Characterization of protein tyrosine kinase activity in murine Leydig tumor cells.
The activity of protein tyrosine kinase (EC 2.7.1.37) was characterized from Leydig tumor cells (M5480A) using the synthetic peptide NH2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH as a substrate. Relatively high tyrosine-specific protein kinase activity (about 135 pmol/mg protein per min) was detected in a particulate fraction (30 000 X g pellet) and was found to be linear as a function of time and protein concentration. The enzymic activity in the particulate fraction was stimulated 1.4-fold by 0.02% Nonidet P-40 as judged by 32PO4 incorporated into the peptide. Phosphorylation of endogenous proteins in M5480A particulate fractions with [gamma-32P]ATP resulted in several alkali-resistant radiolabeled bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Included in this group was a major radiolabeled doublet with an apparent molecular-weight in the range of 50 000-54 000. Phosphoamino acid analysis of hydrolysates of these eluted proteins indicated the presence of phosphotyrosine. Several alkali-resistant radio-labeled bands, including a major doublet with an apparent molecular-weight of 32 000, were also detected after culturing M5480A cells in the presence of 32PO4. These studies demonstrate the presence of high levels of protein tyrosine kinase activity in Leydig tumor cells and of endogenous protein substrates for this enzyme activity.
L J
LJ
Dangott
D
D
Puett
M H
MH
Melner
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003902:Detergents / Q000494:pharmacology; D007984:Leydig Cell Tumor / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D051379:Mice; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis*; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013736:Testicular Neoplasms / Q000201:enzymology*
D000596:Amino Acids; D003902:Detergents; D009363:Neoplasm Proteins; D011506:Proteins; D011505:Protein-Tyrosine Kinases; D008274:Magnesium