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K Kohama

Publications and source records attributed to K Kohama.

At least 91 records · Page 5Linked to original sources
PubMed1991

A protein-bound polysaccharide from Basidiomycetes enhances myosin phosphorylation but inhibits myosin ATPase activity: studies with a crude actomyosin preparation of chicken gizzard smooth muscle.

Chicken gizzard actomyosin, containing the calmodulin-myosin light chain kinase (MLCK) system, was incubated in the presence of various concentrations of PSK, a protein-bound polysaccharide from Basidiomycetes, together with Ca2+ and Mg-ATP. The phosphorylation of myosin was enhanced half-maximally by 10-4 g/ml of PSK. However, a similar concentration of PSK reduced the Mg-ATPase activity of the actomyosin. The former was brought about through stimulation of the MLCK activity and the latter through inhibition of the myosin ATPase activity.

Actomyosin↗
PubMed1991

Identification of plasmid partition function in coryneform bacteria.

We have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. This fragment was able to stabilize the unstable plasmids in cis but not in trans.

Base Sequence↗
PubMed1991

A myosin-like protein from smooth muscle.

A protein was purified from chicken gizzard smooth muscle. It bound ATP and actin. Actin activated the Mg2(+)-ATPase activity of this protein. The Ca2(+)-ATPase activity was lower than K(+)-EDTA ATPase activity. Thus, it appears that this protein is akin to myosin I rather than to conventional myosin. However, ATPase activities of the protein were much lower than those of myosin I. A protein cofactor, such as protein kinase, which would enhance these activities remains to be purified from the smooth muscle.

Actins↗
PubMed1991

[Calcium inhibition, a new mode for Ca2+ regulation].

The intracellular concentration of Ca2+ ([Ca2+]i) in eukaryotic cells is low under resting conditions. Membrane stimulation can produce a transient increase in [Ca2+]i from submicromolar to micromolar levels. Classically, the effects of elevated [Ca2+]i on eukaryotic cell responses are excitatory: they induce muscle contraction or activate metabolic systems. However, micromolar concentration of Ca2+ has been found to inhibit the actin-myosin-ATP interaction of the lower eukaryote Physarum polycephalum, which is the first example of an effect transiently abolished by elevation of [Ca2+]i in response to extracellular stimulation (Kohama, K.: Trends Pharmacol. Sci. 22, 433-435, 1991). The inhibitory effects on the interaction are closely related to myosin phosphorylation. The activities of kinases to phosphorylate myosin was also inhibited by Ca2+. This article describes the inhibitory mode for Ca2+ regulation of the actomyosin system in Physarum and reviews this inhibitory mode discovered subsequently in many different cell types in both plants and animals.

Actins↗
PubMed1991

Inhibitory Ca(2+)-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: role of a Ca(2+)-dependent inhibitory factor.

ATP-dependent interactions between myosin and actin in the lower eukaryote, Physarum polycephalum, are inhibited by micromolar levels of Ca2+. This inhibition is mediated by the binding of Ca2+ to myosin, the phosphorylation of which is required if Ca2+ is to inhibit the activities of myosin (Kohama, K., Trends Pharmacol. Sci. 11, 433-435 (1990)). As the first step to examine whether Ca2+ also regulates phosphorylation in the actomyosin system, we purified myosin light chain kinase (MLCK) of 55 kDa almost to homogeneity. The MLCK activity was high whether or not Ca2+ was present. However, a Ca(2+)-dependent inhibitory factor (CIF) purified from Physarum (Okagaki et al., Biochem. Biophys. Res. Commun. 176, 564-570 (1991)) was shown to reduce the MLCK activity in a Ca(2+)-dependent manner. Using crude preparations, not only MLCK but also myosin heavy chain kinase and actin kinase were shown to be inhibited by Ca2+ half-maximally at micromolar levels. Since CIF is the only Ca(2+)-binding protein in the preparations, we propose that this inhibitory Ca(2+)-regulation of the kinases for actomyosin is mediated by CIF.

Animals↗
PubMed1989

Direct inhibition of contractile apparatus by analogues of amiloride in the smooth muscle of guinea-pig taenia caecum and chicken gizzard.

The relaxant effects of amiloride and its analogues, benzamil, 5-(N,N-diethyl)-amiloride (DEAM) and 5-(N-ethyl-N-isopropyl)-amiloride (EIAM), were investigated using smooth muscle of guinea-pig taenia caeci and chicken gizzard. High K+-induced contractions of intact taenia and gizzard were inhibited by these compounds (1-100 microM) with the order of potency; benzamil greater than or equal to EIAM greater than DEAM greater than amiloride. Contractions of permealized taenia and gizzard were also inhibited by these compounds at concentrations 8-35 times higher than those needed to inhibit the contractions of intact tissues. These compounds inhibited 20 K myosin light chain (MLC) phosphorylation at the concentrations needed to inhibit the contraction in the permealized muscles. Calmodulin (CaM) activity, as monitored by erythrocyte membrane (Ca2+ + Mg2+)-ATPase and phosphodiesterase activities, was inhibited by DEAM and EIAM at similar concentrations as those to inhibit the MLC phosphorylation. Benzamil also inhibited CaM activity at concentrations 4-8 times higher than those required to inhibit MLC phosphorylation. However, amiloride failed to inhibit CaM activity. Among these compounds, amiloride and benzamil inhibited Ca2+/CaM-independent MLC phosphorylation due to trypsin-treated MLC kinase. Taenia tissue gradually accumulated these compounds and the tissue/medium ratio exceeded 3.5-17 after a 3-hr incubation period. These results indicate that amiloride and its analogues inhibit smooth muscle contraction mainly by the direct inhibition of MLC phosphorylation. The inhibitory effect of amiloride may be attributable to the inhibition of MLC kinase, whereas the inhibitory effect of DEAM and EIAM may largely be attributable to the inhibition of CaM. Benzamil may inhibit contraction by the inhibition of both MLC kinase and CaM. Differences in the drug-sensitivity between intact and permealized tissues may be attributable to the difference in drug accumulation by the cell.

Amiloride↗
PubMed1989

Cross-reactivity of an antineurofilament antibody with a troponin-T isoform.

Histochemical examination has revealed that a monoclonal antibody raised against the 200-kilodalton (kd) subunit of neurofilament reacts not only with neural tissue but with type 1 and type 2C fibers of skeletal muscle. To identify the cross-reactive substance(s), rat soleus muscle, which includes type 1 and type 2C fibers, was separated into a soluble fraction and an insoluble myofibrillar fraction. Western blot analysis demonstrated that the antibody reacted with 30-kd and 36-kd polypeptides in the soluble fraction and with a 38-kd polypeptide in the insoluble fraction. We isolated a fraction that contained the 38-kd polypeptide from the insoluble fraction. The material in this fraction bound to F-actin. Two-dimensional electrophoresis of the fraction showed that the 38-kd polypeptide was part of the group of isoforms of troponin-T, which was identified by a monoclonal antibody developed against troponin-T. Thus, we conclude that this antineurofilament antibody recognizes one of the isoforms of troponin-T in type 1 and type 2C fibers.

Animals↗
PubMed1989

Ca2+ activates actin-filament sliding on scallop myosin but inhibits that on Physarum myosin.

The actin-activated ATPase activity of Physarum myosin has been shown to be inhibited by microM levels of Ca2+, the mode of which is in contrast to the activating effect of Ca2+ on scallop myosin (Kohama, K. (1987) Adv. Biophys. 23, 149-182 for a review). To determine if Ca2+ regulates ATP-dependent sliding between actin and the myosins, fluorescent actin-filaments were allowed to move on the myosins fixed to a glass surface. The movement on Physarum and scallop myosins was inhibited and activated, respectively, by Ca2+. For this myosin-linked regulation to occur for Physarum myosin, myosin phosphorylation was shown to be a prerequisite.

Actins↗
PubMed1988

Amino acid sequence of the calcium-binding light chain of myosin from the lower eukaryote, Physarum polycephalum.

We have established a new method for preparing Physarum myosin whose actin-activated ATPase activity is inhibited by micromolar levels of Ca2+. This Ca2+-inhibition is mediated by the Ca2+ binding to the myosin rather than by the Ca2+-dependent modification of the phosphorylated state of the myosin (Kohama, K., and Kendrick-Jones, J. (1986) J. Biochem. (Tokyo) 99, 1433-1446). Ca2+-binding light chain (CaLC) has been suggested to be primary importance in this Ca2+ inhibition (Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, T., and Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). The amino acid sequence of CaLC was determined; it was composed of 147 amino acid residues and the N terminus was acetylated. The molecular weight was calculated to be 16,131. The homology of CaLC in the amino acid sequence with 5,5'-dithiobis-(2-nitrobenzoic acid) light chain and alkali light chain of skeletal muscle myosin were rather low, i.e., 25% and 30%, respectively. Interestingly, however, the CaLC sequence was 40% homologous with brain calmodulin. This amino acid sequence was confirmed by sequencing the cloned phage DNA accommodating cDNA coding CaLC. Northern and Southern blot analysis indicated that 0.8-kilobase pair mRNA was transcribed from a single CaLC gene. This is the first report on the amino acid sequence of myosin light chain of lower eukaryotes and nucleotide sequence of its mRNA.

Amino Acid Sequence↗
PubMed1988

Purification of myxamoebal fragmin, and switching of myxamoebal fragmin to plasmodial fragmin during differentiation of Physarum polycephalum.

We have isolated and purified an activity from amoebae of Physarum polycephalum that reduces the flow birefringence of a solution of F-actin in a Ca2+-dependent manner. The purified activity from 100 g of amoebae consisted of 1 mg of a 40,000 mol. wt protein. DNase I-affinity chromatography demonstrated that the protein binds to Physarum actin in a Ca2+-dependent manner, and the binding is not reversed by excess EGTA. Viscometric measurement indicated that the protein (i) accelerates polymerization of G-actin, and (ii) severs F-actin, in a Ca2+-dependent manner. Thus, the protein appeared functionally similar to the fragmin previously isolated from Physarum plasmodia (plasmodial fragmin). However, the two proteins had slightly different mobilities on urea-SDS-PAGE, and antibodies raised against the two proteins scarcely cross-reacted with each other. Hence, we conclude that the two proteins are closely related to but are different from each other, and we have named the novel protein 'myxamoebal fragmin'. Immunoblot analysis indicated that myxamoebal and plasmodial fragmins are specifically present in amoebae and plasmodia, respectively. Results of immunofluorescence staining suggest that the synthesis of plasmodial fragmin is switched on coordinately with the synthesis of the heavy chain of plasmodial myosin and other plasmodium-specific contractile proteins during the apogamic differentiation of amoebae to plasmodia.

Actins↗
PubMed1988

Physarum myosin light chain interacts with actin in a Ca2+-dependent manner.

Actin-modulating activity was analysed with the 16,131-dalton calcium-binding light chain (CaLc, Kobayashi et al. (1988) J. Biol. Chem. 263, 305-313) of Physarum myosin, which is under an inhibitory Ca-control (Kohama and Kendrick-Jones (1986) J. Biochem. 99, 1433-1446). When skeletal muscle actin was polymerized in the presence of CaLc and Ca2+, increases in both viscosity and birefringence were reduced under high shear conditions. However, CaLc did not inhibit actin polymerization under no or low shearing forces, which was demonstrated by a variety of methods including fluorescence intensity measurements using pyrenyl actin. We propose that actin polymerized in the presence of CaLc and Ca2+ is easily fragmented under high shearing forces to produce the changes in viscosity and birefringence.

Actins↗
PubMed1987

Direct activation by okadaic acid of the contractile elements in the smooth muscle of guinea-pig taenia coli.

Okadaic acid isolated from black sponge (Halichondria okadai), at the concentration of 10 mumol/l, caused contraction in saponin-treated skinned smooth muscle of guinea-pig taenia coli in the absence of Ca2+. In the presence of low concentration (0.3 mumol/l) of Ca2+, okadaic acid induced a greater contraction than in the absence of Ca2+. Okadaic acid potentiated the contractions induced by Ca2+ and pCa2+-tension curve was shifted to the left as well as upward by 1 mumol/l okadaic acid. Native actomyosin preparation (myosin B) containing calmodulinmyosin light chain kinase system and phosphatase was obtained from taenia coli. Okadaic acid (10 mumol/l) increased the actomyosin Mg2+-ATPase activity in the presence or absence of Ca2+. Okadaic acid (1-100 mumol/l) had no effect on calmodulin activity as monitored by Ca2+-calmodulin activated cyclic nucleotide phosphodiesterase activity and the (Ca2+ + Mg2+)-ATPase activity or erythrocyte membranes. These results suggest that okadaic acid directly activates contractile elements of smooth muscle.

Animals↗
PubMed1987

Myosin switching during amoebo-plasmodial differentiation of slime mold, Physarum polycephalum.

We reported previously that myosins from amoebal and plasmodial stages in the life cycle of Physarum polycephalum differ in the primary structure of heavy chains and phosphorylatable 18,000 Mr light chains, while Ca-binding 14,000 Mr light chains are common to both myosins (Kohama & Takano-Ohmuro, Proc Jpn acad 60B (1984) 431; Kohama et al., J biol chem 260 (1986) 8022). We have carried out immunofluorescence microscopical studies upon differentiating cultures of amoebic colonies, which show apogamic amoebo-plasmodial differentiation as follows: Typical amoebae differentiate into mono-nucleate intermediate cells with swollen nuclei and then into two or multi-nucleate young plasmodia (Anderson et al., Protoplasma 89 (1976) 29. Antibodies against plasmodial myosin heavy chain (PMHC) and 18,000 Mr plasmodial myosin light chain (PMLC18) stained intermediate cells and young plasmodia, but not typical amoebae. On the other hand, antibody against amoebal myosin heavy chain (AMHC) stained typical amoebae and intermediate cells--but not young plasmodia. Thus staining was detected using antibodies against both PMHC and AMHC in intermediate cells. Intermediate cells were also stained by antibody against another plasmodium-specific cytoskeletal protein, viz., high molecular weight actin-binding protein (HMWP). We propose that synthesis of myosin subunits switches immediately from amoebal to plasmodial type in mono-nucleate cells with swollen nuclei. This myosin switching is associated with the initiation of HMWP synthesis.

Antibodies↗
PubMed1987

Ca-inhibitory myosins: their structure and function.

Actin-myosin-ATPase interaction of Physarum is under inhibitory Ca-control. Ca-binding to myosin inhibits the interaction, while Ca-release from myosin removes inhibition. However, this myosin-linked control system alone is not sufficient to account for in vivo contraction. A Ca-dependent inhibitory factor, whose active principle appears to be identical with or similar to Ca-Lc of myosin, augments myosin-linked control by interacting with actin. This novel regulatory system may not be specific for the actomyosin system in Physarum plasmodia, because some actomyosin-related processes, i.e., secretion and motility, are subject to inhibitory Ca-control. Accumulation of examples for the inhibitory Ca-control in higher and lower eukaryotes will clarify its phylogenic significance.

Actins↗