Purification and characterization of a protein-tyrosine kinase p72syk from porcine spleen.
We have succeeded in purifying p72syk, 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 phosphocellulose, 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-p72syk 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.mg protein-1) was relatively lower than that of the 40-kDa kinase, a catalytic fragment of p72syk which lacks two src homology regions 2 domains. The p72syk 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 Km value for the peptide was 0.91 mM and that for ATP was 0.48 microM. Mn2+, Mg2+ and Co2+ 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 p72syk may exist as a less active form compared with the 40-kDa kinase and that the part of p72syk 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.
Yang
C
C
Department of Biochemistry, Fukui Medical School, Matsuoka, Japan.
Yanagi
S
S
Wang
X
X
Sakai
K
K
Taniguchi
T
T
Yamamura
H
H
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*; D013552:Swine; D000072377:Syk Kinase
D000596:Amino Acids; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase