Purification and characterization of a rat liver membrane tyrosine-protein kinase, the possible protooncogene c-yes product, p60(c-yes)

K. Azuma, M. Ariki, T. Miyauchi, H. Usui, M. Takeda*, K. Semba, Y. Matsuzawa, T. Yamamoto, K. Toyoshima

*この研究の対応する著者

研究成果: Article査読

11 被引用数 (Scopus)

抄録

A tyrosine-protein kinase was purified more than 270-fold from the rat liver plasma membrane fraction by successive column chromatographies on Sephacryl S-300, wheat germ agglutinin-agarose, casein-Sepharose, and hydroxylapatite, followed by isoelectrofocusing electrophoresis. The enzyme with pI of 6.2 was a 60-kDa single polypeptide which represented 42% of total protein. The enzyme reacted quantitatively with a monoclonal antibody to the amino-terminal sequence (Cys-3 to Ser-66) specific to the human c-yes protein, but not with antibodies to the specific amino-terminal sequences of the c-src, fyn, and lck proteins. The purified enzyme contained almost no phosphotyrosine residue but was autophosphorylated with Mg·ATP exclusively at tyrosine residues with concomitant increase in the kinase activity. The rates of autophosphorylation of the enzyme and phosphorylation of tyrosine-glutamate (1:4) copolymers, catalyzed by the enzyme were proportional to the square of enzyme concentration, suggesting that p60(c-yes) undergoes autophosphorylation through intermolecular catalysis, resulting in stimulation of the enzyme activity. Although the enzyme reaction showed an essential requirement for Mg2+ or Mn2+ with optimal concentrations of 20 and 3 mM, respectively, autophosphorylation significantly activated the enzyme only in the presence of Mg2+. Autophosphorylation of the enzyme reduced the K(m) for tyrosine-glutamate copolymers and tubulin, but not for ATP, and increased the V(max) of copolymer and tubulin phosphorylation.

本文言語English
ページ(範囲)4831-4839
ページ数9
ジャーナルJournal of Biological Chemistry
266
8
出版ステータスPublished - 1991
外部発表はい

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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