Structure and Function of Sarcoplasmic Reticulum

By Sidney Fleischer

Structure and Function of Sarcoplasmic Reticulum is a compendium of papers from an International Conference on Sarcoplasmic Reticulum held in Japan on November 1-4, 1982. Section I is a review of sarcoplasmic reticulum including the "discovery" of the relaxing factor, the calcium binding of relaxing factor, as well as phosphate transfer and calcium transport coupling. Section II involves the chemistry and structure of the calcium pump protein in sarcoplasmic reticulum. One paper describes the role of protein-lipid interactions in the organization and function of biomembranes. Section III considers the kinetics and thermodynamics of the calcium pumping mechanism, particularly the binding of ligands to calcium ATPase of the sarcoplasmic reticulum, as well as the conformational changes of the sarcoplasmic reticulum Ca-ATPase induced by substrate binding and phosphorylation. A paper gives the results of several experimental techniques in substrates binding assays employing millipore filters and a thermostated filtration apparatus. Section IV describes the calcium ions release process such as rapid and reversible actions, while Section V discusses the regulation of calcium ions uptake and release in the ion channel vesicles. This book can be helpful for researchers in biophysical engineering, pharmacologists, and scientists in the fields of biochemistry and biophysics.

• Language: English
• Publisher: Academic Press
• Release date: Oct 22, 2013
• ISBN: 9781483277448

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YUJI TONOMURA 1923 – 1982

YUJI TONOMURA 1923 – 1982

At the height of his career (when he died), Yuji Tonomura belonged to two worlds: that of contractility and that of ion transport, but his first love was contractility. It all began in the early postwar years. Though bedridden with tuberculosis that was nearly fatal, he collaborated with his lifelong friend Watanabe to produce the first analytic treatment of actomyosin ATPase and turbidity. Their paper appeared in Nature, but went unnoticed (it was much too early for enzyme kinetics studies). Thus a pattern that was thereafter to plague Yuji and distress his friends was set: delayed recognition. Tonomura did not suffer mathematical illiterates, and was not fond of popularizing his work. His thoroughly honest criticisms were seldom softened to mollify their targets. His relentless logic, complex graphs, and mathematical reasoning made him difficult to read and to listen to. All these attributes contributed to his delayed recognition. But in time we learned that it was unwise and self-defeating to ignore him. The importance of one of his greatest contributions—the discovery of the ADP·Pi intermediate–escaped the grasp of Western scientists for at least five years. Collectively, we were unable (or unwilling to study his work. Appreciation of his other major contributions was delayed because his experiments were ahead of their time. In 1964 he seemed to be saying, If I can do with organic solvents what you can do with thiol reagents, then we must both be producing a change in conformation. The statement seemed a little mystical at the time, but twenty years and countless papers later, most of us now think that activation results from conformation distortion, not from blocking thiols.

Yuji was an accomplished physical biochemist, but his associations with organic synthesists were exceptionally fruitful. It was with Kubo that he discovered the reactive lysine of myosin and with Ikehara that he produced a wonderful family of ATP analogs tailored to track substrate movement. The ramifications of these discoveries continue to this day. Measurements of fluorescence energy transfer in muscle proteins are now rather fashionable, but one of the cleverest examples remains the Tonomura–Onishi study of the proximity of the tryptophan active site. In the last decade of his life, Tonomura was persuaded to visit America on short sabbaticals, each of which had beneficial results. First, his health invariably improved during each visit (a fact he attributed as much to being able to escape the Osaka tensions as the Osaka weather). Second, a number of beautiful contributions resulted [the first (and only) number average measurement of myosin and the EPR method for tracing the effects across oligomeric systems, experiments arguing for the importance of rotation in free energy transfer, etc.]. Third, a canard, widespread among his students, was repeatedly refuted. Although Yuji often shared a Kirin or a Suntory with his students, they never forgot that he was the Sensei. As is common in such a relationship, legends about him passed from one generation of students to the next. One of these was that although the Sensei was brilliant in direction, he was absolutely hopeless at the bench. It was a constant pleasure to be able to prove to these young students that in fact Yuji was a highly skilled experimenter who produced many neatly filled books with precise data. Tonomura’s irreverence for symposia was unaffected by his mounting international recognition. In 1981 he was invited to a meeting in Paris, attended only by the best of the world’s muscle researchers. Well, it’s my long-awaited chance to see the Louvre, he told his congratulating friends. He himself provided the research community with two notable symposia: the 1980 Myosin Conference in Sapporo, and the 1982 meeting on The Structure and Function of Sarcoplasmic Reticulum, the proceedings of which appear in this volume.

The life of a man who is both admired and loved by his friends always seems to end too soon, particularly in terms of tasks left undone. For several years prior to his death, Tonomura had been grappling with a difficult problem: Are the two heads of a myosin molecule unsymmetrical? Settling the issue would have far-reaching consequences for contractility. As usual, it was Tonomura’s group against a disbelieving world. Progressively, those of opposite beliefs began to relent under the pressure of experimental results. Researchers began to concede that, yes, there are different heavy chains in a myosin preparation. And, having failed to find more than one kind of heavy chain in smooth muscle, Yuji quickly conceded that the nonequivalence, could not be fundamental. At this time it seems that the resolution may lie in isozymes, but it is such a pity that Yuji did not live to settle the issue himself.

Tonomura’s second productive love was ion transport. Early studies in collaboration with Yamamoto described the formation of a phosphorylated enzyme intermediate in the mechanism of ATP utilization by the calcium-dependent ATPase. Thereafter, in collaboration with Yamada, he reported that enzyme phosphorylation with Pi in the presence of a transmembrane calcium gradient is an early event in the reversal of the calcium pump. He then proceeded with Kanazawa to describe the kinetics of formation and breakdown of phosphorylated enzyme intermediate at low temperature, and in collaboration with Sumida noted the phenomenon of calcium translocation accompanying enzyme phosphorylation. His contributions extend to the Na+, K+-ATPase for which he defined, in collaboration with Yamauchi, cation binding parameters.

Tonomura’s work in the field of ion transport and coupled enzyme reactions retained the complexity and sophistication which was inherent in his work. Nevertheless, the tangible and accessible nature of the phosphorylated enzyme intermediate soon led to the acceptance of his work. Many of us have been inspired by his ideas and experiments.

In addition to his direct scientific contributions, Tonomura’s training of illustrious researchers is an example of the extraordinary influence a university professor can have not only in his own nation but internationally. Those of us who were fortunate to work with him in the same laboratory and to discuss ideas, methodology, and experimental results, know well the pervasive extent of his influence, and understand how it was possible for Professor Tonomura to inspire such a large school of excellent students.

Finally we like to recall Yuji, the human being and friend, his keen appreciation for Eastern and Western art, the benevolent pride in his own accomplishments, his serene conversation late at night over a beer, his humor and sense of values. Yuji’s memory will continue to inspire and encourage us in the future.

MANUEL MORALES and GUISEPPE INESI

Section I

Historical Background and Overview

Outline

Chapter 2: THE SARCOPLASMIC RETICULUM CALCIUM PUMP: EARLY AND RECENT DEVELOPMENTS CRITICALLY OVERVIEWED

RELAXING FACTOR, SARCOPLASMIC RETICULUM AND TROPONIN

A HISTORICAL SURVEY

Setsuro Ebashi,     Department of Pharmacology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113

Publisher Summary

This chapter presents the schematic illustration of the effects of ionic strengths and adenosine triphosphate (ATP) concentrations on the response of actomyosin system to ATP in the presence of enough Mg ion. In 1948, Kielley and Meyerhof published a paper entitled A new magnesium-activated adenosinephosphatase, and it was found that the properties of fraction and the new enzyme were very much alike. Both were precipitated by 20 grams per 100 ml of ammonium sulfate (0.35 saturation) and looked opalescent. The preparation obtained by Kielley and Meyerhof was certainly active as the relaxing factor. The papers of Kielley and Meyerhof described nearly all the properties of the microsomal enzyme that is now called Ca-transport ATPase: (1) the enzyme is a particulate fraction, (2) it is activated by Mg ion, its optimum being 4mM and is activated by Mn to a lesser extent, (3) it contains a considerable amount of lipid, particularly lecithin, and (4) Clostridium welchiilecithinase abolishes its activity in parallel with the disappearance of lecithin.

INTRODUCTION

When we look back on the history of biological sciences, there can be seen several cases where important new biological concepts were first developed from studies on muscle and then generalized. Ca ion, which is now accepted as the most fundamental mediator of cellular functions in general, is a typical example.

Fig. 8 Linear relationship between Ca binding capacities and relaxing activities on glycerinated fibers of various chelating agents.

For abbreviations for chelating agents see Table 1. The experiments were carried out in the solution containing 0.15M KC1 0.01M MgCl2 and 0.02M Tris-maleate buffer (pH 6.8). Ca binding capacities were calculated taking the interference by Mg ion into consideration. DTTTA, N’,N- Dimethyltriethylene tetramine-N,N’ -tetraacetic acid, was kindly supplied by Prof. Schwarzenbach, which was not included in the previous experiment shown in Table I (18). DTTTA has virtually no affinity for Mg, weaker than GEDTA, so that, in spite of its rather weak affinity for Ca, it shows a relatively strong Ca binding in the presence of a high concentration of Mg ion (26). (quoted from ref. 18).

In 1950’s almost all muscle biochemists were enthusiastically engaged in the research on the myosin-actin-ATP interaction in vitro. The studies on the relaxing factor were not considered to be on the royal road. This factor, however, identified with the sarcoplasmic reticulum and related to Ca ion. Subsequent effort to clarify the mode of action of Ca ion was led to the discovery of the third factor other than myosin and actin, i.e., the troponin-tropomyosin system, the regulatory system of muscle contraction motivated by Ca ion.

Nearly twenty years have passed since I left the field of sarcoplasmic reticulum research. During this time remarkable progress has been made and now I feel myself quite a stranger. I am thus not qualified to give a true historical survey.* Instead, I will describe my personal experiences on how I became interested in studying the ‘relaxing factor’ and how I left the field of sarcoplasmic reticulum. Emphasis will be placed on my failures rather than my success through good lack.

DISCOVERY OF THE RELAXING FACTOR

In 1947, Professor H. Kumagai, my teacher, suggested that I should work on smooth muscle pharmacology with an electrophysiological approach, and I spent several vain years on this. In the meantime, I had a chance to read a humble mimeographed copy of the book, Chemistry of Muscular Contraction, 1947, by Szent-Györgyi (4), which excited me greatly. Naturally, I wished to start immediately working on the actomyosin system, but my lack of biochemical training compelled me to postpone beginning until early in 1952. At that time I was able to get the second edition of the book (4), in which I found the preparation method of ‘glycerol-extracted psoas’. This experimental material was much more familiar to a pharmacologist than actomyosin itself. So I began preliminary experiments in collaboration with K. Fujita.

For a short while we enjoyed the beautiful contraction of glycerinated rabbit psoas induced by ATP. Soon we noticed that muscle fibers, shortened by ATP, never relaxed even after ATP removal. This was not consistent with the traditional concept of muscle contraction, one of its outstanding characteristics being its reversibility. As a pharmacologist acquainted with the drug action on living muscle, I could not overlook this puzzling fact. Incidentally, there appeared then a very interesting paper of Bozler (5), which showed that the ATP-contracted glycerinated fibers could be relaxed by a higher concentration of ATP.

However, there was no possibility for such a prompt increase of ATP concentration in muscle cells and, therefore, this could not be a physiological mechanism of relaxation. After many failures I became suddenly aware of our daily experiences with fresh glycerinated fibers which tended to relax even with a relatively low concentration of ATP. This might indicate that a soluble intrinsic factor to induce relaxation should have been retained in fresh fibers (we know today that this interpretation was not right; gradual decrease of relaxing tendency was mainly due to the deterioration of the sarcoplasmic reticulum with elapsed time). So we prepared a simple muscle extract and applied it to ATP-contracted glycerinated fibers. As a result, clear relaxation was induced — it was a dramatical success (Figs. 1–4).*

Fig. 1 Relaxation of glycerinated rabbit psoas by ATP in the presence of muscle extract (m.e.)

A piece of muscle was homogenized, centrifuged at a moderate speed, and the resulting supernatant was used as m.e. Glycerinated psoas was soaked in a solution containing 0.15 M KCl and 10 mM MgCl2, and the contraction was started by adding ATP to the solution. Shortening of the fibers was recorded by a light lever on a smoked drum (time in minutes). Note that the relaxation by a moderate ATP concentration in the presence of m.e. is far more pronounced than that by a high ATP concentration without m.e.

Fig. 2 Reversible contraction-relaxation cycle of glycerinated muscle fibers by ATP with the aid of ‘muscle extract’.

a: ATP, 5 mM in final concentration, was added. b: ATP, 5 mM, plus ‘muscle extract’ were added. c: the bath solution was exchanged with the solution containing no ATP and ‘muscle extract’. For others see the legend to Fig. 1.

Fig. 3 Reversal of relaxation into contraction by Ca ion.

a: ATP, 5 mM in final concentration, plus ‘muscle extract’ were added to the solution. b: CaCl2, 1 mM in final concentration, was further added. c: CaCl2, 0.5 mM, was further added. For others see the legend to Fig. 1.

Fig. 4 Enhancement of relaxing action of ‘muscle extract’ by phosphate.

a, b: see the legend to Fig. 2. c: CaCl2, 2 mM in final concentration, was added. d: Sodium phosphate, 30 mM in final concentration, was further added. Oxalate showed a stronger effect, but emphasis was laid on phosphate because we thought at that time that this might be somehow related to physiological mechanism. It is worthy of note that Kamada was interested in phosphate, ascribing some physiological role to it (2). For others see the legend to Fig. 1.

A few months after our observation, however, Prof. Kumagai notified me of a brief communication in Nature, in which Bendall (6) demonstrated an experiment very similar to ours; furthermore, we learned that the factor itself had been discovered already in 1951 by Marsh in Bailey’s laboratory (7).

If it were nowadays, I should have abandoned the whole project, greatly disappointed, regretting my lack in getting up-to-date information in advance. But the situation in which Japanese scientists were placed was terribly depressed at that time, so I instead took some degree of satisfaction even with this now meaningless result and continued the effort to isolate the active principle from the extract with the collaboration of F. Takeda (9, 10).

Our work had thus been almost in vain, but one little fruit was that we have deepened our understanding of the actin-myosin interaction in the presence of ATP as expressed in Fig. 5, which was indebted to original profound thoughts of Szent-Györgyi (4) and Bozler (5).

Fig. 5 Schematic illustration of the effects of ionic strengths and ATP concentrations on the response of actomyosin system to ATP in the presence of enough Mg ion.

AM: contracted state. A + M: relaxed state (myosin and actin are dissociated). At that time we did not know that MgATP, not ATP, was the substrate for the actomyosin system; the presence of Mg ion was considered to be necessary for maintaining the actomyosin system in the active state. So we used ‘ATP’, which was later replaced by ‘MgATP’.

In the meantime, there appeared several papers asserting that the factor should be an ATP-regenerating system such as creatine kinase-phosphocreatine system (11) or myokinase (12). We could not agree with this idea; for instance, the active fraction obtained by ammonium sulfate fractionation was different from the fraction of such an ATP-regenerating system (Fig. 6) (10, 14).

Fig. 6 Cooperation of two fractions derived from ‘muscle extract’ in inducing relaxation of glycerinated psoas.

Experiments were carried out in the solution containing 0.15M KC1 and l0mM MgCl2. Fr. A was the fraction precipitated by ammonium sulfate between 10 and 20 g per 100 ml, the main factor of ‘muscle extract’, being identifical with Kielley-Meyerhof enzyme. Fr. B, precipitated between 30 and 40 g per 100 ml contained myokinase, which explained the effect of Fr. B to some extent, but in the case of fairly well washed glycerinated fibers, myokinase could not replace Fr. B (see Fig. 2 in ref. 13). This puzzling problem has not yet been clarified. (quoted from ref. 13).

KIELLEY-MEYERHOF ENZYME

One day, I happened to come across the papers by Kielley and Meyerhof published in 1948 (14, 15), entitled A new magnesium-activated adenosinephosphatase, and found that the properties of our fraction and the ‘new’ enzyme were very much alike. Both were precipitated by 20 grams per 100 ml of ammonium sulfate (0.35 saturation) and looked opalescent. The preparation obtained by Kielley and Meyerhof was certainly active as the relaxing factor. We prepared the factor only by high speed centrifugation, another important step of purification process of Kielley-Meyerhof enzyme, omitting the step of ammonium sulfate fractionation; the enzyme thus prepared was active as the routine preparation, or sometimes better than the latter, and could retain its activity for a much longer time. We were now convinced of the identity of the two preparations, the relaxing factor and Kielley-Meyerhof’s enzyme — both were the same (Fig. 6) (13, 16).

Indeed, the papers of Kielley and Meyerhof described nearly all the properties of the microsomal enzyme which is now called Ca-transport ATPase:

i) The enzyme is a particulate fraction (14, 15).

ii) It is activated by Mg ion, its optimum being 4mM, and is activated by Mn to a lesser extent (15).

iii) It contains a considerable amount of lipid, particularly lecithin (17).

iv) Clostridium welchii lecithinase abolishes its activity in parallel with the disappearance of lecithin (17) (Fig. 7).

Fig. 7 Relationship between inactivation of ATPase activity of Kielley-Meyerhof enzyme and liberation of phosphate from the enzyme by Clostridium welchii lecithinase. ‘I’ contained the results of five series of experiments and ‘II’ showed those of a single experiment (the curve was shifted to the right side to indicate the uniformity of the results). This was the first experiment to show the involvement of phospholipid in enzymatic action. Later it was shown that Clostridium welchii lecithinase could abolish relaxing activities (16). (Kielley and Meyerhof; quoted from ref. 17).

The Ca-transport enzyme or Ca-Mg-ATPase was thus already discovered in 1948 (14, 17). What we did later was only to relate the enzyme to its physiological roles such as the relaxation (13, 16) or Ca uptake (18–20>). I am puzzled why most research workers in this field have ignored this monumental work by Kielley and Meyerhof.

SOLUBLE RELAXING FACTOR AND EDTA RELAXATION

The next step of the research was naturally to inquire into the mechanism of how the particulate enzyme would exert its physiological activity. Most researchers thought that a soluble ‘true relaxing factor’ of a small molecular weight must have been produced from the particulate fraction (cf. 21). The ambition of muscle biochemists was to be the first to isolate such a fascinating substance. I, however, was for no particular reason not happy with this idea.

In the meantime, Bozler (22) in 1954 and Watanabe (23) in 1955 independently reported that EDTA could induce the relaxation of contracted glycerinated fibers. This should have given a chance to Japanese scientists to reveal the secret of ‘relaxation’, because a pharmaceutical firm in Japan (Dojin Pharmaceutical Company) had been producing various kinds of chelating agents, originally invented by G. Schwarzenbach; GEDTA (glycoletherdiaminetetraacetic acid, EGTA) was naturally included among them. However, my lack of thorough consideration and some erroneous information caused me to miss this favorable opportunity (24) (Table I). I also tried to determine Ca uptake by the Kielley-Meyerhof enzyme in the presence of ATP, but colorimetric analysis by my poor hand did not allow me to detect any positive result (unpublished data).

TABLE I

Relative affinities for Ca and Mg ions and relaxing activities of various chelating agents.

: relaxing action

*this value was derived from erroneous information and should read 0.03. Considering this point and the Mg interference with Ca binding capacities, we then found the linear relationship between Ca binding capacities and relaxing activities as shown in Fig. 8.

(quoted from ref. 24).

This seemingly hopeless situation caused me to become very depressed and I wished to make a change, so early in 1958 I wrote to Prof. Fritz Lipmann in the hope that I could learn up-to-date biochemistry. His answer was that I should continue my work.

One midnight in Lipmann’s laboratory, I was wondering why GEDTA was so effective as a relaxing agent (Table I). Suddenly I felt something was wrong. So I dashed to the library to consult the original book (English version) of Schwarzenbach (25) and found that a previous information about the binding constant of GEDTA for Mg was erroneous. Corrected results now revealed beautiful relationship between Ca binding capacities and relaxing activities (18).

CALCIUM BINDING OF RELAXING FACTOR OR SARCOPLASMIC RETICULUM

There was then no doubt, I thought, that the relaxing factor, the Kielley-Meyerhof enzyme, should bind Ca ion utilizing the energy of ATP. Prof. Lipmann generously gave me permission to test my bold idea. I worked hard to carry through within a rather short term the project suggested by him, i.e., the ATP-ADP exchange reaction of the enzyme (Fig. 9), which reflects its most fundamental nature. In the meantime, I had the pleasure of becoming acquainted with A. Weber, who had detailed experimental evidence which made her sure that the inhibitory action of EDTA on the ATPase of myofibrils was due to its Ca binding (26). Eventually the day came to carry out the experiment, cherished in mind for months, early in June, 1959. The result was just expected; this was only experience in my life in which the result completely lived up to my expectation (Fig. 10).

Fig. 9 Effect of deoxycholate on the ATP-ADP exchange reaction of the relaxing factor (Kielley-Meyerhof enzyme). Prof. Lipmann had predicted the association of rapid ATP-ADP exchange reaction with this membraneous ATPase, which clearly indicated the presence of phosphorylated intermediate of the enzyme. He was interested in the question whether or not deoxycholate, which could abolish the relaxing action and Ca uptake of the enzyme, would uncouple the exchange reaction from the ATPase reaction. Arrow indicates the deoxycholate concentration that abolished the Ca uptake. Since this concentration of deoxycholate partially solubilizes the membrane ATPase, the above result implies many suggestions even nowadays. (quoted from ref. 19).

Fig. 10 Ca uptake of the relaxing factor.

The suspension of the relaxing factor in the solution containing 0.01 mM CaCl2, mixed with 42Ca, was spun down. The radioactivity in the pellet relative to the total was considered to indicate the amount of Ca contained in the relaxing factor, or fragmented sarcoplasmic reticulum. Since I was very anxious to know the results as soon as possible, I was bold enough to carry out this experiment without any preliminary tests. For further detail, see the original paper (19). Inlet: Triad-like structure found in the relaxing factor. The electron micrograph of the relaxing factor was composed almost exclusively of vesicular structures, in which we could occasionally see triad-like structures like the above. (quoted from ref. 19, but modified for this article).

Everything then went very well. Every datum supported the Ca concept (27). Even such a low Ca ion concentration as 0.2 µM could exert a definite effect on the superprecipitation of well-washed natural actomyosin (myosin B) a few µM of Ca ion completely restored its contractility (Fig. 11). It was quite an easy task to construct the whole picture of the physiological contraction-relaxation cycle (27a). I thought that everything would have been solved and everyone would agree with me, but the situation was not so simple.

Fig. 11 Superprecipitation of EDTA-washed natural actomyosin at varied concentrations of Ca ion.

Natural actomyosin prepared by ordinary method was carefully washed with EDTA-containing solutions to remove bound Ca and then with EDTA-free solutions to remove EDTA. Superprecipitation was carried out in the solution containing 0.06 M KC1, 1 mM MgCl2 0.02 M MgCl2, 0.02 M Tris-maleate buffer (pH 6.74) and a certain concentration of Ca ion indicated on the curve in µM. (quoted from ref. 27).

DISCOVERY OF TROPONIN

In 1962, a big symposium was organized by Dr. John Gergely in Dedham (21). All the data (cf. 21) further convinced me as well as A. Weber of the Ca concept, but contrary to our expectation the idea was very unpopular at the Symposium (cf. 21). One of the criticisms against the Ca concept was that Ca ion could certainly exert its effect on crude systems such as glycerinated fibers or natural actomyosin, but not on pure actomyosin; such an agent as being ineffective on the pure system could not be considered to be the true factor. One implication of this criticism might be that such a crude system would be contaminated by fragmented sarcoplasmic reticulum, thereby producing the soluble relaxing factor.

In 1956, Perry (28) showed that EDTA could repress the ATPase of natural actomyosin, but not that of reconstituted actomyosin. After establishment of the Ca concept A. Weber worked out this problem (29) and showed that some reconstituted actomyosin preparations were somehow sensitive to GEDTA, and the act in side was responsible for whether or not the reconstituted system would be sensitive to Ca ion.

I felt that we must solve this enigmatic problem to convince those who would not believe the Ca concept and therefore started this task immediately after coming back from Dedham. My idea at first was that the routine procedure to prepare actin, i.e., acetone treatment, would be too drastic to retain the protein’s subtle nature. I thought once that we had succeeded in obtaining such an actin preparation. Soon we realized, however, that this was not due to the success of preparing ‘native actin’, but the presence of the ‘third factor’ in that actin preparation (30). The factor was rather easily separated from actin and found to resemble tropomyosin, which had been discovered by Bailey long ago but of which the function had long been unknown. Since classical tropomyosin did not exhibit the physiological function of ‘native tropomyosin’ (30), there was no doubt that ‘native tropomyosin’ should contain another factor. Indeed, a new globular protein, the first Ca binding protein of biological importance, was isolated from ‘native tropomyosin’ and named troponin (31–32).

The story of the further work since that time has been described in previous review articles (33–36>), so I will avoid overlapping. However, I would like to refer to two points. First, we were very lucky that troponin was very immunogenic; this enabled us to determine its localization in the myofibril and consequently formulate the thin filament model (Fig. 12). Second, the troponin system represents the most advanced or differentiated mechanism of Ca regulation, not only of muscle but of all cells, of which the secret resides in the function of troponin T, the most enigmatic subunit among three troponin subunits. The work along this line (cf. 37) will eventually solve the mechanism of how Ca ion can control the myosin-actin interaction.

Fig. 12 A model for the fine structure of the thin filament, i.e. the troponin-tropomyosin-F-actin complex.

Lower figure shows the staining of sarcomere by antitroponin, which provided most important information in formulating the above model. (quoted from ref. 34, but modified for this article).

CALCIUM ERA

Establishment of the Ca concept in the contractile mechanism naturally tempted us to look for a Ca-dependent metabolic system. Indeed, Ozawa et al. (38, 39) have found in 1967 that phosphorylase b kinase is as sensitive to Ca ion as the contractile system. It was very impressive to realize that the well-known activation by cyclic AMP of this enzyme also required Ca ion (Fig. 12)