Purification and characterization of a protein tyrosine phosphatase containing SH2 domains.
A protein tyrosine phosphatase (PTP) containing two SH2 domains (PTP1C) was purified to near homogeneity from an adenovirus expression system by a two-step chromatographic procedure with a yield of 67%. The purified enzyme behaves as a monomer of 68 kDa on gel filtration and is totally specific for phosphotyrosyl residues. Its optimal pH is around neutrality for protein substrates such as reduced, carboxyamidomethylated, maleylated (RCM)-lysozyme and myelin basic protein but below 5 for low molecular weight compounds such as para-nitrophenyl phosphate (p-NPP) and phosphotyrosine. Furthermore, with the protein substrates, it displays an activity less than 1% of that obtained with other known PTPs but comparable activities toward p-NPP and phosphotyrosine. Its responsiveness toward the usual PTP activators (e.g. spermine) or inhibitors (e.g. vanadate, molybdate, heparin, or Zn2+) varied considerably with the nature of the substrates involved. Limited digestion with trypsin caused the cleavage of a C-terminal segment of the enzyme, giving rise to a 63-kDa fragment; this cleavage resulted in an approximately 20- and 10-fold activation of the enzyme toward RCM-lysozyme and myelin basic protein, respectively.
Zhao
Z
Z
Department of Biochemistry, University of Washington, Seattle 98195.
Bouchard
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P
Diltz
C D
CD
Shen
S H
SH
Fischer
E H
EH
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D010446:Peptide Fragments; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases