High-yield purification of a pp60c-src related protein-tyrosine kinase from human platelets

Peter Presek, Christoph Reuter, Duygu Findik, Peter Bette

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Abstract

A protein-tyrosine kinase (PTK, EC 2.7.1.112) from human platelets was purified with high yield. Purification of the enzyme involved sequential chromatography on casein-agarose, tyrosine-agarose, heparin-Sepharose and hydroxylapatite. The procedure resulted in substantially enriched 54 52 kDa polypeptides on SDS-polyacrylamide gel electrophoresis and a yield of about 25% in PTK activity. About 250 μg of purified protein could be obtained from 1 g of cell protein. The purification factor varied between 1000 and 1500. Determination of the molecular mass of the purified PTK under nondenaturating conditions by molecular sieve chromatography revealed that the enzyme is a monomer of about 50 kDa. Among various protein substrates tested, casein was most prominently phosphorylated. All substrates were exclusively phosphorylated at tyrosine residues. Autophosphorylation at tyrosine residues of the 54 52 kDa proteins was observed in the presence of Mg2+ or Mn2+. At each purification step, the 54 52 kDa proteins were precipitated by sera from tumor-bearing rabbits immunoprecipitating pp60src, but not by control sera. The amount of the immunoprecipitated purified 54 52 kDa phosphoproteins was directly proportional to the amount of antiserum used. Partial peptide mapping by V8 proteinase showed a 26 kDa tyrosine-phosphorylated fragment for the 54 and the 52 kDa proteins as well as for the pp60c-src molecules of intact platelets. All these data indicated that purified PTK is closely related to pp60c-src of human platelets. Using casein as a substrate for the purified enzyme, the Km for ATP was 4 μM and the Vmax for the reaction was 2.0 nmol/min per mg.

Original languageEnglish (US)
Pages (from-to)271-280
Number of pages10
JournalBBA - Molecular Cell Research
Volume969
Issue number3
DOIs
Publication statusPublished - May 13 1988

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Keywords

  • (Human blood)
  • Autophosphorylation
  • Platelet
  • Protein purification
  • Protein-tyrosine kinase

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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