Structure and function of the two heads of the myosin molecule. II. Separation of the two fractions of subfragment-1 of myosin by affinity column chromatography on immobilized F-actin: direct evidence for acceleration by F-actin of the decomposition of the reactive enzyme-phosphate-ADP complex formed on head B of myosin.

F-Actin (FA) and pyruvate kinase (PK) [EC 2.7.1.40] were immobilized on PAB-cellulose. HMM-Subfragment-1 (S-1) was applied to a column of immobilized FA and PK, and eluted with 1-1.5 muM ATP and 1 mM PEP in 50 mM KCl, 2 mM MgCl2, and 10 mM Tris-HCl at pH 7.8 and 4 degrees. The size of the initial burst of Pi liberation of S-1 applied to the column was 0.5 mole/mole S-1. The burst size of S-1 decreased with increase in the fraction number, and S-1 in later fractions showed a burst size of 0.1-0.3 mole/mole. On the other hand, the rate of the ATPase [EC 3.6.1.3] reaction in the steady state was almost independent of the burst size, and increased slightly with increase in the fraction number. The ATPase activity of S-1 with a burst size of less than 0.2 mole/mole was scarcely activated by FA. Usually, the dependence on the burst size of S-1 of its ATPase activity in the presence of FA was sigmoidal, and marked activation by FA was observed when the burst size was larger than 0.3-0.4 mole/mole. Similar results were obtained with S-1 fractions separated by the ultracentrifugation method described in our previous paper ((1976) J. Biochem. 79, 419-434).     

Structure and function of the two heads of the myosin molecule. III. Cooperativity of the two heads of the myosin molecule, shown by the effect of modification of head A with rho-chloromercuribenzoate on the interaction of head B with F-actin.

Subfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB. The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification. Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80% of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of Pi liberation was retained in S-1(T) dissociated from FA. It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of Pi liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Ca2+ sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB. CMB-S-1(CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2 X 10(4) were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1 : 0.18 : 0.09), indicating that heads A and B have the same subunit structure.     

Separation of myosin subfragment 1 into two fractions, one having the burst site and the other having the non-burst site.

During Mn(II)-ATP hydrolysis by myosin, the predominant intermediate formed at the burst site of the enzyme below 10 degrees is the myosin-ADP complex formed by adding ADP to myosin, while above 10 degrees it is the myosin -ADP-P1 complex generated by ATP hydroolysis (Yazawa, Morita, & Yagi (1973) J. Biochem. 74, 1107; Hozumi & Tawada (1975) Biochim. Biophys. Acta 376, 1; Tawada & Yoshida (1975) J. Biochem. 78, 293). It is suggested that the second (non-burst) site of myosin predominantly forms the myosin-ATP complex (Hozumi & Tawada, ibid.). From these findings, it is expected that (i) myosin subfragment 1 (S1) having the burst site is bound to actin in Mn(II)-ATP solution containing ADP below 10 degrees, because it forms the S1-ADP complex even in the presence of ATP; (ii) the other S1, i.e., that having the non-burst site, is dissociated from actin, because it forms the S1-ATP complex. These two expectations were confirmed by viscosity measurements of acto-S1 solutions, giving a basis for the separation of S1 into two fractions: one having the burst site and the other having the non-burst site. S1 having the non-burst site could be extracted from partially papain [EC 3.4.22.2]-digested myofibrils of rabbit skeletal muscle with a solution containing MnCl2, ATP, and ADP at 0 degrees. S1 having the burst site was extracted from myofibrils already used for the extraction of S1 having the non-burst site, with a solution containing MgCl2 and ATP at 20 degrees. The former S1 fraction had Mg-ATPase [EC 3.6.1.3] activity, but scarcely showed any initial burst of Pi liberation. The latter S1 showed a Pi burst of more than 0.5 (M/M). The steady state ATPase activity of the former S1 was slightly higher than that of the latter. The burst size of normal S1, i.e., that extracted from papain-digested myofibrils with Mg-PPi or Mg-ATP, was 0.5 (M/M). The ultraviolet absorption spectrum of the non-burst type S1 was not changed by ADP but was changed by ATP, though the difference spectrum was distinct from that of normal S1 and the difference molar extinction coefficient at 289 nm was only 20% of that of normal S1. No significant difference was seen in the compositions of these two S1's and normal S1, as determined by SDS gel electrophoresis.     

Temperature-dependence of tension development by glycerinated muscle fibers of rabbit psoas in Mg-ITP solution.

The isometric tension of single fibers isolated from glycerinated rabbit psoas muscle was measured at various temperatures using Mg-ITP as a substrate. The tension developed in Mg-ITP decreased linearly as the temperature was reduced from 24 degrees C to 4 degrees C. Myosin formed the myosin--product complex predominantly via ATP hydrolysis at the burst site during Mg-ATP hydrolysis, irrespective of temperature, and the tension developed in Mg-ATP decreased linearly as the temperature decreased (Yoshida and Tawada (1976) J. Biochem. 80, 861). During Mg-ITP hydrolysis, myosin forms the myosin*-product complex predominantly at the burst site above 20 degrees C, while myosin forms the myosin*-substrate complex below 8 degrees C (Hozumi (1976) Eur. J. Biochem. 63, 241). However, the temperature dependence of tension development in Mg-ITP is linear, as with Mg-ATP, as mentioned above. This temperature dependence is not compatible with some muscle models which assume the formation of the myosin*-product complex by cross-bridges prior to combination with actin during contraction.     

Desensitization of substrate inhibition of acto-H-meromyosin ATPase by treatment of H-meromyosin with rho-chloromercuribenzoate. Relation between the extent of desensitization and the amount of bound rho-chloromercuribenzoate1.

H-Meromyosin (CMB leads to betaME-H-meromyosin) was prepared by tryptic digestion of myosin, which had been treated with CMB bound to H-meromyosin and the extent of desensitization of the substrate inhibition of acto-H-meromyosin ATPase [EC 3.6.1.3.] was investigated. Both the dissociation of acto-H-meromyosin induced by ATP and substrate inhibition decreased with increase in the amount of bound CMB to a minimum value at about 1 mole of CMB bound per mole of H-meromyosin. The substrate inhibition of acto-H-meromyosin ATPase was restored to the original level by complete removal of the bound CMB by further treatment of CMB leads to beta ME-H-meromyosin with a large excess of beta-mercaptoethanol. The dissociation constant of acto-H-meromyosin in the presence of ATP decreased markedly on modification with CMB, while the maximum ATPase activity ar a sufficiently high concentration of F-actin remained essentially unchanged. Acto-H-meromyosin was reconstituted from F-actin and CMB LEADS TO beta ME-H-meromyosin, containing less than the stoichiometric amount of bound CMB. Its ATPase activity and the extent of dissociation of acto-H-meromyosin induced by ATP were explained as those of a mixture of unmodified H-meromyosin and CMB leads to beta ME-H-meromyosin containing 1 mole of CMB per mole of H-meromyosin. Half of the light chains (g2), with a molecular weight of 18,000, were removed from myosin by treatment with CMB and beta-mercaptoethanol. After this treatment, on further incubation of the myosin with a large excess of beta-mercaptoethanol, the myosin contained only half of the g2, but the substrate inhibition of acto-H-meromyosin ATPase was restored completely. The initial burst of P1 liberation and the EDTA-ATPase activity decreased to almost zero on specific modification of the SH1-groups with NEM, while the initial burst decreased to some extent and the EDTA-ATPase activity to 50% of the original value on binding of 1 mole CMB per mole of H-meromyosin. The actomyosin-type of ATPase activity was strongly inhibited by modification with CMB. The extent of the dissociation of acto-H-meromyosin induced by ATP was unaffected by modification with NEM, while it decreased on further treatment of NEM-myosin with CMB FOLLOWED BY BETA-MERCAPTOETHANOL.     

Modeling of the actomyosin ATPase activity

In the rapid “quench” kientics of myosin, the “initial phosphate burst” is the excess inorganic phosphate that is produced during the early time-course of ATP hydrolysis by myosin subfragment-1 (S-1) or HMM. In general, the existence of a Pi burst implies a rapid (i.e., generally an order of magnitude faster than the steady-state hydrolysis rate) lysis of the phospho-anhydride bond within the ATP molecule, followed by one or more slower steps that are rate limiting for the process. Thus, the presence of a Pi burst can provide an important clue to the mechanism of the reaction. However, in the case of actomyosin, this clue as long been the subject of controversy and misunderstanding. To measure the (initial) Pi burst, myosin S-1 (or HMM) is rapidly mixed with ATP and then the mixture is acid quenched after a specific time period. The medium produced contains free Pi generated from hydrolysis of the ATP. The quantitative measure of the phosphate generated in this way has always been significantly greater than that expected by steady-state “release” of Pi alone, and it is that very difference between this measured Pi after the quench and that amount of Pi expected to be released by steady-state considerations in that same time period that has been referred to as the “initial Pi burst”. Recent investigations of the kinetics of Pi release have used an entirely new method that directly measures the release of Pi from the enzyme-product complex. These studies have made reference to the properties of the “initial Pi burst” in the presence of actin, as well as to a new kinetic entity: the “burst of Pi release”, and have been often vague concerning the true nature of the initial Pi burst, as well as the properties of Pi release as predicted by the current models of the actin activation of the myosin ATPase activity. The purpose of the current article is to correct this oversight, to discuss the “burst” in some detail, and to display the kinetics predicted by the current models for the actin activation of myosin. Furthermore, predictions for the kinetics of the new “burst of Pi release” are discussed in terms of its ability to discriminate between the two current competing models for actin activation of the myosin ATPase activity.     

Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.     

Inhibition of actomyosin subfragment 1 ATPase activity by analog peptides of the actin-binding site around the Cys(SH1) of myosin heavy chain

The synthetic heptapeptide, Ile-Arg-Ile-Cys-Arg-Lsy-Gly-ethoxy, an analog of one of the actin binding sites on myosin head (S-site) (Suzuki, R., Nishi, N., Tokura, S., and Morita, F. (1987) J. Biol. Chem. 262, 11410-11412) was found to completely inhibit the acto-S-1 (myosin subfragment 1) ATPase activity. The effect of the heptapeptide on the binding ability of S-1 for F-actin was determined by an ultracentrifugal separation. Results indicated that the heptapeptide scarcely dissociated the acto-S-1 complex during the ATPase reaction. Consistent results were obtained from the acto-S-1 ATPase activities determined as a function of S-1 concentrations in the absence or presence of the heptapeptide at a fixed F-actin concentration. The heptapeptide reduced the maximum acto-S-1 ATPase activity without affecting the apparent dissociation constant of the acto-S-1 complex. The heptapeptide bound by a site on actin complementary to the S-site probably inhibits the activation of S-1 ATPase by F-actin. These results suggest that S-1 ATPase is necessary to rebind transiently with F-actin at the S-site in order to be activated by F-actin. This is consistent with the activation mechanism proposed assuming the two actin-binding sites on S-1 ATPase (Katoh, T., and Morita F. (1984) J. Biochem. (Tokyo) 96, 1223-1230).     

Characterization of the ATPase active site in myosin subfragment-1 with the use of vanadate plus ADP as a reversible "affinity-labeling" reagent: evidence for heterogeneity in the active sites

Our previous work showed that the active site heterogeneity in heavy meromyosin (HMM) becomes evident when highly reactive SH-groups in HMM are modified by thimerosal (Kawamura, Higuchi, Emoto, & Tawada (1985) J. Biochem. 97, 1583-1593). The heterogeneity was revealed by "affinity-labeling" analysis with vanadate plus ADP, which was developed in the previous paper. To see whether this heterogeneity is due to the head-head interaction or two different alkali light chains present in HMM, we carried out similar studies with myosin subfragment-1 (S1) and one of the isozymes, S1(A1), which contains only the alkali light chain 1, and obtained essentially the same results as those previously obtained with HMM. The S1 results are easily explained by the same hypothesis previously used for explaining the HMM results: SH-modified S1 or S1(A1) contains two kinds of active site in a 1:1 ratio with almost the same ATPase activity: one hydrolyzes ATP by a mechanism giving a protein Trp fluorescence enhancement, whereas the other hydrolyzes ATP by another mechanism giving no fluorescence enhancement.     

Amino acid sequence of the amino-terminal 24 kDa fragment of the heavy chain of chicken gizzard myosin

Chicken gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine (IAEDANS) in the presence of ATP and in 0.15 M KCl, where the myosin assumed 10S conformation. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodaltons) was isolated by gel filtration on a Sephadex G-100 column followed by chromatography on a CM 52 column. The amino acid sequence of the fragment was analyzed by conventional methods, and was: (S,Z)K-P-L-S-D-D-E-K-F-L-F-V-D-K-N-F-V-N-N-P-L-A-Q-A-D-W-S-A-K-K- L-V-W-V-P-S-E-K-H-G-F-E-A-A-S-I-K-E-E-K-G-D-E-V-T-V-E-L-Q-E-N-G-K-K- V-T-L-S-K-D-D-I-Q-K-M-N-P-P-K-F-S-K-V-E-D-M-A-E-L-T-C-L-N-E-A-S-V-L- H-N-L-R-E-R-Y-F-S-G-L-I-Y-T-Y-S-G-L-F-C-V-V-I-N-P-Y-K-Q-L-P-I-Y-S-E-K-I- I-D-M-Y-K-G-K-K-R-H-E-M-P-P-H-I-Y-A-I-A-D-T-A-Y-R-S-M-L-Q-D-R-E-D-Q- S-I-L-C-T-G-E-S-G-A-G-K-T-E-N-T-K-K-V-I-Q-Y-L-A-V-V-A-S-S-H-K-G-K. The amino-terminus was blocked, and the fragment was assigned as an amino-terminal part of the heavy chain of gizzard myosin. Position 127 was occupied by epsilon-N-trimethyllysine. Trp-130 of rabbit skeletal myosin heavy chain, which was reported to cross-link to an azide derivative of ATP by Okamoto and Yount (Proc. Natl. Acad. Sci. U.S. 82, 1575-1579 (1985], was replaced by glutamine in gizzard myosin. Cys-93 of the fragment is the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin (Onishi, H. (1985) J. Biochem. 98, 81-86).     

Modification of cardiac and smooth muscle myosins with 2,4,6-trinitrobenzenesulfonate. Evidence for differences in structure around the active sites of cardiac, smooth, and skeletal muscle myosin ATPase.

Myosins purified from cardiac (porcine heart) and smooth (chicken gizzard) muscles were modified with 2,4,6-trinitrobenzenesulfonate (TNBS) and the effects on the kinetic properties of myosin ATPase [EC 3.6.1.3] were studied. The following results were obtained. 1. About 0.5 mol of TNBS per mol of myosin head was incorporated rapidly, irrespective of the presence of PP1 (2mM), into both types of myosin studied. 2. The size of the initial burst of P1 liberation for both myosins was found to be 0.5--0.6 mol/mol head. 3. The rapid incorporation of TNBS into cardiac muscle myosin was accompanied by a rapid decrease in the size of the initial P1 burst, and it was completely lost after modification for 20 min. However, smooth muscle myosin retained its P1 burst. 4. The EDTA (K+)-ATPase activity of both myosins modified in the presence or absence of PP1 decreased sharply with incorporation of TNBS. 5. Superprecipitation and ATPase activity of reconstituted actomyosin from cardiac myosin and skeletal F-actin decreased only after 10 min of modification with TNBS in the absence of PP1. 6. The spectra of TNP bound to myosins from cardiac and smooth muscles were unchanged by the addition of PP1. The above findings are compared with those previously obtained for skeletal muscle myosin [Miyanishi, T., Inoue, A., & Tonomura, Y. (1979) J. Biochem. 85, 747--753], and the structural and functional differences among the myosins derived from skeletal, cardiac, and smooth muscles are discussed.     

Separation of subfragment-1 of H-meromyosin into two equimolar fractions with and without formation of the reactive enzyme-phosphate-ADP complex.

H-Meromyosin (HMM) was digested with insoluble papain [EC 3.4.22.2]. Neither the size of the initial burst of Pi liberation (0.5 mole/mole of myosin head) nor the Mg2+-ATPase [EC 3.6.1.3] activity of HMM in the steady state was affected by this treatment. Acto-S-1 was obtained by mixing F-actin with HMM digested with insoluble papain (HMM-S-1). The size of the initial burst of Pi liberation of acto-S-1 was 0.35 mole/mole of S-l at an ATP concentration of 0.5 mole/mole of S-1, and 0.5 mole/moleof S-1 at ATP concentrations above 1 mole/mole of S-1...     

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.     

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.     

Myosin isoforms in normal and dystrophic chickens

The myosin isoform content in the affected fibers of chickens with inherited muscular dystrophy has been investigated with a new high-performance liquid chromatographic procedure for separation of the tryptic fragments of myosin subfragment 1 (S-1). The results indicate that dystrophic muscle contains substantial amounts of normal adult myosin, together with various myosin species present in normal 5-day posthatch chickens. Confirmation was obtained by comparative peptide mapping of the S-1 tryptic fragments and by N-terminal sequencing of 20-kDa species. Together with data on other contractile proteins and certain metabolic enzymes [Obinata, T., Takano-Ohmura, H., & Matsuda, R. (1980) FEBS Lett. 120, 195-198; Mikasa, T., Takeda, S., Shimizu, T., & Kitaura, T. (1981) J. Biochem. (Tokyo) 89, 1951-1962; Feit, H., & Domke, R. (1982) Cell Motil. 2, 309-315; Cosmos, E. (1966) Dev. Biol. 13, 163-181; Cosmos, E., & Butler, J. (1967) in Exploratory Concepts in Muscular Dystrophy and Related Disorders (Milhorat, A. R., Ed.) pp 197-204, Excerpta Medica, Amsterdam], the results are consistent with the hypothesis that there is a general defect in muscle maturation in avian dystrophy.     

Isolation and characterization of cyclic AMP-independent glycogen synthase kinase from rat skeletal muscle

Glycogen synthase kinase was isolated from rat skeletal muscle. This kinase, which is cyclic nucleotide-independent and calcium-independent, was separated from phosphorylase kinase, cyclic AMP-dependent protein kinase and phosvitin kinase by phosphocellulose chromatography. Gel filtration on Sephadex G-100 resolved the glycogen synthase kinase into two fractions with apparent molecular weights of 68 000 (peak I) and 52 000 (peak II). This step also separated glycogen synthase kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase, which had an apparent molecular weight of 39 000. Peak II glycogen synthase kinase activity was not affected by the addition of calcium, EGTA or a number of cyclic nucleotides. In addition to ATP, dATP would serve as the phosphate donor. Other trinucleotides tested were either poor or ineffective substrates. Activity was about 5-fold greater with Mg2+ than with Mn2+. Glycogen stimulated activity about 25%. Modifications of the methods of Soderling et al. ((1970) J. Biol. Chem. 245, 6317--6328) and Nimmo et al. ((1976) Eur. J. Biochem. 68, 21--30) were developed for purification of glycogen synthease (UDPglucose:glycogen 4-alpha D-glucosyltransferase, EC 2.4.1.11) to specific activity of 35 units/mg of protein. Using this preparation of glycogen synthase as substrate, the phosphorylation and inactivation catalyzed by glycogen synthase kinase was compared to that catalyzed by cyclic AMP-dependent protein kinase or phosphorylase kinase. Each of the kinases had different specificities for phosphorylation sites on glycogen synthase.     

Ca2+ bindings of pig cardiac myosin, subfragment-1, and g2 light chain.

Ca2+ binding to pig cardiac myosin, subfragment-1 (S-1), and g2 light chain were investigated by the equilibrium dialysis method. Two different S-1s, one of which can bind Ca2+ and another which cannot, were prepared. In order to calculate the free Ca2+ concentrations adequately, the amounts of Ca2+ included in various chemicals and proteins were measured by atomic absorption spectroscopy. Ca2+ contamination was greatest in KCl among the chemicals tested. In addition, the Ca2+ strongly bound to myosin and S-1 was released in the presence of Mg2+. When Mg2+ was not added, the Ca2+-binding constant of myosin was 4 x 10(5) M-1 and the maximum binding number was 1.8 mol per mol of myosin. Cooperativity between the 2 Ca2+ bindings could not be demonstrated. Mg2+ strongly inhibited the Ca2+ binding: at a free Ca2+ concentration of 1 x 10(-5) M, 1.3 mol Ca2+ was bound to myosin in the absence of Mg2+, but 0.6 and 0.2 mol were bound in the presence of 0.3 and 4.5 mM Mg2+, respectively. The Ca2+-binding constant of S-1, which contained a 15,000 dalton component, was 8.6 x 10(5) M-1, and the maximum binding number was 0.7 mol per mol of S-1. The 15,000 dalton component could be exchanged with extraneous g2. S-1 which lacked the 15,000 component could not bind Ca2+ at free Ca2+ concentrations less than 0.1 mM. The Ca2+ binding to free g2 light chain was about 100 times weaker than the binding to myosin, as indicated previously for skeletal myosin (Okamoto, Y. & Yagi, K. (1976) J. Biochem. 80, 111--120). The Ca2+-binding constant was obtained as 4.1 x 10(3) M-1 in the absence of added Mg2+. Phosphorylation of g2 light chain did not affect the Ca2+ binding to the free g2 light chain or to myosin. Ca2+ binding to cardiac native tropomyosin was also measured.     

Essential factor of gizzard "troponin" fraction. A new type of regulatory protein.

The physiological activity of gizzard "troponin" fraction ((1975) J. Biochem. 78, 859) was shown to be due to the 80,000 dalton component.     

Production of nitrite ions from trinitrophenyl myosin and from trinitrophenyl subfragment-1

When trinitrophenyl (TNP) myosin of either chicken breast muscle or porcine cardiac muscle was left to stand in an alkaline medium at 20 degrees C for several hours, nitrite ions were found to be gradually produced. The nitrite production from myosin trinitrophenylated in the presence of PPi occurred at the same rate and to the same extent as that from myosin trinitrophenylated in the absence of PPi. The nitrite production was significantly reduced when thiols of myosin were modified with 2-nitro-5-thiocyanobenzoate. Four different preparations of TNP subfragment-1, S1(Aa), S1(Ab), S1(Ba), and S1(Bb), were obtained from chymotryptic digest of chicken breast myosin trinitrophenylated in the absence of PPi. When these preparations of TNP-S1 were left to stand at alkaline pH, a significant amount of nitrite was produced from S1(Ab) and S1(Bb), but very little from S1(Aa) and S1(Ba). In our previous report (J. Biochem. 97, 965-968, 1985), S1(Aa) and S1(Ba) were suggested to correspond to "non-burst" heads of myosin, and S1(Ab) and S1(Bb) to "burst" heads of the myosin molecule (Inoue et al. (1980) Adv. Biophys. 13, 1-194). Therefore, the present findings described above strongly suggest that the nitrite production involves some interaction of TNP groups with thiols, and that it occurs at the "burst" heads.     

Pre-steady-state kinetic evidence for a cyclic interaction of myosin subfragment one with actin during the hydrolysis of adenosine 5'-triphosphate.

A single cycle of adenosine 5'-triphosphate (ATP) hydrolysis by a complex of actin and myosin subfragment one (acto-S-1) was studied in a stopped-flow apparatus at low temperature and low ionic strength, using light scattering to monitor the interaction of S-1 with actin and fluorescence to detect the formation of fluorescent intermediates. Our results show that the addition of a stoichiometric concentration of ATP to the acto-S-1 causes a cycle consisting of first, a rapid dissociation of the S-1 from actin by ATP; second, a slower fluorescence change in the S-1 that may be related to the initial phosphate burst; and third, a much slower rate limiting recombination of the S-1 with actin. This latter step equals the acto-S-1 steady-state adenosine 5'-triphosphatase (ATPase) rate at both low and high actin concentrations, and like the steady-state ATPase levels off at a V max of 0.9s-1 at high actin concentration. Therefore, the release of adenosine 5'-diphosphate and inorganic phosphate is not the rate-limiting step in the acto-S-1 ATPase. Rather, a slow first-order step corresponding to the previously postulated transition from the refractory to the nonrefractory state precedes the rebinding of the S-1 to the actin during each cycle of ATP hydrolysis.