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K Kohama

Publications and source records attributed to K Kohama.

At least 109 records · Page 6Linked to original sources
PubMed1987

Effect of N-ethylmaleimide on Ca-inhibition of Physarum myosin.

We have established a quick method for preparing Physarum myosins whose actin-activated ATPase activities are inhibited by microM levels of Ca2+ (from plasmodial stage: Kohama, K. & Kendrick-Jones, J. (1986) J. Biochem. 99, 1433-1446; and from amoebal stage: Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, Y., & Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). N-Ethylmaleimide alkylates sulfhydryl (SH) groups on the heavy chains in the heads of the plasmodial myosin. The actin-activated ATPase activity of the modified myosin was significantly decreased when assayed in low Ca2+ concentrations. Moreover, the activity remained low even when the Ca2+ concentrations was increased, i.e., the myosin was desensitized. For complete desensitization, about 4 mol SH per mol myosin (500,000 Mr) must be modified. These residues are probably the "reactive thiols" which have been predicted from primary structure studies to be conserved among myosins of higher and lower eukaryotes. Ultraviolet absorption spectra of the modified and intact myosins showed a peak at 277 nm. The height of this peak in intact myosin was reduced when the Ca2+ concentration was increased. This Ca-induced reduction was hardly detectable in the modified myosin although Ca-binding activity to myosin did not appear to be affected by the modification. We interprete these results that Ca2+ may change the conformation of the myosin heavy chain by binding to myosin and speculate that impairment of this process upon modification could cause the desensitization to Ca2+ in the ATPase activity.

Actins↗
PubMed1987

Native pyrophosphate gel analysis of smooth muscle myosin phosphorylated with protein kinase C.

We have shown that the phosphorylation of smooth muscle regulatory myosin light chain (L20) with myosin light chain kinase (MLCK) produces faster moving bands (GMP1: heterodimer myosin with 1 unphosphorylated L20 and 1 mono-phosphorylated L20, GMP2: homodimer myosin with 2 mono-phosphorylated L20S) on native pyrophosphate polyacrylamide gel electrophoresis (PP1 PAGE) (J. Biochem. 100, 259-268, 1986; J. Biochem. 100, 1681-1684, 1986). However, the mobility of the myosin phosphorylated, at its L20, with protein kinase C (PK-C) was the same that of the unphosphorylated myosin (GM) on PPi PAGE. When the myosin prephosphorylated with MLCK was further phosphorylated with PK-C, PPi PAGE analysis showed only one band comigrating with GM, i.e., GMP1 and GMP2 migrated to the same position as GM. Conversely, when the myosin prephosphorylated with PK-C was further phosphorylated with MLCK, GMP1 and GMP2 were not produced. Thus the effect of L20 phosphorylated with PK-C is quite the opposite of that with MLCK, and the former predominated over the latter. We speculate that phosphorylation of L20 with PK-C "freezes" myosin in the inactive state.

Animals↗
PubMed1987

Inhibition by amiloride of contractile elements in smooth muscle of guinea pig taenia cecum and chicken gizzard.

The relaxant action of amiloride was investigated in the smooth muscles of guinea pig taenia ceci and chicken gizzard. Amiloride inhibited the contractions induced by high K+ (45.4 mM) and carbachol (10 microM) in the taenia with the concentrations needed to induce 50% inhibition (IC50) of approximately 41 microM. A prolonged incubation period (greater than 1 hr) was necessary to obtain the full inhibition of these contractions. The taenia gradually accumulated amiloride and the tissue/medium ratio exceeded 2.0 after a 120-min incubation period. Amiloride had no effect on the high K+-stimulated 45Ca++ uptake or the ATP content of the taenia. Amiloride inhibited the Ca++-induced contraction of the saponin-treated taenia with an IC50 of 186 microM. Amiloride (10-1000 microM) also inhibited superprecipitation and Mg++-adenosine triphosphatase activity of the gizzard native actomyosin as well as the phosphorylation of myosin light chain. The inhibition of the phosphorylation was antagonized competitively by ATP. Amiloride (1 mM) had no effect on the dephosphorylation of myosin light chain upon removal of Ca++ from reaction medium. Amiloride, at concentrations up to 1 mM, had not effect on calmodulin activity as monitored by the Ca++-calmodulin-activated erythrocyte membrane (Ca++ + Mg++)-adenosine triphosphatase and phosphodiesterase activities. In contrast to this, trifluoperazine inhibited the calmodulin activity at the concentration needed to inhibit the Ca++-induced contraction of the permeabilized taenia and the superprecipitation and the phosphorylation of myosin light chain of gizzard. We conclude that amiloride, unlike trifluoperazine, may inhibit directly the myosin light chain kinase activity to induce muscle relaxation.

Actomyosin↗
PubMed1987

Calcium-independent phosphorylation of smooth muscle myosin light chain by okadaic acid isolated from black sponge (Halichondria okadai).

Previously, we have shown that okadaic acid (OA), isolated from black sponge (Halichondria okadai) causes contraction even in the absence of Ca++ in the saponin-permealized taenia isolated from guinea pig cecum. In the present study, mechanism of action of OA was examined using native actomyosin extracted from chicken gizzard smooth muscle. In the absence of Ca++, OA (0.1-1 microM) induced superprecipitation and increased the Mg++-adenosine triphosphatase activity. The OA-induced superprecipitation was enhanced by Ca++ at a concentration (greater than 0.1 microM) which did not activate the calmodulin-dependent myosin light chain (MLC) kinase. The effect of OA was not affected by the calmodulin inhibitor, trifluoperazine, at a concentration (100 microM) needed to inhibit the Ca++-induced response, but was inhibited markedly by the nonselective kinase inhibitors, amiloride (1 mM) and K-252a (5 microM). The OA-induced superprecipitation in the absence of Ca++ was accompanied by phosphorylation of the 20 K dalton MLC, which also was enhanced by low concentration of Ca++ (greater than 0.1 microM). OA did not change the phosphatase activity which dephosphorylates the phosphorylated MLC. An activator of Ca++- and phospholipid-dependent protein kinase, 12-O-tetradecanoylphorbol 13-acetate (1 microM), did not modulate superprecipitation or phosphorylation of MLC in the presence and absence of OA. Furthermore, inhibitors of Ca++ and phospholipid-dependent protein kinase, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (400 microM) and polymyxin B (100 micrograms/ml), affected neither superprecipitation nor phosphorylation of MLC induced by OA. With a reconstituted system containing purified myosin and MLC kinase, OA induced only slight phosphorylation of MLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Isolation and characterization of myosin from amoebae of Physarum polycephalum.

Myosin was isolated from amoebae of Physarum polycephalum and compared with myosin from plasmodia, another motile stage in the Physarum life cycle. Amoebal myosin contained heavy chains (Mr approximately 220,000), phosphorylatable light chains (Mr 18,000), and Ca2+-binding light chains (Mr 14,000) and possessed a two-headed long-tailed shape in electron micrographs after rotary shadow casting. In the presence of high salt concentrations, myosin ATPase activity increased in the following order: Mg-ATPase activity less than K-EDTA-ATPase activity less than Ca-ATPase activity. In the presence of low salt concentrations, Mg-ATPase activity was activated approximately 9-fold by skeletal muscle actin. This actin-activated ATPase activity was inhibited by micromolar levels of Ca2+. Amoebal myosin was indistinguishable from plasmodial myosin in ATPase activities and molecular shape. However, the heavy chain and phosphorylatable light chains of amoebal myosin could be distinguished from those of plasmodial myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunological studies, suggesting that these are different gene products. Ca2+-binding light chains of amoebal and plasmodial myosins were found to be identical using similar criteria, supporting our hypothesis that the Ca2+-binding light chain plays a key role in the inhibition of actin-activated ATPase activity in Physarum myosins by micromolar levels of Ca2+.

Adenosine Triphosphatases↗
PubMed1986

The mobility of gizzard myosin in pyrophosphate gel electrophoresis is increased by its light chain phosphorylation.

Phosphorylation of the 20,000 Mr light chain (L20) of gizzard myosin reversibly increased the mobility of myosin in pyrophosphate polyacrylamide gel electrophoresis (PP1 PAGE). Gizzard heavy meromyosin (HMM) with phosphorylated L20 also moved faster than that with unphosphorylated L20. This mobility increase of HMM is large enough to account for that of intact myosin. Scallop myosin, desensitized by removing its regulatory light chain, was combined with L20 and subjected to PPi PAGE. Hybrid myosin with the phosphorylated light chain moved faster than that with the unphosphorylated light chain. No such effect of light chain phosphorylation was observed with phosphorylatable light chain from breast or ventricular myosin. Thus, gizzard, but not breast or ventricular phosphorylatable light chain is furnished with the 'regulatory' property that is phosphorylation increases myosin mobility in PPi PAGE.

Animals↗
PubMed1986

Analysis of smooth muscle myosin phosphorylation with native pyrophosphate gels and its application to studies on myosin phosphorylation in contracting smooth muscle.

Gizzard smooth muscle myosin, the 20,000 Mr light chain (L20) of which had been phosphorylated in vitro with a calmodulin-myosin light chain kinase system, was separated into 5 isolated bands in a pyrophosphate polyacrylamide gel. Their mobilities were in the following order: myosin with 2 unphosphorylated L20 (GM) less than myosin with 1 unphosphorylated and 1 mono-phosphorylated L20 (GMP1) less than myosin with 2 mono-phosphorylated L20 (GMP2) less than myosin with 1 mono-phosphorylated and 1 di-phosphorylated L20 (GMP3) less than myosin with 2 di-phosphorylated L20 (GMP4). We used this pyrophosphate polyacrylamide gel electrophoresis to analyze the phosphorylated state of taenia coli smooth muscle during K+-induced contraction. During the initial 2 min contraction, phosphorylated forms corresponding to GMP1 and GMP2 were detected in addition to the unphosphorylated form.

Animals↗
PubMed1986

The inhibitory Ca2+-regulation of the actin-activated Mg-ATPase activity of myosin from Physarum polycephalum plasmodia.

Myosin was rapidly prepared from the slime mould, Physarum polycephalum to a high level of homogeneity (greater than 95%), in a high yield (about 10 mg/100 g tissue) and in a phosphorylated state (about 5 mol phosphate/mol of 500,000 Mr myosin). Actin activated the Mg-ATPase activity of this myosin in the absence of Ca2+ about 30-fold, and this actin-activated ATPase activity was reduced to about 20% of the original activity when Ca2+ concentration was increased to 50 microM, i.e., the actin-myosin-ATP interactions show Ca-inhibition. The Ca2+ concentration giving half-maximum inhibition was 1-3 microM. The Ca-inhibition was clearly observed at physiological concentrations of Mg2+ but was obscured at both lower and higher concentrations of Mg2+. The Ca-inhibitory effect on ATP hydrolysis by actomyosin reconstituted from skeletal actin and Physarum myosin was quick and reversible. Ca-binding measurement showed that myosin bound Ca2+ with half-maximal binding at 2 microM Ca2+ and maximum binding of 2 mol per mol myosin, indicating that Ca2+ may inhibit the ATPase activity by binding to myosin. The involvement of this myosin-linked regulatory system in the Ca2+ -control of cytoplasmic streaming is discussed.

Actins↗
PubMed1986

Superprecipitation is a model for in vitro contraction superior to ATPase activity.

We investigated which activity of myosin B (natural actomyosin), super-precipitation or ATPase, is better suited as a parameter for measuring in vitro contraction with emphasis on the response of myosin B to Sr2+. The Sr2+ concentration giving half-maximum (KSr2+) binding of Sr2+ to cardiac troponin was not different from that to skeletal troponin, whether the troponins were contained in the actomyosin system or in the isolated state. However, KSr2+ for the ATPase activity of cardiac myosin B is slightly smaller than that of skeletal myosin B. This difference in KSr2+ is more marked when superprecipitation was used as the indication for in vitro contraction. If the above results were compared with those of Kitazawa (1976) using glycerinated fibers of cardiac and skeletal muscle, the differences in KSr2+ between cardiac and skeletal muscle increased in the order: Sr-binding to troponin less than ATPase activity less than superprecipitation less than contraction of glycerinated fibers. The increase in the effect of pH on Ca2+-related properties is also in the order: Ca-binding to troponin less than or equal to ATPase activity less than superprecipitation less than contraction of glycerinated fibers. All the results coincide with one another that the results with super-precipitation are invariably more akin to those with glycerinated fibers than to those with the ATPase activity. It is concluded that superprecipitation is a better parameter for representing in vitro contraction than ATPase activity.

Actomyosin↗
PubMed1982

Regulatory mechanism in smooth muscle: actin-linked regulation.

Our view about the Ca regulation of smooth muscle contraction can be summarized as follows: the regulatory system is actin-linked and consists of tropomyosin and leiotonin; the latter is a complex of two proteins, leiotonin A, the regulatory moiety, and leiotonin C, the Ca-binding moiety. Leiotonin resembles troponin in some respects, but is clearly different in that it has no affinity for tropomyosin and is effective at a leiotonin/actin molar ratio of less than 1:50. The methods of preparing leiotonin were critically reviewed and a suggestion was provided for the development of a new procedure. References were also made to our idea that the myosin light chain phosphorylation may not play an essential role in smooth muscle contraction. With reference to a new type of actin-linked factor from Physarum polycephalum through which Ca2+ represses the actin-myosin-ATP interaction, the diversity of the modes of action of Ca2+ was discussed.

Actins↗
PubMed1981

Purification and characterization of a 43,000 dalton "DNAase binding protein" distinct from actin.

"DNase binding protein" of 43K daltons as determined by SDS-polyacrylamide gel electrophoresis, was purified from the 0.1 M KCl-soluble (non-structural) fraction of chicken skeletal muscle. The protein was distinct from actin in amino acid composition and physicochemical properties. "DNase binding protein" was also isolated from other kinds of muscle and non-muscle cells. The ratio of the amino acid incorporation rate of "DNAase binding protein" to that of actin is different skeletal and smooth muscle and non-muscle cells.

Actins↗
PubMed1980

Heterogeneity of amino acid incorporation rate in adult skeletal muscle actin.

An actin preparation extracted at 25 degree C for 2 h from the residue which had previously been subjected to the routine extraction procedure, i.e. at 4 degree C for 15 min, showed markedly increased rates of amino acid incorporation, i.e. 48% higher than routinely extracted actin. The nature of the actin having higher amino acid incorporation rates was discussed in relation to 10S-actinin.

Actins↗
PubMed1979

Divalent cation binding properties of slow skeletal muscle troponin in comparison with those of cardiac and fast skeletal muscle troponins.

1. New methods of preparing troponins from slow skeletal and cardiac muscle of the chicken have been developed. The electrophoretic mobilities of slow skeletal muscle troponin subunits were different from those of the corresponding fast skeletal muscle subunits. 2. A new method for determining the amount of divalent cations bound to troponin was developed. The principle of the method is to immobilize troponin by conjugating it with Sepharose 4B resin, thus making it readily sedimentable. 3. The numbers of Sr and Ca ions bound to slow muscle troponin at concentrations sufficient to produce maximum contraction were 1.73 and 1.36 mol per mol, respectively, being nearly equal to those of cardiac troponin but half of those of fast muscle troponin. 4. The concentrations of Sr and Ca ions giving half-maximal ion binding to slow muscle troponin (K50%) were 5.5 X 10(-6) M and 4.6 X 10(-7) M, respectively. 5. K50% for Sr of cardiac troponin was significantly higher than that of slow muscle troponin. Although K50% for Sr of cardiac troponin was the same as that of fast muscle troponin, cardiac troponin bound more Sr ions than fast muscle troponin at lower Sr ion concentrations. The mechanism underlying the high sensitivity of cardiac muscle contraction to Sr ions is discussed in comparison with that of slow muscle.

Animals↗