Two different preparations of subfragment-1 from chicken skeletal myosin and from porcine cardiac myosin

Two different subfragment-1 preparations were obtained from either skeletal or cardiac myosin. They were identical in the heavy chain and light chain compositions but different in the pH dependence of the Ca-ATPase activity and in the relationship with "reactive lysine residues" (RLR).     

Amino-acid sequence of the short subfragment-2 in adult chicken skeletal muscle myosin

The amino-acid sequence of a short subfragment-2 in the amino-terminal portion of subfragment-2 (S-2) derived from adult chicken skeletal muscle myosin was completely determined. Peptides cleaved by cyanogen bromide and by lysyl endopeptidase of S-carboxymethylated S-2, and hydrolytic peptides obtained with trypsin or dilute acetic acid of larger CNBr fragments were isolated and sequenced. This region was composed of 257 amino-acid residues, and hydrophobic and charged residue repeat units were found highly conserved and with a periodicity in 7 or 28 residues. This sequence of the short S-2 fragment of chicken skeletal muscle myosin was compared with the sequence of chicken and rat embryonic skeletal muscle myosins, rabbit skeletal and rabbit cardiac muscle myosin (alpha-myosin heavy chain), and 95.3%, 86.8%, 89.9% and 94.2% sequence identities were observed, respectively.     

Complete amino-acid sequence of subfragment-2 in adult chicken skeletal muscle myosin

Although the complete amino-acid sequence of the short subfragment-2 (short S-2) and the partial sequence of the hinge region derived from adult chicken skeletal muscle myosin have been reported previously, the sequence of the N-terminal portion of subfragment-2 (S-2) and the connective portion between the above two regions could not be determined. In this study, the amino-acid sequence of these undetermined portions were completely sequenced. Furthermore, overlaps of cyanogen bromide (CNBr) peptides in the hinge region were also isolated and sequenced. Peptides obtained by hydrolysis with dilute formic acid and by digestion with lysyl endopeptidase of S-2 were purified and sequenced. These results established the complete amino-acid sequence of S-2 composed of 429 amino-acid residues. This sequence of adult chicken skeletal muscle myosin was compared with that of chicken embryonic skeletal muscle, chicken gizzard muscle and rabbit cardiac muscle myosin (alpha-myosin heavy chain) and shows degrees of 96%, 38% and 84% sequence identities, respectively. The frequency with which hydrophobic residues are present at position "a" in seven-residues repeats of the hinge region was markedly reduced when compared to the short S-2 sequence of the chicken skeletal muscle myosin.     

Amino-acid sequence of the hinge region in chicken myosin subfragment-2

The CNBr fragments of the hinge region in the carboxyterminal portion of long subfragment-2 derived from adult chicken pectoralis muscle myosin were isolated and sequenced by conventional methods. The alignment of these fragments was deduced from the homology of their sequences with those of other myosins, so that the sequence of the hinge region consisting of 127 amino-acid residues was determined. A comparison of this sequence with that of chicken embryonic skeletal muscle, chicken gizzard muscle and rabbit cardiac muscle (alpha-myosin) shows degrees of 95%, 36% and 82% sequence identities, respectively. Furthermore, the frequency with which hydrophobic residues are present at position "a" in seven-residues repeats of this region was significantly lower than the other portions of the rod.     

The primary structure of skeletal muscle myosin heavy chain: II. Sequence of the 50 kDa fragment of subfragment-1

The complete amino acid sequence of the 50 kDa fragment of subfragment-1 from adult chicken pectoralis muscle myosin was determined. It contained 431 residues including an epsilon-N-trimethyllysine at position 346. The 431-residue sequence corresponds to the sequence of residues 206 to 639 of chicken embryonic breast muscle myosin heavy chain which was predicted from the nucleotide sequence of the cDNA by Molina et al. [Molina, M. I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488]. Comparing the two sequences, 23 amino acid substitutions and three deletions/insertions are recognized.     

Preparation of subfragment-1 from abalone smooth muscle myosin

Myosin subfragment-1 (S1), which has one heavy chain (HC) (93 kDa) and two light chains (LC1 and LC2), was prepared by papain digestion of myosin from abalone-smooth muscle in the presence of Ca2+. The Ca-sensitivity of abalone S1 itself was not lost completely (about 30%). The tryptic digestion of S1 showed that in the presence of EDTA, S1 HC was split into 68, 55, and 23 kDa fragments, as in the presence of Ca2+, but 23 kDa was further degraded into 19 kDa. In contrast to the result in the presence of Ca2+, LCs disappeared in the early stage of reaction and Ca-ATPase activity decreased rapidly to about 70% of that of intact S1. This rapid decrease of Ca-ATPase activity seemed to be accompanied with the digestion of LCs. Therefore, LCs contribute to the protection of 23 kDa fragment from further digestion, to the maintenance of Ca-ATPase activity by stabilizing the structure of S1 to some extent in the presence of Ca2+. Since F-actin suppressed the cleavage of S1 HC to 68 and 23 kDa during tryptic digestion, it might be that 23 and 68 kDa corresponded to 20 kDa (C-terminal fragment) and to 50 + 25 kDa (N-terminal fragment) of skeletal myosin S1, respectively.     

Interaction of a new fluorescent ATP analogue with skeletal muscle myosin subfragment-1

A new fluorescent ribose-modified ATP analogue, 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic]-ATP (NBD-ATP), was synthesized and its interaction with skeletal muscle myosin subfragment-1 (S-1) was studied. NBD-ATP was hydrolysed by S-1 at a rate and with divalent cation-dependence similar to those in the case of regular ATP. Skeletal HMM supported actin translocation using NBD-ATP and the velocity was slightly higher than that in the case of regular ATP. The addition of S1 to NBD-ATP resulted in quenching of NBD fluorescence. Recovery of the fluorescence intensity was noted after complete hydrolysis of NBD-ATP to NBD-ADP. The quenching of NBD-ATP fluorescence was accompanied by enhancement of intrinsic tryptophan fluorescence. These results suggested that the quenching of NBD-ATP fluorescence reflected the formation of transient states of ATPase. The formation of S-1.NBD-ADP.BeF(n) and S-1.NBD-ADP.AlF(4)(-) complexes was monitored by following changes in NBD fluorescence. The time-course of the formation fitted an exponential profile yielding rate constants of 7.38 x 10(-2) s(-1) for BeF(n) and 1.1 x 10(-3) s(-1) for AlF(4)(-). These values were similar to those estimated from the intrinsic fluorescence enhancement of trp due to the formation of S-1.ADP.BeF(n) or AlF(4)(-) reported previously by our group. Our novel ATP analogue seems to be applicable to kinetic studies on myosin.     

Thermal stability of myosin subfragment-1 decreases upon tryptic digestion in the presence of nucleotides

Myosin subfragment-1 (S-1), digested with trypsin in the presence of ATP, rapidly loses its ATPase activity upon mild heat treatment even if ATP or ADP is present. The heat-treated molecule is very sensitive to further tryptic digestion. Undigested S-1 and S-1 digested in the absence of ATP are protected by nucleotides. The loss of the protective effect of nucleotides correlates with the tryptic splitting of the 25 kDa amino-terminal fragment between Arg 23 and Ile 24.     

Caldesmon inhibits skeletal actomyosin subfragment-1 ATPase activity and the binding of myosin subfragment-1 to actin

Smooth muscle contraction is controlled in part by the state of phosphorylation of myosin. A recently discovered actin and calmodulin-binding protein, named caldesmon, may also be involved in regulation of smooth muscle contraction. Caldesmon cross-links actin filaments and also inhibits actin-activated ATP hydrolysis by myosin, particularly in the presence of tropomyosin. We have studied the effect of caldesmon on the rate of hydrolysis of ATP by skeletal muscle myosin subfragment-1, a system in which phosphorylation of the myosin is not important in regulation. Caldesmon is a very effective inhibitor of ATP hydrolysis giving up to 95% inhibition. At low ionic strength (approximately 20 mM) this effect does not require smooth muscle tropomyosin, whereas at high ionic strength (approximately 120 mM) tropomyosin enhances the inhibitory activity of caldesmon at low caldesmon concentrations. Cross-linking of actin is not essential for inhibition of ATP hydrolysis to occur since at high ionic strength there is very little cross-linking as determined by a low speed sedimentation assay. Under all conditions examined, the decrease in the rate of ATP hydrolysis is accompanied by a decrease in the binding of myosin subfragment-1 to actin. Furthermore, caldesmon weakens the equilibrium binding of myosin subfragment-1 to actin in the presence of pyrophosphate. We conclude that caldesmon has a general weakening effect on the binding of skeletal muscle myosin subfragment-1 to actin and that this weakening in binding may be responsible for inhibition of ATP hydrolysis.     

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. I. Tilt angle of myosin subfragment-1 in the rigor complex

A three-dimensional image of the "rigor" complex of actin and chymotryptic myosin subfragment-1 was reconstituted from electron micrographs of negatively stained specimens. Data went out to 20 A radially and 26 A axially. The reconstituted images allowed us to deduce the angle between the major axis of the main part of myosin subfragment-1 and the axis of the actin helix. The subfragment-1 molecules were attached to the actin filament in a configuration in which they were tilted by only about 15 degrees from the plane perpendicular to the axis of the actin helix. The implication of the smaller tilt angle than the commonly accepted value is discussed.     

Crosslinking of tropomyosin to myosin subfragment-1 in reconstituted rabbit skeletal thin filaments

Rabbit skeletal tropomyosin was labeled with the bifunctional photoactivatable crosslinker N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate. After irradiating the rigor complex composed of myosin subfragment-1, crosslinker-labeled tropomyosin, and F-actin, a crosslinked product was formed. This product was identified as a 1:1 adduct of tropomyosin and subfragment-1. This finding is in support of recent structural studies which suggest that tropomyosin and subfragment-1 are in close proximity to each other, and may be relevant to the mechanism of thin filament regulation.     

Dinitrophenylated thiols in tryptic fragments of the heavy chain from chicken gizzard myosin

Trypsin digestion of phosphorylated and 3H-labeled dinitrophenylated chicken gizzard myosin released major fragments of Mr 29,000, 50,000 and 66,000 in a ratio of close to one to one. They contained 58% of the label bound to thiols of the heavy chains; 28% of the label was bound to the light chains. The heavy chain fragments of Mr 29,000 and Mr 66,000 were dinitrophenylated when the enzyme activity was inhibited. The 3H-labeled dinitrophenylated myosin alone followed a somewhat different pattern in that the label was bound to the light chains predominantly. Thiolysis of the phosphorylated and dinitrophenylated myosin with 2-mercaptoethanol restored the K+ -ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity and the dinitrophenyl group was removed from the N-terminal fragment of Mr 29,000 of the heavy chain, predominantly. In contrast, restoration of the enzymic activity occurred in thiolyzed dinitrophenylated myosin alone when the label was removed from the light chains rather than the tryptic fragments of the heavy chain. Phosphorylation induced conformational changes in gizzard myosin that altered the reactivity of the thiols in fragments of the globular heavy chain region.     

Tryptophan-130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment-1

Rabbit skeletal muscle myosin subfragment-1 (S-1) was reacted with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S-1. In order to assign the most reactive tryptophan of the 5 S-1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS-treated S-1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS-treated S-1 and was further cleaved at a single cysteine residue by 2-nitro-5-thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122-204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S-1.     

Elementary steps of the actin-activated ATPase reaction of cardiac muscle myosin subfragment-1

The rates of the elementary steps of the actomyosin ATPase reaction were measured using the myosin subfragment-1 of porcine left ventricular muscle. The results could be explained only by the two-route mechanism for actomyosin ATPase (Inoue, Shigekawa, & Tonomura (1973) J. Biochem. 74, 923-934), in which ATP is hydrolyzed via routes with or without accompanying dissociation of actomyosin. The dependence on the F-actin concentration of the rate of the acto-S-1 ATPase reaction in the steady state was measured in 5 mM KCl at 20 degrees C. The maximal rate, Vmax, and the dissociation constant for F-actin of the ATPase, Kd, were 3.0 s-1 and 2.2 mg/ml, respectively. The Kd value was almost the same as that determined from the extent of binding of S-1 with F-actin during the ATPase reaction. The rate of recombination of the S-1-phosphate-ADP complex, S-1ADPP, with F-actin, vr, was lower than that of the ATPase reaction in the steady state. Thus, ATP is mainly hydrolyzed without accompanying dissociation of acto-S-1 into S-1ADPP and F-actin. In the cardiac acto-S-1 ATPase reaction, the rate of the ATPase reaction in the steady state and that of recombination of S-1ADPP with F-actin were about 1/5 those of the skeletal acto-S-1 ATPase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the actin interface of skeletal myosin subfragment-1 by treatment with dibromobimane

Recently, by treating the head portion of skeletal myosin subfragment-1 (S1) with the bifunctional agent dibromobimane, we introduced an intramolecular covalent cross-link which resulted in the stabilisation of an internal loop in the heavy chain structure of the head [Mornet et al. (1984) Proc. Natl Acad. Sci. USA 82, 1658-1662]. In order to define the functional properties of this new S1 conformational state, we have first determined the experimental conditions for the optimum modification of S1 by dibromobimane. We finally settled on a 60% yield of cross-linked S1. Because the modification occurs between the 50-kDa and the 20-kDa tryptic heavy chain fragments which have been postulated to be involved in the interaction of native S1 with actin, we have investigated the association of dibromobimane-treated S1 with actin, using chemical cross-linking of their rigor complex with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linked species obtained were analyzed by polyacrylamide gel electrophoresis and compared with those known for unmodified S1. The carbodiimide-catalyzed linkage between actin and dibromobimane-modified S1 led to a singlet protein band migrating with an apparent molecular mass of 155 kDa, in contrast to the usual doublet bands of 175 kDa and 185 kDa produced with native S1. This result suggests that a change has occurred at the actin interface on the dibromobimane-treated S1 heavy chain. The covalent complex generated by carbodiimide cross-linking between actin and dibromobimane-modified S1 (27-kDa + 50-kDa + 20-kDa fragments) was submitted to chemical hydrolysis with hydroxylamine. The nature of the products identified is consistent with the conclusion that the internal freezing of the heavy chain structure by dibromobimane induces the loss of the ability to cross-linkage of the actin site on the 20-kDa domain but does not affect the conformation of the second site on the 50-kDa segment, which becomes the unique actin region cross-linkable by actin.     

Interaction of myosin subfragment-1 with actin. II. Location of the actin binding site in a fragment of subfragment-1 heavy chain

The heavy chain of subfragment-1 prepared by chymotrypsin treatment had a molecular weight of about 96K. The heavy chain was split into 26 K, 50 K, and 21 K fragments by trypsin. When the trypsin-treated subfragment-1 was cross-linked with dimethyl suberimidate, cross-linked products of 26 K, 50 K, and 21 K fragments and of 50 K and 21 K fragments appeared, but there was little cross-linked product of 26 K and 50 K fragments or of 26 K and 21 K fragments. When the cross-linking experiments were carried out in the presence of actin, a new band appeared and the amount of cross-linked product of 26 K, 50 K, and 21 K fragments decreased by about 50%. The molecular weight of the new band was lower than that of the cross-linked product of 26 K, 50 K, and 21 K fragments, and higher than that of the dimer of actin. Based on this and some other results, we suggest that this band represented a cross-linked product of actin and the 50 K fragment. We also suggest that the decrease in the amount of cross-linked product of 26 K, 50 K, and 21 K fragments reflected the conformational change in subfragment-1 due to the binding of actin.     

Effect of limited trypsin digestion on the biochemical kinetics of skeletal myosin subfragment-1.

We have investigated the effect of limited trypsin digestion of chymotryptic myosin Subfragment-1 (S-1) on its kinetic properties. We find that Vmax (i.e., the extrapolated maximal ATPase activity at infinite actin) remains approximately constant, independent of the period of digestion. We also find that the apparent actin activation constant, KATPase, and the apparent dissociation constant, Kbinding, are both significantly weakened by trypsin digestion of S-1, and that these kinetic parameters change in concert. In addition, we investigated the effect of limited trypsin digestion on the initial phosphate burst. We find that trypsin digestion has no effect on the rate of the tryptophan fluorescence enhancement that occurs after ATP binds to digested S-1, but that the magnitude of the fluorescence enhancement falls approximately 40% with digestion. Digested S-1 also showed anomalous behavior in that the fluorescence magnitude increased and the fluorescence rate dropped in the presence of actin. Trypsin digestion also decreased the magnitude of the chemically measured Pi burst approximately 35%, but this magnitude was essentially unaffected by actin. A possible explanation for this behavior is discussed.