Characterization of two tyrosine-specific protein kinases from pig spleen. Substrate-specific effect of autophosphorylation.
Spontaneously active tyrosine-specific protein kinases I and II (designated TyrK I and TyrK II) have been purified to electrophoretic homogeneity from a particulate fraction of porcine spleen based on an assay that used poly(4Tyr, Glu) as a substrate. SDS/polyacrylamide gels revealed a doublet of bands of about Mr 51,000 for TyrK I and two protein bands of Mr 55,000 and 54,000 for TyrK II. After incubation in the presence of [gamma-32P]ATP, the bands corresponding to both protein kinases contained phosphotyrosine. The two tyrosine protein kinases showed high activities with poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu) as substrates and lower activity with angiotensin II. Neither histone, phosvitin, casein nor bovine serum albumin were phosphorylated. Both protein tyrosine kinases were activated by millimolar concentrations of Mg2+ whereas Mn2+ was less effective. The effects of various polyanionic and polycationic substances depended on the nature of the peptide substrate. With poly(Tyr, 4Glu) as a substrate, the substances either inhibited the activities of TyrK I and TyrK II or had no effect. However, activation was observed with angiotensin II as substrate in the presence of polylysine, polyornithine, protamine sulfate, and heparin as effectors. When angiotensin II was used as substrate, activation also occurred by autophosphorylation, in parallel to the phosphate incorporation into the protein kinases. Activation by autophosphorylation was not observed with the synthetic peptide substrates, poly(Tyr, 4Glu) and poly(Tyr, 3Ala, 6Glu).
Faculty of Biology, University of Konstanz, Federal Republic of Germany.
A G
AG
Batzer
S
S
Kirsch
H W
HW
Hofer
D000818:Animals; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine; D011099:Polyglutamic Acid / Q000494:pharmacology; D011107:Polylysine / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D010455:Peptides; D019000:Phosphotyrosine; C008973:polyornithine; D011107:Polylysine; D011099:Polyglutamic Acid; D014443:Tyrosine; D008345:Manganese; D011505:Protein-Tyrosine Kinases; D008274:Magnesium