Purification and initial characterization of the lymphoid-cell protein-tyrosine kinase p56lck from a baculovirus expression system.
The lymphocyte-specific protein-tyrosine kinase p56lck has been purified 90-fold to approximately 30% purity in 30% yield from a baculovirus expression system by a two-column purification procedure. At least two forms of p56lck were isolated, differing in the extent of phosphorylation and migrating as 56- and 59-kDa species on SDS/PAGE but as a single 56-kDa band after treatment with potato acid phosphatase. Autophosphorylation of purified p56lck occurred at a rate of 25 fmol/min to a maximum incorporation of approximately 2 mol of phosphate per mol of p56lck with tyrosine-394 (but not tyrosine-505) and other, unidentified tyrosine residue(s) being the major sites of phosphorylation in vitro. Phosphorylation of tyrosine-containing peptides was monitored using an automated HPLC system. Although peptide substrate Km values were in the 1-5 mM range, the Vmax for the 13-amino acid peptide RRLIEDAEYAARG (modified p60src autophosphorylation site) was 120 min-1 (350 min-1 when adjusted for p56lck purity), suggesting that the enzyme purified from recombinant baculovirus-infected Sf9 cells has a high catalytic turnover compared with other tyrosine kinases.
Ramer
S E
SE
Department of Biological Chemistry, Harvard Medical School, Boston, MA.
Winkler
D G
DG
Carrera
A
A
Roberts
T M
TM
Walsh
C T
CT
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008214:Lymphocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D009842:Oligopeptides; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)