Properties of a tyrosine protein kinase from calf thymus. Response to ionic strength and divalent cations.
A tyrosine protein kinase activity has been partially purified from calf thymus using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Detergent extracts of calf thymus possessed only low levels of specific peptide phosphorylating activity when assayed at low ionic strength. The inclusion of NaCl at a concentration of 2 M stimulated endogenous tyrosine protein kinase activity, while the activity of other endogenous kinases was inhibited. This sensitivity to NaCl was retained following partial purification of the enzyme. The phosphorylation of other substrates such as casein or the R-R-SRC peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) by the tyrosine protein kinase was less sensitive to NaCl. Phosphorylation of the PK-1 peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) by the purified catalytic subunit of cAMP-dependent protein kinase was inhibited by NaCl. The effect of NaCl on angiotensin I phosphorylation could be mimicked by KCl or sodium acetate. The principal effect of NaCl was to increase the Vmax of the enzyme for the phosphorylation of angiotensin I. At low ionic strength, Mn2+ and Co2+ were the preferred required divalent cations. At elevated NaCl concentrations Mg2+ was preferred, with half-maximal activation occurring at 35 mM Mg2+. By conducting peptide phosphorylation assays in the presence of elevated levels of Mg2+ and NaCl, tyrosine protein kinase activity can readily be detected in extracts from cell lines that express low levels of the enzyme.
D000803:Angiotensin I; D000818:Animals; D000838:Anions; D002413:Cations, Divalent; D002417:Cattle; D066298:In Vitro Techniques; D009994:Osmolar Concentration; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology; D013950:Thymus Gland / Q000201:enzymology*
D000838:Anions; D002413:Cations, Divalent; D012965:Sodium Chloride; D000803:Angiotensin I; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases