Purification and enzymatic characterization of pp60c-src from human platelets.
The purification of pp60c-src has been hampered by the low levels of protein it represents in most cells and its tendency to undergo proteolysis during purification. The discovery that the platelet expresses unusually high levels of pp60c-src has made large-scale purification from a normal source feasible. We have developed a method for the purification of intact pp60c-src to near homogeneity from human platelets and have determined the enzymatic properties of this purified protein in vitro. Rapid, high yield purification of pp60c-src from isolated platelet membranes was achieved in a two-step protocol involving sequential chromatography on an anti-pp60c-src immunoaffinity matrix and phenyl-Sepharose. This protocol yielded 0.5 mg of pp60c-src from 30 units of platelets. Using enolase as an exogenous substrate, the specific activity of the enzyme was 25 nmol P.min-1.mg-1. The Km for MnATP2- for enolase phosphorylation (2.2 microM) was higher than for the autophosphorylation of pp60c-src (0.6 microM). Maximal enzyme activity required either Mn2+ or Mg2+, and both ATP and GTP could be utilized as the phosphate donor. Evidence is shown which indicate that the autophophorylation of pp60c-src in vitro occurs through an intramolecular mechanism and that this reaction is reversible.
Feder
D
D
Department of Microbiology and Immunology, University of California Medical Center, San Francisco 94143.
Bishop
J M
JM
D000255:Adenosine Triphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000097:blood*; D016392:Proto-Oncogene Proteins pp60(c-src)
D011518:Proto-Oncogene Proteins; D008345:Manganese; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D010751:Phosphopyruvate Hydratase; D008274:Magnesium