[Tyrosine protein kinase from cattle cerebral cortex: purification, characteristics, protein substrates for phosphorylation and inhibitors of activity].
Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted and chromatographically fractionated. The activity associated with tyrosine protein kinases was fully extracted from the membranes by 1% sodium cholate and eluted in two peaks (I and II) during chromatography of protein extracts on DEAE-Toyopearl in the presence of sodium cholate. The predominant in cerebral cortex membrane tyrosine protein kinase of peak I (about 75% of the total activity) was purified 1930-fold by gel filtration on Sephacryl S-300, chromatography on hexyl- and phenyl-Sepharose and by rechromatography on DEAE-Toyopearl. The amount of the enzyme prepared from 250 g of bovine brain was 20 micrograms, the enzyme yield and specific activity being 3.8% and 3.9 nmol/mg protein/min, respectively. The purified protein kinase of peak I represents a protein with Mr of 62-63,000 (p62) capable of being autophosphorylated in the presence of [gamma-32P]. Protein kinase p62 phosphorylates enolase, tubulin and calpactin I as well as model substrates in the series: histone H5 greater than poly(G, T)n greater than or equal to histone H2A greater than poly(G, A, T)n, histone H4 greater than caseins, histones H1 and H2B, poly(G, A, L, T)n. The enzyme is specific for Mn2+ at the optimal concentration about 1 mM. The KmMn-ATP is 0.3 microM; Km for histone H5 and poly(G, T)n are 0.45 mg/ml and 0.06 mg/ml, respectively. The protein kinase p62 activity is inhibited by NaCl (IC50 approximately 75-100 mM) as well as by quercetin, adriamycin and lasalocid (IC50 approximately 14-34, 23 and 90 microM, respectively). It is concluded that protein kinase p62 is analogous to the c-src gene protein kinase.
Petukhov
S P
SP
Chibalin
A V
AV
Kovalenko
M V
MV
Bulargina
T V
TV
Severin
E S
ES
D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity
D011505:Protein-Tyrosine Kinases
Tirozinovaia proteinkinaza iz kory mozga byka: ochistka, kharakteristika svoĭstv, belkovye substraty fosforilirovaniia i ingibitory aktivnosti.