Escherichia coli

Abstract

Several biochemical properties of a 43 kDa v-abl-encoded tyrosine-specific protein kinase (p43V-abl) expressed in Escherichia coli were examined. p43v-abl is a fragment of a 60 kDa v-abl-encoded precursor, p60 v-abl, and could be generated by limited proteolysis of a purified p60v-abl with trypsin. Tryptic cleavage of p60v-abl was prevented in the presence of ATP. These results suggest that the catalytic kinase domain of v-abl-derived protein can be separated from other (regulatory) domains by limited proteolysis. p43v-abl readily phosphorylated tyrosine residues on several different protein and peptide substrates, including peptides containing only two amino acid residues. However, the local sequence of the tyrosine-containing peptide substrate significantly affected its rate of phosphorylation. Thus the primary structure and local conformation at the tyrosine acceptor site can play an important role in determining the substrate specificity of v-abl-derived kinase. Phosphorylation by p43 v-abl requires Mn2", Co2" or Mg2" and exhibits a strong preference for ATP as phosphate donor. Analogues of ATP and the thiol-reactive reagent N-ethylmaleimide inhibited p43 v-abl kinase activity. Purified p43 v-abl is intrinsically thermolabile (t1 = 5 min at 40 °C) and phosphorylates glycerol inefficiently (Km = 1.4 M). approximately equal amounts of p6Ov-abl p43v-abl as immunoblot analysis or Coomassie molecular sizes of p43v-abl estimated protein