Biomedical subjects

K Kohama

Publications and source records attributed to K Kohama.

At least 55 records · Page 3Linked to original sources

PubMed1996

Modulatory role of drebrin on the cytoskeleton within dendritic spines in the rat cerebral cortex.

Morphological changes in the dendritic spines have been postulated to participate in the expression of synaptic plasticity. The cytoskeleton is likely to play a key role in regulating spine structure. Here we examine the molecular mechanisms responsible for the changes in spine morphology, focusing on drebrin, an actin-binding protein that is known to change the properties of actin filaments. We found that adult-type drebrin is localized in the dendritic spines of rat forebrain neurons, where it binds to the cytoskeleton. To identify the cytoskeletal proteins that associated with drebrin, we isolated drebrin-containing cytoskeletons using immunoprecipitation with a drebrin antibody. Drebrin, actin, myosin, and gelsolin were co-precipitated. We next examined the effect of drebrin on actomyosin interaction. In vitro, drebrin reduced the sliding velocity of actin filaments on immobilized myosin and inhibited the actin-activated ATPase activity of myosin. These results suggest that drebrin may modulate the actomyosin interaction within spines and may play a role in the structure-based plasticity of synapses.

Actins↗

PubMed1996

Myosin light chain kinase: an actin-binding protein that regulates an ATP-dependent interaction with myosin.

Myosin light chain kinase (MLCK) is a key regulator of smooth muscle contraction. The most conspicuous form of regulation is achieved by phosphorylation of the myosin light chain, allowing myosin to interact with actin. This interaction is regulated by actin-binding proteins that modulate actin filaments. In this review Kazuhiro Kohama and colleagues consider MLCK as an actin-binding protein and attempt to shed light on the cross-talk between the different kinds of regulation of the actin-myosin interaction in smooth muscle. An understanding of these mechanisms will assist the development of compounds with therapeutic importance in muscular disorders.

Actins↗

PubMed1996

Inhibition by drebrin of the actin-bundling activity of brain fascin, a protein localized in filopodia of growth cones.

The purification of drebrin, an actin-binding protein that is specifically expressed in embryonic rat brain, was described previously. During the purification of drebrin, we found that an actin-binding protein of 54 kDa was also expressed at high levels in embryonic brain, and this protein was identified by immunoblotting as fascin. To explore the roles of fascin in brain development, we purified fascin from brains of infant rats and characterized it. We found that the actin-binding activity of fascin was strongly inhibited by drebrin. Fascin caused formation of actin bundles, a process that was inhibited in the presence of drebrin, as confirmed by electron microscopy and a low-speed centrifugation assay. In PC12 cells, fascin was localized in the filopodia of growth cones, whereas drebrin was localized in the basal region of growth cones. Our results suggest that fascin might play an important role in the organization of actin in filopodia and that this organization might be regulated by drebrin.

Actins↗

PubMed1995

The fastest actin-based motor protein from the green algae, Chara, and its distinct mode of interaction with actin.

The endoplasmic streaming in Characean cells is an actin-dependent movement. The motor protein responsible for the streaming was partially purified and characterized. It was soluble at low ionic strength, an ATPase of a molecular mass of 225 kDa and activated more than 100 times by muscle F-actin. Surprisingly, in an in vitro motility assay, the motor protein moved muscle F-actin at 60 microns/s, which is similar to the velocity of streaming in a living cell and 10 times faster than muscle myosin. Proteolytic cleavage of actin impaired movement crucially on muscle myosin, but did not affect movement at all on the Chara motor protein, suggesting that the Chara motor protein would interact with actin via a set of sites different from those of muscle myosin.

Actins↗

PubMed1995

Purification of an ATP-dependent actin-binding protein from a lower eukaryote, Physarum polycephalum.

A novel protein with a molecular mass of 55 kDa, as determined by SDS-PAGE, was purified from plasmodia of Physarum polycephalum. The protein bound to actin filaments with a stoichiometry of 0.27 moles per mole of actin with an apparent dissociation constant of 4 x 10(-8) M. In the presence of ATP, the protein dissociated from actin filaments. Adenosine 5-(gamma-thio)triphosphate and adenyl-5'-yl imidodiphosphate also abolished the actin-binding activity of the protein, but GTP did not. Because the cytoplasmic concentration of ATP oscillates in association with the shuttle streaming of the cytoplasm, it is possible that this protein might be involved in the actin-linked regulation of cytoplasmic streaming.

Actins↗

PubMed1995

Reversible effects of okadaic acid and microcystin-LR on the ATP-dependent interaction between actin and myosin.

Okadaic acid, a toxin from black sponge, and microcystin-LR, a toxin from blue-green algae, were found to stimulate and inhibit, respectively, the actin-activated ATPase activity of skeletal muscle myosin. These effects were confirmed by monitoring the sliding movement of actin-filaments on myosin. This technique also enabled us to demonstrate the reversibility of these effects, a property that is essential for their use as a pharmacological tool for the analysis of the mechanochemical characteristics of muscular contraction. The sites of action of both toxins were within the myosin molecule, as demonstrated by monitoring (i) their effects on the intrinsic tryptophan fluorescence of heavy meromyosin and (ii) their ATP-dependent effects on the ATPase activity. The former effects further suggest that myosin heads are their actual sites of action, and the latter effects suggest that they interact with the ATPase active sites located within the heads.

Actins↗

PubMed1995

Myosin light chain kinase from vascular smooth muscle inhibits the ATP-dependent interaction between actin and myosin by binding to actin.

Myosin light chain kinase (MLCK) was prepared from smooth muscle of bovine aorta. MLCK inhibited the ATP-dependent movement of actin filaments on a glass surface coated with smooth muscle myosin that had been phosphorylated. The inhibitory effect was abolished by calmodulin in the presence of Ca2+ (Ca-CaM). The abolition was also observed when the concentration of actin filaments was increased. The inhibitory effect and its abolition were related to the actin-binding activity of MLCK, that is antagonized by Ca-CaM.

Actins↗

PubMed1994

Drebrin, a development-associated brain protein from rat embryo, causes the dissociation of tropomyosin from actin filaments.

Drebrin is a development-associated neuroprotein whose cDNA into fibroblasts causes the formation of dendrite-like structures (Shirao, T., Kojima, N., and Obata, K. (1992) Neuroreport 3, 109-112). To explore molecular functions of drebrin during brain development, we purified drebrin from brains of rat embryos. Drebrin bound to actin filaments at a stoichiometry of 1:5 with a dissociation constant (Kd) of 1.2 x 10(-7) M. It strongly inhibited the actin binding activity of tropomyosin. Excess amounts of tropomyosin also inhibited the drebrin binding to actin filaments, suggesting that drebrin and tropomyosin competitively bind to actin filaments. Further, drebrin inhibited not only the actin binding activity of alpha-actinin but also the actin cross-linking activity of alpha-actinin. Gene transfection experiments revealed that tropomyosin was dissociated from actin filaments in drebrin-overexpressing fibroblasts. Thus we hypothesize that drebrin may destabilize actin filaments by dissociating tropomyosin and alpha-actinin from actin filaments, resulting in the formation of axon and dendrites during neuronal development.

Actin Cytoskeleton↗

PubMed1994

Bundling of actin filaments by myosin light chain kinase from smooth muscle.

Myosin light chain kinase has an inhibitory effect on the interaction of actin filaments with phosphorylated smooth muscle myosin. Myosin light chain kinase binds to actin filaments, and the inhibition is attributable to the actin-binding activity and not the kinase activity of myosin light chain kinase [Kohama et al. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211]. We now report that myosin light chain kinase is able to assemble actin filaments into thick bundles, which can be visualized by optical and electron microscopy and can be monitored by measuring the sedimentation and flow birefringence of actin filaments. The bundling activity of myosin light chain kinase is abolished by calmodulin in the presence of Ca2+. The possibility is discussed that myosin light chain kinase has multiple actin-binding sites through which it can cross-link actin filaments.

Actins↗

PubMed1994

Stimulation of the ATP-dependent interaction between actin and myosin by a myosin-binding fragment of smooth muscle caldesmon.

We reported previously that smooth muscle caldesmon stimulates the ATP-dependent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurement and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784-21790]. The N-terminal (myosin-binding) fragment and the C-terminal (actin-binding) fragment were purified from digests of caldesmon. The effects of the myosin-binding fragment and the actin-binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin-binding domain and actin-binding domain of caldesmon, respectively. The effect of the myosin-binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin-binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin-binding domain predominates over the inhibitory effect of the actin-binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin-binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon-binding fragment of myosin.

Actins↗

PubMed1994

Local environmental change from the G- to F-form of the actin molecule detected on anisotropy decay measurement.

The fluorescence intensity has been reported to increase 10 to 25 times when N-(1-pyrene)-iodoacetamide (PIAA)-conjugated actin polymerizes from the G- to the F-form. To elucidate the molecular mechanism underlying this process, we measured the time-averaged anisotropy of PIAA-actin in both the G- and F-forms. The anisotropy ratio of PIAA-G-actin (0.137 +/- 0.008) was smaller than that of PIAA-F-actin (0.221 +/- 0.008). Similar results were obtained when N-(1-pyrene)-3-iodopropionamide (PIPA), a PIAA analogue with an extra carbon-chain in its reactive group, was conjugated with actin. The anisotropy ratio increased from 0.096 +/- 0.042 to 0.199 +/- 0.035 when PIPA-actin was transformed from the G- to the F-form. Further more, we measured the anisotropy decay of PIAA-actin in both the G- and F-forms. Least-square fitting revealed that the decay pattern was well fitted the wobbling-in-cone model. In the G-form, the pyrene of PIAA actin diffused in a cone region with a vertical half angle of 33.4 degrees. This value decreased to 25.9 degrees when the actin was transformed to the F-form. Because PIAA and PIPA were conjugated at Cys-374 of actin, our results suggest that a small cleft exists in the actin molecule in the vicinity of Cys-374, and this cleft becomes narrow upon polymerization, resulting in an increase in fluorescence intensity.

Actins↗

PubMed1994

The regulatory role of myosin light chain kinase as an actin-binding protein.

Myosin light chain kinase (MLCK) is present in muscle cells including those of smooth muscle as an actin-binding protein. By avoiding complication introduced as a result of kinase activity of MLCK, we have demonstrated regulatory role of MLCK through its actin-binding activity [Kohama et al. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211]. To analyze such a regulatory role of MLCK, we compared the effects of MLCK on the velocity of the movement of actin filaments on a surface coated with smooth muscle myosin with those of another actin-binding proteins in smooth muscle, namely, caldesmon (CaD) and calponin (CaP). Both CaD and CaP stimulated movement when their concentrations were low, but they inhibited movement as their concentrations were increased. Calmodulin (CaM) in the presence of Ca2+ (Ca-CaM) antagonized the inhibition but hardly affected the stimulation. The effect of MLCK, by contrast, was simply inhibitory when Ca-CaM was not present. No stimulation was observed until Ca-CaM was added. The inhibitory ability of these actin-binding proteins increased in the following order: CaD < CaP < MLCK. The effect of MLCK and CaD on movement was further examined on surfaces coated with skeletal muscle myosin. The basic effect was similar to that observed with smooth muscle myosin. However, 10-fold greater concentrations of MLCK and CaD were required for a comparable effect. Such an increase in the required concentration was also observed when the velocity of movement was increased by elevation of the temperature during the assay with smooth muscle myosin. Thus, it is the velocity of movement itself that determines the required concentrations of MLCK and CaD.

Actins↗

PubMed1994

[Studies on agonists and antagonists of smooth muscle contraction by the use of an actomyosin preparation].

The sites of action of many chemical agents that modify the contraction of smooth muscle are in the smooth muscle membrane. However, a few agents, such as calmodulin inhibitors and protein kinase inhibitors, interact directly with contractile elements of the actomyosin system so as to modify smooth muscle contraction. Here, we describe experimental procedures that are applicable for the screening of smooth muscle relaxants with this mode of action. Myosin B was extracted from chicken gizzard smooth muscle. Because myosin B was a crude preparation of smooth muscle actomyosin, it consisted of regulatory proteins of calmodulin, myosin light chain kinase and protein phosphatase in addition to the contractile proteins of actin and myosin. Interaction of chemical agents with these proteins could be detected by measuring the Mg-ATPase activity of the myosin B preparation. Then we examined whether the agents that altered the ATPase activity was associated with changes in phosphorylation of myosin light chain. If the levels are altered, the agents may interact with the regulatory protein(s). If not, the site of their action was in the contractile proteins. The analysis with these respective proteins will be also described.

Amiloride↗

PubMed1993

Calvasculin, as a factor affecting the microfilament assemblies in rat fibroblasts transfected by src gene.

Cell transformations accompany alterations in cell morphology and microfilament patterns. Calvasculin encodes mRNA termed pEL-98, 18A2, 42A, p9Ka, or mts1, found to be elevated in several metastatic cell lines. We report the elevation of calvasculin expression in SR-3Y1 cells, which show disappearance of ordered microfilaments, compared to that in 3Y1 cells and that the similar distribution of calvasculin to that of actin filaments. Interestingly, calvasculin co-sediments with F-actin and bundles actin filaments in a Ca(2+)-dependent manner. This activity, along with the elevation of calvasculin following transformation, suggests that the disorganization of filaments in SR-3Y1 cell is due to the cross-linking activity of calvasculin.

Actin Cytoskeleton↗

PubMed1993

Stimulatory effect of calponin on myosin ATPase activity.

Calponin, a calmodulin-binding protein of smooth muscle that inhibits the actin-myosin interaction by binding to actin, was shown to bind to myosin and to stimulate the ATPase activity of myosin to some extent. Actin abolished this myosin-linked, stimulatory effect of calponin. Ca(2+)-calmodulin affected neither the myosin-binding activity nor the stimulatory effect of calponin. We further presented a few data which suggest that calponin may exert regulatory activity toward myosin in quite a different way from caldesmon, another smooth muscle protein that binds to myosin, actin, and calmodulin.

Animals↗

PubMed1993

Role of myosin in the stimulatory effect of caldesmon on the interaction between actin, myosin, and ATP.

We have previously shown that caldesmon at low concentrations stimulates the interaction between actin, myosin, and ATP, while at high concentrations it inhibits the interaction [Ishikawa, R., Okagaki, T., Higashi-Fujime, S., & Kohama, K. (1991) J. Biol. Chem. 266, 21784-21790]. When the effect of caldesmon at low concentrations was monitored by measuring myosin ATPase activity in the absence of actin, the effect was slightly but significantly stimulatory; and at higher concentrations no inhibitory effect was observed. Therefore, we related the stimulatory effect with the myosin-binding property of caldesmon. In the presence of actin, a low concentration of caldesmon was not enough to evince the stimulatory effect: myosin concentration must also be low. This is because the stimulatory effect was obscured when myosin concentration was elevated. Ca(2+)-calmodulin abolished the stimulatory effect of caldesmon. However, the concentration of calmodulin required to abolish the stimulation was higher than that required to abolish the inhibition.

Actins↗

PubMed1993

[New trends in studying the regulatory mechanism of smooth muscle contraction].

In this paper, we briefly review current topics about smooth muscle with regards to Ca2+ release, Ca2+ sensitization, and Ca2+ regulation of contraction. Inositol 1,4,5-trisphosphate releases Ca2+ from the sarcoplasmic reticulum, where Ca(2+)-dependent immediate feedback control may work. However, the involvement of this feedback control in the Ca(2+)-induced Ca2+ release mechanism remains to be elucidated. Either agonist or GTP gamma S is known to increase the Ca2+ sensitivity of myofilaments. The agonist-induced Ca2+ sensitization could be explained by the up-regulation due to myosin light chain kinase or by the down-regulation due to myosin light chain phosphatase. The GTP gamma S-induced Ca2+ sensitization seems to be mediated by rho A p21, a small G protein. Thus, myosin phosphorylation is not the obligatory way to regulate the actin-myosin interaction. We propose that cross-linking between actin and myosin may work as an alternative way to regulate the interaction from biochemical studies. The candidates for the cross-linkers are caldesmon, calponin and myosin light chain kinase. The inhibitory effect of Ca2+ on the interaction, which is observed under the specific conditions for measuring smooth muscle contraction, may hold the key to finding the physiological significance of the cross-linking activity.

Actins↗

PubMed1993

Analysis of the biotin biosynthesis pathway in coryneform bacteria: cloning and sequencing of the bioB gene from Brevibacterium flavum.

The biotin biosynthetic pathway of three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum, and Corynebacterium glutamicum were analysed by cross-feeding experiments using several Escherichia coli biotin-requiring mutants. The three strains of coryneform bacteria tested were able to convert 7-keto-8-aminopelargonic acid to biotin, through a biotin synthetic pathway identical to that from E. coli. The biotin biosynthetic gene, bioB, of B. flavum was cloned by phenotypic complementation of E. coli bioB mutants. The bioB gene was located on a 1.7 kb HindIII-SacI DNA fragment. Nucleotide sequence analysis of this fragment revealed that the bioB gene of B. flavum consists of a 1005 bp open reading frame. Its deduced amino acid sequence is 35.7% and 31.5% identical to that of the E. coli and Bacillus sphaericus bioB gene products, respectively. B. flavum mutants obtained by in vivo disruption of the bioB gene lost their ability to grow on minimal medium containing dethiobiotin, indicating that the bioB gene product is necessary for the conversion of dethiobiotin to biotin.

Amino Acid Sequence↗