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Biomedical subjects

E Nishida

Publications and source records attributed to E Nishida.

At least 217 records · Page 12Linked to original sources
PubMed1986

Binding of microtubule-associated protein 2 and tau to the intermediate filament reassembled from neurofilament 70-kDa subunit protein. Its regulation by calmodulin.

Two major brain microtubule-associated proteins (MAPs), MAP2 and tau, were found to bind to the intermediate filaments reassembled from neurofilament 70-kDa subunit protein (= 70-kDa filaments). The binding was saturable. The apparent dissociation constant (KD) for the binding of MAP2 to the 70-kDa filaments was estimated to be 4.8 X 10(-7) M, and the maximum binding reached 1 mol of MAP2/approximately 30 mol of 70-kDa protein. The apparent KD for the tau binding was 1.6 X 10(-6) M, and the maximum binding was 1 mol of tau/approximately 3 mol of 70-kDa protein. It was also found that MAP2 and tau did not compete with each other for binding to the 70-kDa filaments. Most interestingly, calmodulin, a ubiquitous Ca2+-binding protein in eukaryotic cells, was found to inhibit the binding of MAP2 and tau to the 70-kDa filaments. The inhibition by calmodulin was regulated by changes in Ca2+ concentration around 10(-6) M, and was canceled by trifluoperazine, a calmodulin inhibitor.

Animals↗
PubMed1986

Two mammalian heat shock proteins, HSP90 and HSP100, are actin-binding proteins.

Two high molecular weight heat shock proteins, HSP90 (Mr, 90,000) and HSP100 (Mr, 100,000), were separately purified from extracts of cultured cells of a mouse lymphoma cell line, L5178Y. Both of the HSPs exist in homodimeric form under physiological conditions. Their physicochemical properties are quite similar to each other. Each of the purified HSPs was shown to coprecipitate with rabbit skeletal muscle actin under actin-polymerizing conditions. Both HSP90 and HSP100 increased the low-shear viscosity of filamentous actin solutions in a dose-dependent manner, which suggests that these HSPs cross-link actin filaments. Although some molecular properties and the effects described above on actin solution of HSP90 and HSP100 resemble those of alpha-actinin, the HSPs were distinguished from alpha-actinin by various means, including visualization of molecular shapes by electron microscopy with the aid of the low-angle rotary shadowing technique. Immunofluorescence staining by specific antisera against HSP90 revealed that HSP90 was localized in ruffling membranes in addition to the cytoplasmic space.

Actins↗
PubMed1986

On the mechanism of calmodulin-induced inhibition of microtubule assembly in vitro.

Binding of calmodulin to microtubule-associated proteins (MAPs) was analyzed by the equilibrium gel filtration method. The apparent dissociation constant (Kd) of calmodulin binding was found to be 2 microM for tau, and 5 microM for MAP2. These Kd values were similar to the Kd previously determined for calmodulin binding to tubulin. The inhibitory effect of increasing concentrations of calmodulin on the kinetics of microtubule assembly from tau and tubulin was not mimicked by decreasing the concentration of tau alone or tubulin alone. These results suggest that calmodulin inhibits microtubule assembly by its binding to both MAPs and tubulin.

Animals↗
PubMed1985

pH control of actin polymerization by cofilin.

Cofilin, a 21,000 molecular weight actin-regulatory protein (Nishida, E., Maekawa, S., and Sakai, H. (1984) Biochemistry 23, 5307-5313), was here shown to be capable of reversibly controlling actin polymerization and depolymerization in a pH-sensitive manner. When cofilin was reacted with F-actin at different pH, the depolymerized actin concentration (= monomeric actin concentration) was higher at elevated pH. At pH less than 7.3, the monomeric actin concentrations did not exceed approximately 1 microM even in the presence of excess amounts of cofilin, whereas at pH greater than 7.3 it increased in proportion to the concentration of cofilin added, and complete depolymerization of F-actin occurred by the addition of an excess amount of cofilin. Moreover, in the presence of cofilin, rapid interconversion of monomeric and polymeric forms of actin can be induced by simply changing the pH of the medium. Thus, this study provides a new possible mechanism regulating actin polymerization, pH control.

Actin Depolymerizing Factors↗
PubMed1985

An actin-depolymerizing protein (destrin) from porcine kidney. Its action on F-actin containing or lacking tropomyosin.

An Mr 19 000 protein (destrin) that has the ability to rapidly depolymerize F-actin in a stoichiometric manner was purified from porcine kidney by sequential chromatography on DNase I-agarose, hydroxyapatite, and Sephadex G-75. Its actin-depolymerizing activity is reversibly controlled by changes in KCl concentration but is insensitive to Ca2+ concentration. The rate of depolymerization of F-actin by destrin is much faster than that of spontaneous depolymerization induced by dilution and is not markedly decreased by the addition of end-blocking reagents such as cytochalasin B. These results suggest that destrin depolymerizes F-actin by interacting directly with F-actin protomers. Binding of muscle tropomyosin to F-actin slows down the rate of destrin-induced depolymerization of F-actin by about 30-fold. The data suggest that the destrin-induced depolymerization occurs from the ends of F-actin when F-actin is complexed with tropomyosin, but it takes place from the entire length of F-actin in the absence of tropomyosin.

Actin Depolymerizing Factors↗
PubMed1985

Calmodulin inhibits interaction of actin with MAP2 and Tau, two major microtubule-associated proteins.

We have previously shown that microtubule-associated protein 2 (MAP2) and Tau, two major microtubule-associated proteins, interact with actin differently as measured by low-shear viscosity and that their activities are modified by phosphorylation (Nishida, E., Kotani, S., Kuwaki, T., and Sakai, H. (1982 in Biological Functions of Microtubules and Related Structures (Sakai, H., Mohri, H., and Borisy, G. G., eds) pp. 297-309, Academic Press, Japan). In the present study we further examined their interaction using turbidimetry, electron microscopy, low- and high-shear viscometry. MAP2 increased the low-shear viscosity of actin filament but had weaker effect on high-shear viscosity and turbidity of actin filaments. In contrast, Tau reduced high-shear viscosity of actin filaments and enhanced the turbidity which were due to formation of actin filament bundles as shown by electron microscopy. We conclude that MAP2 is a gelation factor, while Tau is a bundling factor. A well-known Ca2+-dependent regulatory protein, calmodulin, inhibited both MAP2-actin and Tau-actin interaction in a Ca2+-dependent manner. The calmodulin-dependent inhibition was canceled by higher concentrations of MAP2 or Tau, and calmodulin had no effect on the viscosity of actin filament alone, indicating that this inhibition is based on the stoichiometric interaction of calmodulin with MAP2 or Tau.

Actins↗
PubMed1985

Phosphorylation of tubulin and microtubule-associated proteins by the purified insulin receptor kinase.

The purified insulin receptor kinase catalyzed the phosphorylation of native tubulin and microtubule-associated proteins (MAPs; MAP2, tau) on tyrosine residues. Insulin (10(-7) M) stimulated the reaction by 4-10-fold by increasing Vmax with little change in Km. alpha-Tubulin was preferred as a substrate for the kinase compared to beta-tubulin. MAP2 was found to be the best substrate among the cytoskeletal proteins tested; in the presence of insulin, the Vmax for MAP2 was 6.3 nmol/min/mg, its Km was 5.1 microM, and 1.7 mol of phosphate were incorporated per mol of MAP2. Under the same conditions used for this phosphorylation of tubulin and MAPs, actin and tropomyosin were very poorly phosphorylated. These data, coupled with previous evidence for potential functional relationships between insulin action and microtubules, raise the possibility that microtubule proteins may be cellular targets for the insulin receptor kinase.

Humans↗
PubMed1985

Phosphorylation of fodrin (nonerythroid spectrin) by the purified insulin receptor kinase.

Fodrin (nonerythroid spectrin) from porcine brain was found to be phosphorylated on tyrosine residues by the purified insulin receptor kinase. The phosphorylation occurred in an insulin-sensitive manner with a physiologically relevant km. The beta(235 K) subunit of fodrin, but not the alpha(240 K) subunit, was phosphorylated by the kinase. Neither the alpha(240 K) subunit nor the beta(220 K) subunit of erythrocyte spectrin was phosphorylated under the same conditions. Fodrin phosphorylation by the purified insulin receptor kinase was markedly inhibited by F-actin. These data raise the possibility that tyrosine phosphorylation of fodrin plays some roles in the regulation of plasma membrane-microfilament interaction.

Actins↗
PubMed1985

Opposite effects of cofilin and profilin from porcine brain on rate of exchange of actin-bound adenosine 5'-triphosphate.

Cofilin, an actin-binding protein isolated from porcine brain that reacts with actin in a 1:1 molar ratio [Nishida, E., Maekawa, S., & Sakai, H. (1984) Biochemistry 23, 5307-5313], decreases the rate of exchange of ATP bound to G-actin with 1,N6-ethenoadenosine 5'-triphosphate in solution. From analyses of the dependence of the exchange rate on the cofilin concentration under different KCl concentrations, dissociation constants (KD) for the cofilin-actin binding at 0, 50, and 140 mM KCl were determined to be 0.12, 0.15, and 0.25 microM, respectively. In contrast to cofilin, profilin isolated from porcine brain increases the rate of exchange of G-actin-bound ATP, like Acanthamoeba profilin. The kinetic analyses gave KD values for the profilin-actin binding of 1.1 and 1.5 microM, respectively, at 50 and 200 mM KCl.

Actin Depolymerizing Factors↗
PubMed1985

Purification of cofilin, a 21,000 molecular weight actin-binding protein, from porcine kidney and identification of the cofilin-binding site in the actin sequence.

Cofilin, a 21,000 molecular weight protein originally purified from porcine brain that is capable of binding to actin filaments in a molar ratio of the protein to actin monomer of 1:1 in the filament (Nishida et al. (1984) Biochemistry 23, 5307-5313), was purified from porcine kidney in the present study. The two cofilins from brain and kidney were indistinguishable from each other with respect to the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the one-dimensional peptide map, and the mode of interaction with actin. Treatment of the actin-cofilin complex with a zero-length cross-linker, 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC), generated a cross-linked product with an apparent molecular weight of 63,000. Analysis of this product by peptide mapping (Sutoh (1982) Biochemistry 21, 3654-3661) showed that cofilin was cross-linked with the N-terminal segment of actin containing residues 1-12.

Actin Depolymerizing Factors↗
PubMed1984

Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin.

Cofilin, a 21 000 molecular weight protein of porcine brain, reacts stoichiometrically with actin in a 1:1 molar ratio. Upon binding of cofilin, the fluorescence of pyrene-labeled actin under polymerizing conditions is changed into the monomer form, irrespective of whether cofilin is added to actin before or after polymerization. Cofilin decreases the viscosity of actin filaments but increases the light-scattering intensity of the filaments. The centrifugation assay and the DNase I inhibition assay demonstrate that cofilin binds to actin filaments in a 1:1 molar ratio of cofilin to actin monomer in the filament and that cofilin increases the monomeric actin to a limited extent (up to 1.1-1.5 microM monomer) in the presence of physiological concentrations of Mg2+ and KCl. Cofilin is also able to bind to monomeric actin, as demonstrated by gel filtration. Electron microscopy showed that actin filaments are shortened and slightly thickened in the presence of cofilin. No bundle formation was observed in the presence of various concentrations of cofilin. The gel point assay using an actin cross-linking protein and the nucleation assay also suggested that cofilin shortens the actin filaments and hence increases the filament number. Cofilin blocks the binding of tropomyosin to actin filaments. Tropomyosin is dissociated from actin filaments by the binding of cofilin to actin filaments. Cofilin was found to inhibit the superprecipitation of actin-myosin mixtures as well as the actin-activated myosin ATPase. All these results suggest that cofilin is a new type of actin-associated protein.

Actin Depolymerizing Factors↗
PubMed1984

Isolation of low molecular weight actin-binding proteins from porcine brain.

Three new actin-binding proteins having molecular weights of 26,000, 21,000, and 19,000 were isolated from porcine brain by DNase I affinity column chromatography. These proteins were released from the DNase I column by elution with a solution of high ionic strength. They were further purified by column chromatographies using hydroxyapatite, phosphocellulose, and Sephadex G-75. All of these actin-binding proteins behaved as monomeric particles in the gel filtration chromatography. After elution of the three actin-binding proteins, actin and profilin were recovered from the DNase I column with 2 M urea solution. The eluted was further purified by a cycle of polymerization and depolymerization and finally by gel filtration. Little difference in polymerizability was detected between the purified brain actin and muscle actin. After sedimentation of the polymerized brain actin, profilin was purified by DEAE-cellulose and gel filtration column chromatographies. In the assay of the action of these actin-binding proteins, the 26K protein was found to cause a large decrease in the rate of actin polymerization, while showing little effect on the extent of polymerization. The 21K protein decreased the steady-state viscosity of actin solution in a concentration-dependent manner irrespective of whether it was added before or after actin polymerization. It reacted with actin at a 1:1 molar ratio.

Actins↗
PubMed1984

Action of a 19K protein from porcine brain on actin polymerization: a new functional class of actin-binding proteins.

A 19K protein isolated from porcine brain not only inhibits actin polymerization but depolymerizes actin filaments quickly. The protein reacts stoichiometrically with actin in a 1:1 molar ratio. When actin is induced to polymerize with salts in the presence of the brain 19K protein, the lag phase is prolonged, and the extent of polymerization is decreased, but the half-polymerization time is not increased. This can be explained by assuming that the 19K protein severs growing actin filaments and thus causes an increase in the number of filament ends during the polymerization process, thereby accelerating the overall polymerization. Moreover, the low-shear viscosity of actin filaments is reduced much more than the high-shear viscosity by the 19K protein, suggesting that actin filaments become shorter in the presence of the 19K protein than in its absence. Actin filament depolymerization by the 19K protein is much faster than that by brain profilin or than spontaneous depolymerization. This indicates that the 19K protein depolymerizes actin filaments not only by sequestering actin monomers but also by directly attacking the filaments. The number of actin filaments, measured by assaying the nucleating ability, is increased by substoichiometric concentrations of the 19K protein, irrespective of whether the protein is added to actin monomers before polymerization or added to performed actin filaments. These results suggest that the brain 19K protein not only stabilizes actin monomers but also cuts actin filaments, thereby decreasing the extent of actin polymerization and also changing the filament length. The action on actin of the actin-depolymerizing protein from starfish oocytes resembles that of the brain 19K protein, although the molecular weight of the starfish protein is slightly smaller. The brain 19K protein and starfish protein should be classified into a new functional group of actin-binding proteins.

Actin Depolymerizing Factors↗
PubMed1984

Characterization of the action of porcine brain profilin on actin polymerization.

When porcine brain actin is polymerized in either KCl/MgCl2 or KCl alone, porcine brain profilin prolongs the lag phase and inhibits the rate and extent of polymerization in a concentration-dependent manner. Profilin also decreases the elongation rate in a concentration-dependent manner. Moreover, addition of profilin to steady-state actin filaments causes slow depolymerization. All these actions of profilin are explainable by a monomer sequestering mechanism. The inhibition by profilin of both the extent of polymerization and the elongation rate is stronger in KCl alone than in KCl/MgCl2. Moreover, it was found that brain profilin inhibits the polymerization of brain actin more strongly than that of muscle actin. Brain 88K protein/actin complex (88K/A), which has been shown to cap the barbed end of actin filaments, potentiates the inhibitory action of profilin; i.e. the extent of polymerization is much more reduced by profilin in the presence of 88K/A than in its absence.

Actins↗