On the Molecular Weight of Myosin. II<sup>*</sup>

Tonomura, Yūji; Appel, Pearl; Morales, Manuel F.

doi:10.1021/bi00866a017

Cited by 268 publications

(129 citation statements)

References 31 publications

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“…Myosin and actin were prepared from back and leg muscles of rabbit according to Tonomura et al [27] and Spudich and Watt [28], respectively. S1 was prepared by digesting myosin filaments with chymotrypsin [29] and was further purified by gel filtration on a Sephadex G-100 column.…”

Section: Proteinsmentioning

confidence: 99%

Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment‐1

Mühlrád

Chaussepied

1990

European Journal of Biochemistry

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Myosin subfragment-1 from rabbit skeletal muscle was digested by thermolysin at 25", 12" and 0°C. Thermolysin cleaves subfragment-1 heavy chain into two stable fragments, 28 kDa and 70 kDa, aligned in this order from the N-terminus [Applegate, D. & Reisler, E. (1983) Proc. Nut1 Acad. Sci. U S A 80, 7109-71121. The rate of digestion at 25°C was significantly increased in the presence of MgATP and somewhat less in the presence of MgADP, or magnesium pyrophosphate. This activating effect of the nucleotides was decreased at 12°C and completely eliminated at 0 "C. The results can be explained by assuming that there are two subfragment-1 conformers [Shriver, J. W. & Sykes, B. D. (1981) Biochemistry 20, 2004, and that both the addition of ATP or its analogs, and lowering the temperature, shift the conformational equilibrium in the direction that is more susceptible to thermolysin. Actin inhibited thermolysin digestion of subfragment-I at all three temperatures studied. Actin inhibition can be explained either by shifting the equilibrium of the conformers in the direction of the less susceptible form or by direct interference of actin with the binding of thermolysin to subfragment-1 . Actin inhibition of thermolysin digestion also prevailed when subfragment-1 was in a ternary complex with nucleotide and actin, in both the strongly and weakly attached states. Similarly, actin inhibited the digestion of subfragment-1 modified by 1,4-phenylenediamine, which also forms a weakly attached complex with actin. No difference could be found in the accessibility of the thermolysin-susceptible site of subfragment-l at the 28-70 kDa junction in either rigor, strongly or weakly attached states, which indicates the similarity of the structure proximal to this specific site in the three attached states.Myosin subfragment-I (SI) is the head segment of myosin which contains two separate sites responsible for the hydrolysis of ATP and for actin binding [l]. Binding ligands to their respective sites induces localized structural changes in S1. The structural changes accompanying the binding and transformation of ligands are essential for myosin function. According to the hypothesis of Shriver and Sykes [2], S1 exists as two conformers in equilibrium with each other. This equilibrium depends on ambient factors such as temperature or pH and is perturbed by the binding of nucleotides or actin. A number of results including 19F-NMR spectra measurements of S1 whose SH1 thiol was modified by a fluorine-containing probe [3], ultraviolet absorption spectrum [4], H'/H2 exchange [5] and limited trypsinolysis studies [6] support the two-conformer hypothesis.When both binding sites of S1 are occupied by nucleotide and actin, they have opposing effects on its structure, as found in limited-proteolysis studies [7 -121. The binding of the intermediates of the ATP cycle decreases the affinity of actin for its binding site and vice versa. Altogether, the results imply the existence of inter-site communication in S1 [13, 141. On the basis of stability, the...

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Section: Proteinsmentioning

confidence: 99%

Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment‐1

Mühlrád

Chaussepied

1990

European Journal of Biochemistry

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“…The methods used for the preparation were partly variants of earlier work (6,12,13) and are summarized below.…”

mentioning

confidence: 99%

Isolation and partial renaturation of proteolytic fragments of the myosin head.

Mühlrád¹,

Morales²

1984

Proc. Natl. Acad. Sci. U.S.A.

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Methods have been devised for isolating two of the tryptic fragments (those termed "20K" and "50K") of myosin "subfragment 1" in pure form. Fragment 20K was examined for renaturation after removal of denaturants used in its preparation. It generated a CD spectrum corresponding to ca. 64% formed structure (roughly what would be expected from its amino acid sequence) and a red-shifted UV spectrum such as arises when phenylalanine and tyrosine are perturbed by structural interactions. Actin affinity of fragment 20K was tested by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking, inhibition of the actin-activated ATPase of subfragment 1 containing light chain 3, cosedimentation with actin, and light scattering; the affinity exceeded 5 x 106 M-'.The foregoing suggests that moiety 20K has a sovereign existence in (i.e., is a domain of) myosin subfragment 1. Preliminary work indicates that fragment 50K also binds actin, but with a lesser affinity."Subfragment 1" (S-1) is the segment of the myosin molecule on which the ATPase site and the actin-binding site (1) and perhaps "communicating" apparatus also involved in energy transduction in muscle (2) reside. To elucidate the substructure of S-1 in relation to transduction is therefore of manifest importance, and a first step is to search for domains among which functionalities may be distributed. Mornet et al. (3) have reported that not only trypsin (4) but also a variety of proteases cut S-1 into very nearly the same three heavy chain fragments (termed "27K," "50K," and "20K"). Their result shows that open enzyme-vulnerable segments of heavy chain exist between the fragments and thus that a necessary condition for regarding the fragments as domains is satisfied. Together, their result and ours begin to approach what is necessary and sufficient for concluding that the fragments are domains. Stimulated by the experiments of Kassab and Mornet (5, 1) on partial purification and actin-binding ability of 20K, we have attempted to renature it and have obtained a 20K preparation that has several of the properties expected of the "native" particle having some sovereign existence in S-1. We have also obtained a few analogous results with 50K.MATERIALS AND METHODS Chemicals. Ultrapure urea, sucrose, and guanidine'HCl were from Schwarz/Mann. Thermolysin, ATP, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-acetyltyrosine amide were from Sigma. L-1-Tosylamido-2-phenylethyl chloromethyl ketone (Tos-PheCH2Cl)-trypsin and a-chymotrypsin were from Millipore. All common chemicals were reagent grade.Proteins. Rabbit skeletal muscle actin and myosin were prepared by standard methods (6, 7). S-1 was obtained by digesting myosin filaments with a-chymotrypsin (8) tKassab, R

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“…Myosin was routinely prepared from back muscle of rabbits according to Tonomura et al (3). S-1 was prepared from myosin digested by insoluble papain according to Lowey et al (4).…”

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confidence: 99%

4,4'-Bis (1-anilinonaphthalene 8-sulfonate) (bis-ANS): a new probe of the active site of myosin.

Takashi¹,

Tonomura²,

Morales³

1977

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT. The interaction of myosin subfragment-1 (S-1) with 4,4'-bis(1-anilinonaphthalene 8-sulfonate) (bis-ANS) has been studied by monitoring the fluorescence of the latter when the two components form a complex. Because ATP and ATP analogs partially displaee complexed bis-ANS it has also been possible to study interactions of S-1 and nucleotides by using the displacement effect. Ap roximate values of the parameters of these various interactions have been measured. Some possible applications of bis-ANS have been explored. For example, it provides a very convenient method for obtaining the Michaelis constant, K., in steady-state S-1 nucleoside triphosphatase; this particular application has also provided some evidence for inferring that in Ca2+ (but not in Mg2+) adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) 5-1 behaves like a mixture of two components, each with its own Km. Clear energy transfer occurs between tryptophan residues and bound bis-ANS. The fluorescence also suggests that S-1 undergoes some slow relaxations following substrate binding.Weber and his associates have recently produced 4,4'-bis(lanilinonaphthalene 8-sulfonate) (bis-ANS), a compound that fluoresces intensely in hydrophobic environments and practically not at all in water (1). Speculating from observations with NAD enzymes (2), Weber (personal communication) suggested to us that bis-ANS, because of its shape, might have a general affinity for ATP-binding sites. We have investigated this possibility, using as enzyme myosin subfragment-l (S-1), and have found that indeed some of the bis-ANS in binding equilibrium with S-1 is bound to the active (adenosinetriphosphatase) site of this enzyme. The fluorescence of bis-ANS so bound disappears upon displacement by ATP or its derivatives. This finding has allowed us to explore some properties of the active site in a preliminary but encouraging way. Complications in the use of bis-ANS with the myosins are that it binds also to sites other than the active site, and that the active site may be damaged at high levels of such other binding. Myosin concentration was measured by absorbance at 280 nm (AV70m = 5.70); S-1 concentration was also measured by absorbance at 280 nm (AVOm = 7.70) (5). In each case an appropriate correction for light .scattering was made. The molecular weights employed were 480,000 for myosin (3) and 115,000 for S-1 (4). MATERIALS AND METHODSAbbreviations: S-1, subfragment-I of myosin; 8-Br-ATP, 8-bromoadenosine 5'-triphosphate; AMP-P[NHJP, 5'-adenylyl imidodiphosphate; bis-ANS, 4,4'-bis(1-anilinonaphthalene 8-sulfonate); ATPase, adenosinetriphosphatase. * On leave from: Department of Biology, Osaka University, Osaka, Japan. 2334 Bis-ANS (1) was a gift from G. Weber of the University of Illinois; 8-bromoadenosine 5'-triphosphate (8-Br-ATP) (6) was a gift from H. Takenaka of Osaka University, Osaka. The formulas for these compounds are shown in Fig. 1. 5'-Adenylyl imidodiphosphate (AMP-P[NH]P) was obtained from International Chemical and'Nuclear Corp., Chemical an...

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On the Molecular Weight of Myosin. II^*

Cited by 268 publications

References 31 publications

Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment‐1

Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment‐1

Isolation and partial renaturation of proteolytic fragments of the myosin head.

4,4'-Bis (1-anilinonaphthalene 8-sulfonate) (bis-ANS): a new probe of the active site of myosin.

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On the Molecular Weight of Myosin. II*

Cited by 268 publications

References 31 publications

Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment‐1

Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment‐1

Isolation and partial renaturation of proteolytic fragments of the myosin head.

4,4'-Bis (1-anilinonaphthalene 8-sulfonate) (bis-ANS): a new probe of the active site of myosin.

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On the Molecular Weight of Myosin. II^*