title: Comparison of tyrosine protein kinases in membrane fractions from mouse liver and Ehrlich ascites tumor.
abstract: Tubulin was phosphorylated mainly at tyrosine residues by membranes from mouse liver and Ehrlich ascites tumor with ATP in the presence of MnCl2, ZnCl2, NaVO3, and Nonidet P-40 in an epidermal growth factor (EGF)- and insulin-independent manner. The tyrosine tubulin kinase activity in the tumor membranes is comparable to the activity in liver membranes. Two tyrosine tubulin kinases, namely I (Mr = 64,000) and II (Mr = 46,000), were purified 5-17-fold and were free of the EGF receptor and the insulin receptor. Phosphorylation of endogenous proteins produced major alkali-resistant phosphoproteins of 56 and 53 kDa in kinase I preparations and of 46 and 37 kDa in kinase II preparations. These kinases phosphorylated tubulin stoichiometrically at tyrosine and had a preference of alpha-subunit to beta-subunit of tubulin. Apparent Km values of kinases I and II for tubulin were about 4 and 8 microM and for ATP were about 2 and 4 microM, respectively. Thiol reagents inhibited their reactions. N alpha-p-Tosyl-L-lysine chloromethyl ketone stimulated the reactions at 1 mM but inhibited them at 5 mM. Although kinases I and II also phosphorylated angiotensin II, tyrosine-glutamate copolymers, and heavy chains of anti-pp60src IgG, they differed from each other in preferences for the substrates. More than 95% of the enzyme activities was not immunoprecipitated with the anti-pp60src IgG which cross-reacts with pp60c-src. In comparison with the corresponding liver enzymes, tumor enzymes were more stable to heat and incorporated more phosphate into tubulin, but showed lower activity toward angiotensin II and anti-pp60src IgG.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D006358:Hot Temperature; D007074:Immunoglobulin G / Q000032:analysis; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008970:Molecular Weight; D015688:Oncogene Protein pp60(v-src); D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012191:Retroviridae Proteins / Q000276:immunology; D012995:Solubility; D014404:Tubulin / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D007527:Isoenzymes; D011506:Proteins; D012191:Retroviridae Proteins; D014404:Tubulin; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src)