Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus.
Sequences termed v-abl, which encode the protein-tyrosine kinase activity of Abelson murine leukemia virus, have been expressed in Escherichia coli as a fusion product (ptabl50 kinase). This fusion protein contains 80 amino acids of SV40 small t and the 403 amino acid protein kinase domain of v-abl. We report here the purification and characterization of this kinase. The purified material contains two proteins (Mr = 59,800 and 57,200), both of which possess sequences derived from v-abl. Overall purification was 3,750-fold, with a 31% yield, such that 117 micrograms of kinase could be obtained from 40 g of E. coli within 6-7 days. The specific kinase activity is over 170 mumol of phosphate min-1 mumol-1, comparable to the most active protein-serine kinases. Kinase activity is insensitive to K+, Na+, Ca2+, Ca2+-calmodulin, cAMP, or cAMP-dependent protein kinase inhibitor. The Km for ATP is dependent on the concentration of the second substrate. GTP can also be used as a phosphate donor. The enzyme can phosphorylate peptides consisting of as few as two amino acids and, at a very low rate, free tyrosine. Incubation of the kinase with [gamma-32P]ATP results in incorporation of 1.0 mol of phosphate/mol of protein. This reaction, however, cannot be blocked by prior incubation with unlabeled ATP. Incubation of 32P-labeled kinase with either ADP or ATP results in the synthesis of [32P]ATP. This suggests the phosphotyrosine residue on the Abelson kinase contains a high energy phosphate bond.
Foulkes
J G
JG
Chow
M
M
Gorka
C
C
Frackelton
A R
AR
Baltimore
D
D
D000011:Abelson murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D002472:Cell Transformation, Viral; D004279:DNA, Viral / Q000032:analysis; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli; D007700:Kinetics; D009052:Leukemia Virus, Murine / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009994:Osmolar Concentration; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification*; D011505:Protein-Tyrosine Kinases; D014443:Tyrosine / Q000378:metabolism
D004279:DNA, Viral; D000804:Angiotensin II; D014443:Tyrosine; D008345:Manganese; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D008274:Magnesium