing superprecipitation of the actoqyosin system, the nucleotide bound to actin will be released and will pass into solution. We now think that there is a possibility--and this is a bare figment of speculation--that somehow the movement during contracticn may be mediated at the places of interaction of myosin with actin. Thus a local change in the structure of actin causes the thin filament to stretch after interaction with an active site of myosin and allows a neighboring active site to get in contact with myosin which can then also undergo this cycle of change.

In rather general terms, I could describe contraction in the following fashion: regardless of the actual function or actual mechanism of contraction muscle is really not well-informed in physics. It hasn't learned its lesson, and if you keep a chair or you try to hold a chair in an outstretched position, you get tired pretty fast, even if you don't do any work. The chair is not rnoving since you just hold it, but you still get very tired in a very short time. What it means is that even under isometric conditions when you don't do any work, the contraction process is going on. That is to say, cycles of interactions are occurring. The explanation of this phenomenon is unclear as is that of another even more surprising phenomenon, so-called fat effect, which says that the total energy mobilization of the muscle depends on the work it will have to do or, in other words, on the load which is put at the end of the muscle. The muscle will perform work to lift this load. It mobilizes a greater amount of energy than is required if it were to shorten in an unloaded condition. This has been shown physiologically in recent experiIzents on ATP liberation and proton liberation under such conditions. It is a very surprising thing that the molecules appear to know what is intended sometimes several lengths of molecules away. One possible explanation is the following. Let's assume that myosin filaments and actin filaments have different periodici- ties. It doesn't matter what the periodicity is just so long as they are not the same. To accomplish contraction, cross links will have to be established at certah sites. Regadless of what the other conditions are, these cross links will drive the two filaments apart. There is a structural change in the myosin or in the actin which may be of short duration, but in order to have contraction, the site from the myosin and the site fromthe actin must come together. The number of sites established will depend on the rate of the shortening. If there is more time available for establishing the site and the reaction, then the two systems move in respect to each other with a slower speed. This in turn means that more sites can be established. If you have an unloaded situation, the muscle will shorten quickly. In loaded condition it will shorten slower. So if the muscle is loaded, there will be time enough for more cross links or active sites to be established, and these active sites which will liberate more energy by means of ATPase cleavage of ATP.

I think I have exhausted my time, and you will have to forgive me if I was a little bit unclear for when you talk of too many things and you say too little about each, this is often the result. However, I thought I would try to bring you up to date from the very beginning, indicating how we think about these problems and how the physical characteristics and 137. chemical reactions will ultimately decide both the appearance and consistency of muscle, or if you want to call it, meat. Thank you very much.

DR. GOLL: Thank you, Dr. Szent-Gyorgyi, for that very excellent resume of the properties of the muscle lqyofibrillor proteins and some of the properties of muscle contraction. Dr. Szent-Gyorgyi made reference briefly in his talk to some of the problems which are inherent in the study of the muscle proteins. It appears that in many cases the methods available for the preparation and purification of the muscle proteins form the major obstacle to their study. Fibrous proteins in general present unique problems in methods for separation, purification and study. We are happy to have with us this mrning Dr. Harry Snyder of Iowa State University who will discuss some of the methods which are available for preparation, purification and isolation of fibrous proteins. Harry got his doctoral degree at the University of California at Daes and is now interested in the study of post-mrtern changes in muscle. The title of Dr. Snyder's paper this morning is "Methods Available to Separate, Isolate and Study Fibrous Boteins " .

DR. SNYIER: Thank you, Darrel.

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