Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an Escherichia coli expression system.
A 350 amino acid soluble fragment of the intracellular catalytic domain of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase has been purified 17-fold to greater than 90% purity from an Escherichia coli expression vector in quantities sufficient for kinetic and structural characterization. To assess substrate specificity, phosphotyrosine peptides corresponding to autophosphorylation sites of the two major classes of tyrosine kinases have been synthesized. Thus 6-12-residue phosphotyrosine peptides of the insulin receptor and epidermal growth factor receptor kinase domains and of the autophosphorylation and C-terminal regulatory sites of p60src and p56lck have been analyzed for kcat and KM by using a nonradioactive chromogenic assay for Pi release. The catalytic domain of LAR PTPase shows kcat values of 20-70 s-1 for phosphotyrosine peptides and affinities that vary 150-fold from 27 microM to 4.1 mM.
Cho
H J
HJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Ramer
S E
SE
Itoh
M
M
Winkler
D G
DG
Kitas
E
E
Bannwarth
W
W
Burn
P
P
Saito
H
H
Walsh
C T
CT
D000595:Amino Acid Sequence; D000943:Antigens, Differentiation / Q000032:analysis*; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006649:Histocompatibility Antigens / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D017493:Leukocyte Common Antigens; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000943:Antigens, Differentiation; D006649:Histocompatibility Antigens; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D015688:Oncogene Protein pp60(v-src); D017493:Leukocyte Common Antigens