Structure and Functions of Contractile Proteins: Revisions, Additions, and a Foreword to the English-Language Edition Prepared by the Author

By Boris F. Poglazov

Structure and Functions of Contractile Proteins focuses on the analysis of problems on the structure and functions of contractile proteins in which substantial progress has been achieved. The book first offers information on the protein constitution of myofibrils and myosin, including adenosinetriphosphatase activity, reaction with actin, and myosin molecule. The text also ponders on the polymerization of actin, tropomyosin, and the theory of contraction. Discussions focus on model experiments and molecular basis of contraction; structural interrelations of muscle proteins; features of the process of polymerization of actin; and size of the actin molecule. The text elaborates on the contractile proteins of the elementary motor structures of cells, as well as the chemical composition and physicochemical and enzymic properties of flagella and cilia; achromatin apparatus and movement of chromosomes; and structure of the flagella and cilia. Motor apparatus of bacteriophage and features of the movement of protoplasm and the mechanism of permeability are also discussed. The manuscript is a reliable source of data for readers interested in the structure and functions of contractile proteins.

• Language: English
• Publisher: Academic Press
• Release date: Jun 28, 2014
• ISBN: 9781483270869

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Introduction

For nearly a quarter of a century intensive research has been carried out to elucidate the mechanism of motor reactions in living organisms. An enormous, mass of factual data has been accumulated and definite progress has been made in the investigation of the energetics of the contractile process and of the relevant structural molecular changes. Most of the present knowledge relates to the motor function of animal muscles. It has been possible to isolate in purified state the contractile muscle proteins (actin, myosin, tropomyosin, etc.). The use of hydrodynamic methods, electron microscopy and x-ray structural analysis has made it possible to accurately define many properties of the molecules of these proteins and their interrelations. Investigation of the interaction of myosin and actin with ATP has received the most attention. The discovery by Engelhardt and Lyubimova of the ATP-ase activity of myosin initiated a major trend in biochemistry. It led to the enzymic cleavage of ATP by myosin and detailed study of the relation of the physicochemical properties of contractile protein to the presence of ATP. Research on another protein, actin, has been less active, and only in recent years have certain properties been found which appear to be important not only for the process of its polymerization but also for the contraction mechanism. We refer to the correlation of the polymerization process with the process of ATP cleavage and the manifestation by actin of the properties of a polyenzyme. The concept of the existence of a partially cleaved form of F-actin, formed during a study of the action of ultrasound on protein, led to the assumption that reversible transitions of a semicleaved and a restored form participate in the shortening of the sarcomere.

Investigation of the enzymic and physicochemical properties of muscle proteins in solutions has prepared the way for the study of their structural interrelations within the myofibril. The development of this trend was substantially advanced by the use of the electron microscope, with its high resolving power, and by parallel observation of the structure of proteins in muscle slices and in isolated state. Due to the brilliant work of Huxley, Hanson and others carried out at a high technological level, the exact location of myosin and actin in the myofibril has been ascertained, and additional data have been obtained on the location of tropomyosin and on the quaternary structure of actin and myosin. However, interpretation of the mechanism of the changes induced in protein structures during contraction presents certain difficulties which have not yet been overcome. Huxley’s hypothesis that the shortening of myofibrils is based on the sliding of actin threads along the myosin threads has been generally accepted. However, many facts are difficult to reconcile with this seemingly orderly scheme, especially since the studies of some authors give proof of a shortening of protein threads during muscular contraction. According to Podolsky, shortening of the sarcomere and disappearance of the I-disk occur as a result of the spiralization of actin threads. The divergences which emerge in the explaining of the experimental data cannot yet be removed inasmuch as the muscles represent a rather complex system in which it is difficult to grasp some of the details. Researchers are therefore turning increasingly to a more simply constructed motor apparatus in which it is easy to interpret the entire process. The organelles of motion of individual cells, namely flagella and cilia, are becoming objects of investigation. Astbury, performing an x-ray structural analysis of flagella, which he termed a monomolecular muscle, was the first to establish that the contraction is associated with transition of the α-configuration of protein into the β-configuration and the formation of transverse folds. In our opinion, the caudal covering of the bacteriophage, which represents the simplest possible motor system, is a true monomolecular muscle. Investigations carried out in our institute and in the Institute of Crystallography of the USSR Academy of Sciences have shown that the contraction of the caudal covering of bacteriophage T2 is due to shortening of the spi rally-wound protein strand and is associated with a specific rearrangement of the molecules. This process reflects the conversion of the α-configuration of protein into the transverse (×)β-configuration. There is every reason to suppose that molecular rearrangements of this type also occur in other motor apparatuses including the animal muscles, especially since, as has been shown by the findings of many investigators, the basis for supply of energy for all contractile movements is invariably the reaction of cleavage of ATP by a protein similar to actomyosin. Thus, while the problem of the structural changes occurring during contractions and of the need for ATP for the execution of work is somewhat clearer, the next task must be a study of the mechanism of energy transfer from ATP to the contractile protein. Particular attention should be given to the intimate reaction coupling ATP to the active center of the enzyme. The SH groups of myosin and a quaternary magnesium chelate of adenosine triphosphoric acid participate in this reaction. This leads to the formation of a mobile system whose constitution includes double bonds of purine with their labile π-electrons and free pairs of electrons of oxygen atoms in phosphate groups, which creates preconditions for enzymic hydrolysis. This process has so far been studied only rather cursorily, and therefore any new data are of great interest.

Contractile proteins are widely distributed in nature and appear to be present not only in motor organs but in any living cell. They have recently been isolated from brain, liver, thyroid, pancreas, etc. Of course, the contractile proteins present in the different animal and plant cells are not identical on account of differences in the conditions and in the specific nature of the process in which they participate, but they are similar in the main enzymic and physicochemical properties. They can thus be discussed as a group, termed actomyosin-like proteins.

The function of the actomyosin-like proteins concerns not only the motor process but also the regulation of phenomena of tissue and membrane permeability. Evidence of this is seen in studies on the swelling and dehydration of mitochondria, on the isolation of a contractile protein from kidneys and on the active transport of ions across membranes in tissues and individual cells.

Due to improvements in electron-microscopic technique and the ever increasing use of x-ray analytical methods in biochemical investigations, questions of the structural organization of contractile proteins at the molecular level are now of primary importance. The greatest advances have been achieved in biochemical investigations associated with study of structure. The question of structural organization is therefore given special attention in this book, and the illustrations are from the most recent electron microscopic investigations.

Some years ago two excellent Russian monographs were published (Ivanov, 1950; Ivanov and Yur’ev, 1961) relating to investigations of contractile systems. The present work is the logical continuation of these two papers.

1

The Protein Constitution of the Myofibrils

Publisher Summary

This chapter focuses on the development in the understanding of the protein constitution of the myofibrils. On many instances, the protein has been isolated by different authors but given different names. Contractin, Δ-protein, meta-myosin, Y-protein, extra protein, and others are similar in many respects, although they have certain distinguishing features. The discovery of these often very similar proteins was probably because of the ability of the muscle proteins to form complexes. In one case, there is predominance of one component, while there is less of it in another. The properties of these proteins are also influenced to some extent by their interaction with myosin, actin, and tropomyosin.

Investigations of muscle proteins and study of their role in muscular contraction started in the middle of the 19th century. The ability of the muscle proteins to form complexes possessing diverse properties depending on the ratio of their constituent components at first led to great confusion, and all sorts of discoveries and rediscoveries of muscle proteins followed.

For a long time the belief prevailed among scientists that muscle proteins were identical with the fibrin of the blood. In 1864 Kühne proposed a method of extracting the myoplasm from frozen muscles with solutions of neutral salts (10% NaCl). This made possible the study of the properties of the extractable muscle protein which Kühne called myosin. Other authors, engaged in investigating muscle proteins, made extensive use of the technical procedures of Kühne, but erroneously identified muscle proteins with blood fibrin and used the inadequate method of protein separation by coagulation at various temperatures. As a result, by the early 20th century, about ten muscle proteins had been found (Il’in, 1900). It was subsequently revealed that many of these proteins differed only in name and were in actual fact the same substance.

A great contribution to the development of muscle biochemistry was made by Danilevskii. In contrast to Kühne, who looked for similarities between the muscle and the blood proteins, Danilevskii made a study of the muscle proteins as vehicles of the specific function of muscles–contractility (Danilevskii, 1881, 1882, 1888; Danilevskii and Shipilova, 1881). By extraction of muscle proteins with water, solutions of salts, alkalis and acids, he distinguished three fractions of muscle proteins: (1) proteins extractable with water; (2) the protein myostromin, extractable with solutions of acids or bases; (3) the protein myosin, extractable with solutions of salts (6-12% NH4Cl).

While performing extraction of muscle proteins, Danilevskii studied the associated changes in the histologic picture of muscle. He found that myosin is a component part of the anisotropic disks. The light birefringence (LBR) of these disks disappeared progressively as the myosin was extracted. Danilevskii then treated the stroma remaining after extraction of myosin with solutions of alkalis or acids. In this process the protein which Danilevskii termed myostromin entered into the solution. Studying the quantitative relations of myosin and myostromin in muscles possessing diverse contractile ability, he reached the conclusion that myostromin played the main role in the contraction process. This exaggeration of the importance of myostromin was due to the incompleteness of data available at that time, and with the subsequent expansion of knowledge about the properties of muscle proteins, particularly the interrelation of myosin with ATP, this viewpoint was abandoned.

In 1942-1943 Straub found that the myosin obtainable from muscles by prolonged extraction occurred in combination with actin, another muscle protein. This combination was called actomyosin. It was found that actin can occur in two forms: a globular form (G-actin) and a fibrillar form (F-actin). Fibrillar actin is formed as a result of polymerization of G-actin. The monomers are the kinetic units of G-actin, and during polymerization they group into dimers (Tsao, 1953a). A characteristic property of actin is the ability to interact with myosin.

Subsequently (1948) Bailey isolated another protein component of muscle and named it tropomyosin on the supposition that it was a prototype of myosin. In some ways (amino acid composition, LBR) this substance resembles myosin, but it has a number of properties which differentiate it substantially from myosin and probably has independent significance.

The proteins myosin, actin and tropomyosin are the main muscle proteins. Quantitatively they constitute 80-90% of the total content of proteins in the myofibrils (Perry, 1956a; Hanson and Huxley, 1955; Ivanov et al., 1959). A number of investigators have attempted to isolate and characterize other protein fractions of myofibrils. Dubuisson, using the method of electrophoretic partition, found that if extraction is performed with saline solutions on muscles in the relaxed state, the proteins actomyosin and myosin enter the solution (Dubuisson, 1948). On electrophoretic partition they give two well defined peaks: an α-peak (actomyosin) and a β-peak (myosin). If the extraction is performed on muscles that have been preliminarily contracted by means of monobromoacetate or other means, the amount of myosin and actomyosin entering into solution decreases markedly, and in their place one finds in the extracts a new slowly moving protein fraction which Dubuisson (1948, 1950a) called contractin. During contraction the binding of myosin with the myofibril becomes stronger, while the interaction of contractin with the stroma weakens (Crepax, Jakob and Seldeslachts, 1950). Kay and Pabst in 1962, using the procedure of sedimentation analysis and light scattering, found the molecular weight of contractin (γ-myosin) to be 49,000. Their investigation of the dispersion of optical rotation of contractin solutions showed that 85% of the molecules of this protein have a double-stranded spiral structure. Keeping in mind that contractin’s properties are similar to those of L-meromyosin and that its content is measured in cases of muscular dystrophy (Azzone, Aloisi, 1958; Azzone, 1958), Kay and Pabst suggested that it might be a product of degradation of myofibrillar proteins. Contractin is apparently identical with γ-myosin (Dubuisson, 1950b), which was isolated at about the same time. In succeeding studies the properties of contractin were examined in greater detail. Schapira, Marcaud-Raeber and Dreyfus in 1957 determined the constants of its electrophoretic mobility and viscosity. It was shown that, unlike myosin, it has no ATP-ase activity and does not react with actin.

Also similar to contractin is metamyosin, isolated by Raeber, Schapira and Dreyfus in 1955. The sole difference consists in its lower electrophoretic mobility, which could be due to its degree of nativity or to the presence of bound impurities. When relaxed muscles are treated with a solution of potassium chloride in concentrations greater than 0.5 M, Y-protein is extracted in addition to myosin and actomyosin (Dubuisson, 1950b, 1950c). On the electrophoregrams the peak corresponding to this protein clearly lags behind that of myosin. The Y-protein was separated from myosin and actomyosin by fractionation with ammonium sulfate. A 35% saturation with (NH4)2SO4 precipitates actomyosin and myosin, while a 37-40% saturation precipitates Y-protein. It is insoluble in distilled water, but will dissolve in solutions with μ = 0.005 at pH 7.9. Solutions of Y-protein have low viscosity; Y-protein lacks the LBR characteristic of many muscle proteins; its optical properties do not depend on the ionic strength. The fact that a hundred times greater concentration of KCl solutions is needed for extraction of this protein than for its entry into solution when already in isolated state indicates its firm link with the muscular stroma. This link is still further strengthened if the muscle happens to be contracted. Under conditions of monobromacetate-induced contraction or postmortem contracture of muscles, neither Y-protein nor myosin is extractable by KCl solution; KI or pyrophosphate must be used to release Y-protein from the myofibrils.

The family of fibrillar proteins of the α, β, and γ series has been extended by the discovery of Δ-protein by Amberson et al., in 1957. Δ-Protein was isolated in pure form by alcoholic fractionation or by the use of Salyrgan for cleavage of the complex of Δ-protein with myosin. The final stage was preparative electrophoretic partition. Nevertheless, the protein fraction so obtained was not homogeneous. Electrophoregrams showed two or sometimes three different peaks (White et al., 1957). The authors suggested that one of them corresponds to tropomyosin, and the other to Δ-protein. The latter is soluble at low ionic strengths, and high concentrations of salts cause it to precipitate. Solutions of Δ-protein have high viscosity and LBR. Electron microscopic observations have shown that filaments prepared from Δ-protein have a periodicity of 165 Å. One of the most characteristic properties of Δ-protein is its ability to form a complex with myosin (Δ-myosin). The presence of the complex was demonstrated by electrophoresis and ultracentrifugation; the electrophoretic mobility and the sedimentation constant of Δ-myosin were intermediate between the values for mypsin and Δ-protein (Bensusan et al., 1957). The addition of Δ-protein to a solution of actomyosin causes a decrease in viscosity because of dissociation of actomyosin and the formation of another complex, namely Δ-myosin. In many respects, Δ-protein is similar to tropomyosin. There is reason to suppose that Δ-protein is a polymerized tropomyosin. Both proteins have identical electrophoretic mobility, a similar absorption spectrum and close isoelectric points, are readily soluble in water and extractable with high concentrations of salts, and are relatively thermostable. However, certain differences between them indicate that Δ-protein and tropomyosin are qualitatively different proteins. For example, a solution of Δ-protein possesses LBR under conditions where tropomyosin does not; Δ-protein can form a complex with myosin, and tropomyosin cannot. The properties of Δ-protein also differ from those of actin and myosin.

Szent-Györgyi, Mazia and Szent-Györgyi (1955) used KCl solutions of high ionic strength in conjunction with ATP or pyrophosphate for extraction of proteins from myofibrils. Under these conditions there occurred extraction of myosin and somesort of protein which according to Corsi (1957) accounted for 7% of the total amount of proteins of the myofibrils. This so-called extra protein extracted from muscles was soluble in KCl in a wide range of concentrations. Given a low ionic strength of the medium, the extra protein is capable of polymerization accompanied by increase in the viscosity of the solution. Villafranca (1956) showed that a change in concentration of KCl from very low values to 0.6 M altered the characteristic viscosity of the protein 10- to 16-fold. He found that the molecular weight of a particle was 447,000 for the polymerized protein and 155,000 for the depolymerized protein. Corsi (1957) performed electrophoretic analysis of the extra protein fraction and discovered three components. The most rapidly moving and quantitatively largest fraction apparently corresponded to tropomyosin, since they had identical solubility and electrophoretic mobility. The nature of the other two components is unknown, but it may be supposed that one of them corresponds to the γ-myosin of Dubuisson (Dubuisson, 1946, 1950a) and the other to Y-protein (Dubuisson 1950b, 1950c). The conditions of their extraction, solubility, etc, were similar. For more precise conclusions it is necessary to compare the amino acid composition of the individual components of the extra protein with that of tropomyosin, γ-myosin and Y-protein.

In contrast to the authors just mentioned, who used solutions of high ionic strength for extraction of proteins, Perry in 1953 performed a prolonged extraction of isolated myofibrils with 0.078 M borate buffer of pH 7.1. A protein fraction consisting of two components, viz. tropomyosin and an unknown protein, entered into the solution. The viscosity of the protein was low (Perry, 1956a, b). Perry postulated that this was some inactive form of actin. A very similar protein was isolated by Tsao et al. (1959), but the method used for isolating it was different (extraction at pH 5.1 in the presence of 0.5 M NaCl followed by precipitation of the protein with ammonium sulfate at 80% saturation and dialysis). The degree of asymmetry of the isolated protein was 1/6; this molecule was less asymmetric than the molecule of myosin, F-actin or tropomyosin. Its sedimentation constant was 6.3 S. The protein was soluble in water. Like the protein isolated by Perry, it had lower electrophoretic mobility than tropomyosin. This protein differed from actin in its properties, but its amino acid composition was similar.

This sums up our knowledge of the protein composition of myofibrils. All the proteins just discussed (except for myosin, actin and tropomyosin), occur in muscle in small quantity and have not so far been very completely studied. It is clear that in many instances the same protein has been isolated by different authors but given different names. Contractin, Δ-protein, metamyosin, Y-protein, extra protein and others are similar in many respects, although they have certain distinguishing features. Probably the discovery of these often very similar proteins was due to the ability of the muscle proteins to form complexes. In one case there is predominance of one component, while in another there is less of it, etc. The properties of these proteins are also influenced to some extent by their interaction with myosin, actin and tropomyosin. These three latter substances account for the main bulk of myofibrils, their properties have been fairly thoroughly investigated, and we now have some knowledge about their role in muscular contraction. In this treatise, therefore, attention will be directed mainly to analysis of the properties of these proteins and consideration of their interaction during the contractile