Open AccessPurification and characterization of a cytosolic protein‐tyrosine kinase from porcine spleen
Author(s) -
KOBAYASHI Tomoko,
NAKAMURA Shunichi,
TANIGUCHI Takanobu,
YAMAMURA Hirohei
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb15433.x
Subject(s) - cytosol , tyrosine kinase , tyrosine , spleen , chemistry , microbiology and biotechnology , biochemistry , biology , immunology , signal transduction , enzyme
A cytosolic protein‐tyrosine kinase has been highly purified from porcine spleen using [Val 5 ]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 K m value for ATP was relatively low, 0.35 μM; Mn 2+ was slightly preferred to Mg 2+ as divalent cation. [Val 5 ]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.