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 10000‐fold with an overall yield of about 7%. It had autophosphorylation activity which was carried out by intramolecular catalysis. The stoichiometory 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 μM; 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. Copyright © 1990, Wiley Blackwell. All rights reserved