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E Nishida

Publications and source records attributed to E Nishida.

251 records · Page 14Linked to original sources
PubMed1980

A new protein factor that modulates assembly of microtubules in vitro. I. Purification and characterization.

A new protein factor that modulates microtubule assembly in a Ca2+ or Mg2+ concentration-dependent manner was extracted from porcine brain and purified by ammonium sulfate fractionation, acetone fractionation, chromatography on an affinity column fixed with microtubule proteins, and high speed liquid chromatography. The isolated protein was nearly homogeneous on SDS-polyacrylamide gel, appearing as a 94,000-dalton polypeptide. The protein gave a single symmetric peak with a relatively large Stokes radius on Sepharose 4B gel filtration. Moreover, it sedimented as a single homogeneous component with a sedimentation constant of nearly 5 S on sucrose density gradient centrifugation. These analyses indicated the homogeneity of the isolated protein and the asymmetry of its conformation. The purified protein factor slightly inhibited microtubule assembly under standard assembly condition. Increase in the concentration of either free Ca2+ or free Mg2+ markedly enhanced its inhibitory effect. Neither Ni2+ nor Mn2+ potentiated the inhibitory effect of the protein. The inhibition was reversible in a fashion dependent on the concentration of Ca2+ or Mg2+ and appeared to be stoichiometric rather than catalytic. The inhibitory activity was totally destroyed by trypsin digestion, but was very heat stable and was not lost on treatment with N-ethylmaleimide. This new protein factor may play an important role in regulation of microtubule assembly and/or function.

Animals↗
PubMed1979

Microtubule assembly in the presence of adenosine triphosphate.

Microtubule (MT) assembly was investigated in the presence of ATP and Ca ions using both crude extract (CE) and purified microtubular proteins (PMP) prepared from porcine brains. ATP inhibited MT assembly from CE prepared with the reassembly buffer containing 1 mM GTP. Half-maximal inhibition occurred at an ATP concentration of 0.4-0.5 mM. Calcium ions, on the contrary, cancelled the ATP-induced inhibition, 1-2 microM calcium ions supporting maximal MT assembly. The ATP-induced inhibition in PMP was not so prominent as in CE, but occurred significantly in the presence of RNA. In PMP dissolved in the reassembly buffer containing ATP and yeast tRNA, the content of the ring fraction decreased significantly as compared with PMP containing only RNA. Furthermore, microtubule-associated proteins were found to be capable of binding ATP. The significance of the ATP-induced inhibition of MT assembly and the release of the inhibition by Ca2+ was discussed.

Adenosine Triphosphate↗
PubMed1979

The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. I. Effect of calcium-dependent regulator protein on the calcium sensitivity of microtubule assembly.

We examined the effect of porcine brain Ca2+-dependent regulator (CDR) protein on microtubule (MT) assembly from microtubular proteins isolated from porcine brain by temperature-dependent cycles of assembly-disassembly. CDR exhibited a potent inhibitory effect on MT assembly in the presence of Ca2+, whereas it had little or no effect on the extent of MT assembly in the absence of Ca2+. The increase in KCl concentration greatly potentiated the Ca2+-dependent inhibitory effect of CDR. The effect of CDR was reversible in a Ca2+ concentration-dependent manner, and the extent of inhibition by CDR at a fixed concentration of free Ca2+ was roughly proportional to the concentration of CDR. Moreover, the Ca2+ concentration required for the half-maximal inhibition of MT assembly from a fixed concentration of purified microtubular proteins (PMP) decreased with increasing CDR concentration. On the basis of these results, together with data on the Ca2+-dependent association of CDR and tubulin (J. Biochem., accompanying paper), we propose the following model; Ca2+ + CDR in equilibrium Ca2+-CDR Ca2+-CDR + tubulin in equilibrium Ca2+-CDR-tubulin (nonpolymerizable).

Animals↗
PubMed1979

The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. II. Association of calcium-dependent regulator protein with tubulin dimer.

The Ca2+-dependent regulator protein (CDR) of cyclic nucleotide phosphodiesterase (PDE) was reported to be a Ca2+-dependent regulator of microtubule (MT) assembly in the preceding paper. In this paper, the binding of Ca2+-CDR complex to tubulin dimer was investigated in order to elucidate the Ca2+-dependent inhibitory action of CDR on MT assembly. Purified microtubular proteins (PMPs) isolated from porcine brain did not affect the ability of CDR to activate Ca2+-activatable PDE, and did not include any inhibitory protein of Ca2+-activatable PDE. The binding of CDR to the tubulin dimer was observed on Sephadex G-200 gel filtration and ammonium sulfate fractionation in a Ca2+-dependent manner. CDR did not bind to microtubule associated proteins. We now assume that Ca2+-dependent inhibition of MT assembly by CDR is due to the binding of CDR to tubulin dimer in a Ca2+-dependent manner.

Animals↗
PubMed1977

Relationship between tubulin SH groups and bound guanine nucleotides.

Guanine nucleotides bound to both the non-exchangeable sites (N sites) and exchangeable sites (E sites) of tubulin were completely released after 7 moles of SH groups per tubulin subunit (55,000 molecular weight) had reacted with PCMPS. The blockage of 2 moles of SH groups in the glycerol-reassembly buffer or 1 mole of SH groups in glycerol-free reassembly buffer resulted in complete loss of tubulin polymerizability. However, under both sets of experimental conditions, the amount of guanine nucleotides released from the E sites was less than 8% and the loss of total guanine nucleotides was only 5%. Addition of GSH did not induce reassociation of released guanine nucleotides, although it restored tubulin polymerizability. These results indicate that the loss of tubulin polymerizability on blockage of the SH groups was not due to dissociation of bound guanine nucleotides and that the binding sites of the nucleotides were independent of the SH groups in tubulin required for polymerization. Furthermore, blockage of SH groups did not change the ratio of GTP to GDP bound to tubulin.

4-Chloromercuribenzenesulfonate↗
PubMed1977

Calcium-sensitivity of the microtubule reassembly system. Difference between crude brain extract and purified microtubular proteins.

Microtubule reassembly in crude extracts of porcine brain was inhibited by 10(-5) M Ca2+, wherease 10(-3) M Ca2+ was required for inhibition of the reassembly from purified microtubular proteins. This accounts for the apparent discrepancies between the results reported by other investigators. Furthermore, the Ca-sensitivity of the purified microtubular proteins was nearly completely recovered on addition of a fraction obtained from crude brain extract.

Animals↗
PubMed1972

[Use of oxytocin].

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Administration, Oral↗