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H Onishi

Publications and source records attributed to H Onishi.

18 recordsLinked to original sources
PubMed1979

Chicken gizzard heavy meromyosin that retains the two light-chain components, including a phosphorylatable one.

A method was developed to obtain a preparation of chicken gizzard heavy meromyosin (HMM) that retains the two light-chain components of parent myosin: the 20,000-dalton and 17,000-dalton light-chains. The HMM preparation was also shown to retain two characteristics of the ATPase activity of the parent myosin: the characteristic effect of phosphorylation of the 20,000-dalton light-chain component on the ATPase activity, and the characteristic dependence of the ATPase activity on the KCl concentration. 1. Two distinct stages were observed in the Mg-ATPase reaction catalyzed by gizzard HMM and rabbit skeletal actin in the presence of gizzard "native" tropomyosin (NTM) and Ca2+ ions: an early lag phase, in which the reaction rate gradually increased, and a subsequent steady state, in which the reaction proceeded at a high, constant rate. Urea-gel electrophoresis revealed that the 20,000-dalton light-chain component was gradually phosphorylated in the lag phase, and was fully phosphorylated in the steady state. It was also observed that addition of EGTA (to remove Ca2+ ions) at various times in the lag phase caused neither a further increase nor a decrease in the reaction rate, and that addition of EGTA in the steady state caused no change in the reaction rate. These observations imply that the ATPase activity increased as the amount of phosphorylated 20,000-dalton light-chain component increased, and also that Mg-ATPase of acto-phosphorylated HMM was no longer calcium-sensitive. 2. The Mg-ATPase activity of HMM in the presence of gizzard NTM and Ca2+ ions or EGTA was studied as a function of the concentration of rabbit skeletal actin. The maximal activity (Vmax) and the apparent affinity constant of acto-HMM (KA) were thus estimated from the double-reciprocal plot of Eisenberg-Moos: the Vmax and KA values for phosphorylated HMM (in the presence of Ca2+ ions) were 5 S(-1) and 5.5 mg/ml actin, respectively, and the Vmax value for unphosphorylated HMM (in the presence of EGTA) was 0.3 S(-1), assuming that the KA value with unphosphorylated HMM is equal to that with phosphorylated HMM.

Actins↗
PubMed1979

Calcium regulation in chicken gizzard muscle and inosine triphosphate-induced superprecipitation of skeletal acto-gizzard myosin.

Inosine triphosphate (ITP) does not serve as a substrate for myosin light-chain kinase from gizzard muscle. That is to say, myosin light-chain is not phosphorylated in ITP media. Nevertheless, at pH 6.8, 1 mM or 5 mM ITP induces superprecipitation of skeletal acto-gizzard myosin. The ITP-induced superprecipitation occurs in the absence or presence of calcium ions, and regardless of whether gizzard myosin is phosphorylated or not. On the other hand, at pH 8, 5 MM ITP induces practically no superprecipitation of skeletal acto-gizzard unphosphorylated myosin, whereas it does induce a strong superprecipitation of skeletal acto-gizzard phosphorylated myosin. Superprecipitation is also independent of the presence or absence of calcium ions.

Actins↗
PubMed1979

A medium for commercial production of the halophilic Micrococcus nuclease.

A simple synthetic medium (glutamate-sucrose medium) was devised for production, during growth in shaken flasks, of extracellular halophilic nuclease (nuclease H) by a moderate halophile, Micrococcus varians subsp. halophilus. A simple medium consisting of 0.7% ammonium sulfate, 1.0% glucose, minerals, three vitamins, and 2 M NaCl gave good growth and excellent production of nuclease H in a jar fermentor when the pH was adjusted to 7.5 to 8.0 during cultivation.

Culture Media↗
PubMed1978

The ATPase reaction in the steady state and in the initial burst catalyzed by chicken gizzard myosin in 0.6 M KCl1.

On studying the steady-state activity in 0.6 M KCl, it was found that Mg-ATPase of chicken gizzard myosin was identical with that of rabbit skeletal myosin in the pH-activity profile, Michaelis-Menten constant, and maximum velocity. As regards the "initial burst" of ATP splitting in the presence of Mg (0.6 M KCl), it was found that gizzard and skeletal myosins were identical both in the size of the initial burst and in the velocity-ATP concentration relationship. The only difference we observed was that the Ca- and EDTA-ATPase activities of gizzard myosin were, as reported by other investigators, approximately one-half to one-third of those of skeletal myosin, although the pH-activity profiles for the ATPase of gizzard myosin was essentially the same as that of skeletal myosin.

Adenosine Triphosphatases↗
PubMed1978

Adenosine triphosphatase activity and "thick filament" formation of chicken gizzard myosin in low salt media.

The ATPase activity of chicken gizzard myosin was studied by varying the KCl concentration in the reaction medium. The following was thus found: (a) A sharp depression of the activity occurred when the KCl concentration was reduced to less than 0.3 M, showing the minimum activity around 0.15 M KCl. (b) The activity depression was removed by addition of urea or bay papain-digestion, but not by addition of p-chloromercuribenzoate. (c) In the KCl concentration where the activity depression occurred, the ATPase reaction proceeded in two distinct phases; the activity was relatively high in the early phase of the reaction and declined into the later phase where the steady state reaction took place. (d) In the KCl concentrations higher than that particular concentration or in the presence of urea, the ATPase reaction proceeded in one phase. (e) The temperature dependence of the ATPase activity in the early phase was of an ordinary magnitude being approximately equal to that of the ATPase activity in 0.6 M KCl. In contrast, the temperature dependence of the activity in the later phase was unusually small. Gizzard myosin in various concentrations of KCl was also examined by measuring the turbidity and the light-scattering intensity, and by observation under an electron microscope. The following was thus found: (a) In the KCl concentration where the activity depression occurred, there was a stagnation in the turbidity decrease as the KCl concentration was gradually increased and also the formation of "thick filaments," each of which was approximately 0.6-0.9 micron in length and 20-30 nm in diameter with no central "bare zone." (b) Addition of ATP induced dissociation of the thick filaments, and the dissociation occurred during the early phase of the ATPaseeaction. (c) Moreover, the temperature dependence of the ATP-induced dissociation rate was approximately equal to that of the ATPase activity in the early phase. On the basis of the findings mentioned above, it is concluded that the activity depression results from the ATP-induced dissociation of myosin filaments. Moreover, since high concentrations of KCl or urea also caused dissociation of myosin filaments and yet did not produce the activity depression, it was strongly suggested that gizzard myosin in the ATP-dissociated form must be different from that in the urea- or KCl-dissociated form, probably in the physical state of some myosin aggregates which were not detectable by the physical methods we used. As a side-observation, gizzard myosin filaments formed in the presence of ADP were found to be unusually long (longer than 2 micron), and they looked very similar to the particular filaments of skeletal myosin that were reported, by Moos, to be formed in the absence of the C protein.

Adenosine Diphosphate↗
PubMed1978

Calcium sensitivity of contractile proteins from chicken gizzard muscle.

Actin, myosin, and "native" tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same wasy as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca2+. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca2+. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg2+) in the presence of Ca2+, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg2+) in the presence of Ca2+. In either case, the light-chain phosphorylation required Ca2+. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca2+ and NTM or tropomyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca2+ or in its absence, but did superprecipitated in the presence of NTM and Ca2+. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca2+ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca2+. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.

Actins↗
PubMed1978

Structure and function of chicken gizzard myosin.

In our previous study (Onishi, H., Susuki, H., Nakamura, k., and Watanabe, S. J. Biochem. 83, 835-847, 1978), we found it to be characteristic of chicken gizzard myosin that thick filaments of gizzard myosin are readily disassembled by a stoichiometric amount of ATP (3 mol of ATP per mol of myosin), and that the ATPase activity of gizzard myosin in the ATP-disassembled state is much lower than that of gizzard myosin disassembled by a high concentration of KCl. We now report the following findings: (1) Thick filaments of (unphosphorylated) gizzard myosin can be in a bipolar structure or in a non-polar structure, depending on the method of preparing the thick filaments. (2) Thick filaments of (unphosphorylated) gizzard myosin in either the bioplar or the non-polar structure are readily disassembled by ATP. (3) Addition of rabbit skeletal C-protein does not confer ATP resistance on thick filaments of (unphosphorylated) gizzard myosin. (4) Unphosphorylated) gizzard myosin in the ATP-disassembled state is in a dimeric form as determined by ultracentrifugation. Moreover, 0.2 M KCl-dissociated gizzard myosin in monomeric form is converted to a dimeric form by ATP. (5) The Mg-ATPase activity of (unphosphorylated) gizzard myosin is much lower in its dimeric form (less than one-tenth) than in its monomeric form. The activity depression observed around 0.15 M KCl is therefore due to the formation of myosin dimers. (6) Skeletal L-meromyosin can increase the very low activity of (unphosphorylated) gizzard myosin ATPase at low ionic strength (0.13 M KCl) by forming ATP-resistant hybrid filaments with (unphosphorylated) gizzard myosin, preventing the formation of myosin dimers. (7) Gizzard myosin in which one of the light-chain components is phosphorylated by myosin light-chain kinase can form thick filaments which are resistant to the disassembling action of ATP. (8) Even in the presence of ATP, thick filaments of phosphorylated gizzard myosin do not disassembled into myosin dimers. Accordingly, the ATPase activity of phosphorylated gizzard myosin does not show activity depression at low ionic strength.

Adenosine Triphosphatases↗
PubMed1978

Properties of the halophilic nuclease of a moderate halophile, Micrococcus varians subsp. halophilus.

The halophilic nuclease of Micrococcus varians ATCC 21971 hydrolyzed thymidine 5'-monophospho-p-nitrophenyl ester at a rate that increased with the NaCl concentration up to saturation. The nuclease attacked RNA and DNA exonucleolytically and processively, producing 5'-mononucleotides. The molecular weight of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 99,000, approximately the same as that previously determined for the native enzyme. Examination of amino acid composition showed that acidic amino acids were in high excess over basic amino acids.

Amino Acids↗
PubMed1978

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.

Adsorption↗
PubMed1978

Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp.

A moderately halophilic Acinetobacter sp., capable of producing dextrinogenic amylase, was isolated from sea-sands. Maximum enzyme production was obtained when the bacterium was cultivated aerobically in media containing 1 to 2M NaCl or 1M KCl. Two kinds of amylase, amylases I and II were purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, DEAE-Sephadex A-50 chromatography, and Sephadex G-200 gel filtration. Both enzymes had maximal activity at pH 7.0 in 0.2 to 0.6 M NaCl or KCl at 50 to 55 degrees C. The activities were lost by dialysis against distilled water. Molecular weights for amylases I and II were estimated to be 55 000 and 65 000 respectively by SDS-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose and maltotriose.

Acinetobacter↗
PubMed1977

Phosphorylation and dephosphorylation of a light chain of the chicken gizzard myosin molecule.

Chicken gizzard myosin was incubated with ATP and/or "native" tropomyosin (NTM) of gizzard muscle in the presence or absence of calcium ions. One of the two light chains of the myosin molecule was phosphorylated in the presence of Ca, but not in its absence. The phosphorylated gizzard myosin was dephosphorylated by a crude preparation of myosin light-chain phosphatase obtained from gizzard muscle. In a superprecipitation test in the presence of EGTA, actomyosin reconstituted from dephosphorylated gizzard myosin did not superprecipitate, whereas actomyosin reconstituted from phosphorylated gizzard myosin showed superprecipitation activity which was inhibited by skeletal NTM and reactivated by Ca.

Actomyosin↗