Preliminary characterization of phosphotyrosine phosphatase activities in human peripheral blood lymphocytes: identification of CD45 as a phosphotyrosine phosphatase.
A preliminary characterization of the protein phosphotyrosine phosphatase (PTPase) activity in human peripheral blood lymphocytes (PBL) has been made using two tyrosine-containing peptides and the epidermal growth factor receptor from A-431 cells as substrates. High PTPase activity with a pH optimum near 7.4 was observed in both the membrane and the cytosolic fractions. At least three distinct fractions with PTPase activity were separated on DEAE cellulose columns, indicating that the enzyme is heterogeneous. Vanadate, molybdate, and salts of zinc, copper, and mercury were all effective enzyme inhibitors, although the inhibition was generally incomplete and showed some variation with the enzyme preparation. The difficulty in completely inhibiting PTPase activity in lymphocytes may help explain the variation between laboratories in studies of tyrosine phosphorylation in these cells. Studies with highly purified T lymphocytes obtained by filtration of PBL through nylon wool columns indicated that the activity is present in T cells. Absorption with agarose containing anti-HLe-1, a mouse monoclonal IgG1 antibody specific for the leukocyte-specific surface protein T-200 (CD45), removed up to 40% of the PTPase activity. Enzyme activity was recovered on the immunoadsorbent after extensive washing, confirming that the enzyme was being bound to the beads. Immunoabsorbents containing other mouse IgG1 antibodies failed to bind PTPase activity, indicating that the binding to beads with anti-HLe-1 antibody is specific. Further characterization of the CD45 and PTPase activities in lymphocytes may provide a better understanding of the role of protein tyrosine phosphorylation in the regulation of proliferation and differentiation in these cells.
George
R J
RJ
Howard Hughes Medical Institute, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Parker
C W
CW
D000943:Antigens, Differentiation / Q000032:analysis / Q000378:metabolism*; D006649:Histocompatibility Antigens / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017493:Leukocyte Common Antigens; D008214:Lymphocytes / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases; D013347:Subcellular Fractions / Q000201:enzymology
D000943:Antigens, Differentiation; D006649:Histocompatibility Antigens; D010749:Phosphoprotein Phosphatases; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases