Purification and characterization of a cytosolic protein-tyrosine kinase from porcine spleen.
A cytosolic protein-tyrosine kinase has been highly purified from porcine spleen using [Val5]angiotensin II as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, Sephacryl S-200, casein-Sepharose 4B, heparin-Sepharose CL-6B and anti-(4-aminobenzyl phosphonic acid)--Sepharose 4B. Analysis of the most highly purified preparation by SDS/PAGE revealed a major silver-stained band of molecular mass 40 kDa. The 40-kDa cytosolic protein-tyrosine kinase was purified approximately 10,000-fold with an overall yield of about 7%. It had autophosphorylation activity which was carried out by intramolecular catalysis. The stoichiometry of phosphate incorporation was about 1 mol phosphate/mol enzyme. In the autophosphorylation reaction, the apparent Km value for ATP was relatively low, 0.35 microM; Mn2+ was slightly preferred to Mg2+ as divalent cation. [Val5]Angiotensin II phosphorylation activity of the 40-kDa kinase increased with the amount of phosphate incorporated into the enzyme. A phosphate exchange reaction was observed during the autophosphorylation. These results suggest that the 40-kDa kinase described here is a different type of protein-tyrosine kinase than the enzymes so far reported.
Kobayashi
T
T
Department of Biochemistry, Fukui Medical School, Japan.
Nakamura
S
S
Taniguchi
T
T
Yamamura
H
H
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000804:Angiotensin II / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D002845:Chromatography / Q000379:methods; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013154:Spleen / Q000201:enzymology*; D013552:Swine
D000804:Angiotensin II; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D008274:Magnesium