Protein kinase activity associated with Fc gamma 2a receptor of a murine macrophage like cell line, P388D1.
The properties of protein kinase activity associated with Fc receptor specific for IgG2a (Fc gamma 2aR) of a murine macrophage like cell line, P388D1, were investigated. IgG2a-binding protein isolated from the detergent lysate of P388D1 cells by affinity chromatography on IgG-Sepharose was found to contain four distinct proteins of Mr 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [gamma-32P]ATP. The autophosphorylation of Fc gamma 2a receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Casein was found to be a much better phosphate acceptor than histone in this system, as casein incorporated about 32-fold more 32P than histone did. Phosphorylation of casein catalyzed by Fc gamma 2a receptor complex was dependent on casein concentration (maximum phosphate incorporation being at 0.5 mg/mL), increased with time or temperature, was dependent on the concentration of ATP and Mg2+, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn2+ (greater than 25 mM) or KCl (greater than 100 mM) or by a small amount of heparin (greater than 10 units/mL) and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc gamma 2a receptor used ATP as substrate with an apparent Km of 2 microM as well as GTP with an apparent Km of 10 microM. Prior heating (60 degrees C for 15 min) or treatment with protease (trypsin or Pronase) of Fc gamma 2a receptor complex almost totally abolished casein kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Hirata
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Y
Department of Microbiology, University of Kansas Medical Center, Kansas City 66103.
Suzuki
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T
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D008264:Macrophages; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011961:Receptors, Fc / Q000302:isolation & purification / Q000378:metabolism*; D017452:Receptors, IgG
D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG; D011494:Protein Kinases