Purification and characterization of a protein‐tyrosine kinase p72 syk from porcine spleen

We have succeeded in purifying p72 syk , a non‐receptor‐type protein‐tyrosine kinase carrying high susceptibility to proteolysis [Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S. and Yamamura, H. (1991) J. Biol. Chem. 266 , 15790–15796] from porcine spleen. The purification procedure involves a sequential column chromatography, following extraction with 0.5 M NaCl from spleen homogenate, on phosphocellose, Sephacryl S‐200, heparin‐Sepharose CL‐6B, Mono Q and Mono S. SDS/PAGE of the final purified sample revealed a 72‐kDa protein band with about 95% purity and immunodepletion analysis showed immunological cross‐reactivity with anti‐p72 syk antibody which does not recognize ZAP‐70. It was purified approximately 3000‐fold with an overall yield of 0.54% according to [Val5]angiotensin II phosphorylation activity and the specific activity of the final sample (30 nmol phosphate min –1 ) was relatively lower than that of the 40‐kDa kinase, a catalytic fragment of p72 syk which lacks two src homology regions 2 domains. The p72 syk had an autophosphorylation activity that was performed by intramolecular catalysis accompanied by a phosphate exchange reaction, and could efficiently phosphorylate tubulin, myelin basic protein and H2B histone. Employing [Val5]angiotensin II as a substrate, the apparent K m value for the peptide was 0.91 mM and that for ATP was 0.48 μM. Mn 2+ , Mg 2+ and Co 2+ were effective divalent cations and optimum pH was around 8.0–8.5 for the expression of the activity. These results suggest that the purified p72 syk may exist as a less active form compared with the 40‐kDa kinase and that the part ofp72 syk containing two src homology region 2 domains may participate in the regulation of its activity though the enzymic character is quite similar to that of the 40‐kDa kinase.