The development and application of a radioimmunoassay for rat phosphoenolpyruvate carboxykinase.
We employed a newly developed radioimmunoassay to quantitate P-enolpyruvate carboxykinase protein directly in liver and kidney of intact rats. The radioimmunoassay was dependent upon the presence of sodium dodecyl sulfate in the competitive binding reaction mixture. In conjunction with assayable activity measurements, the radioimmunoassay results also made it possible to assess the average specific activity of the enzyme. In the fed state, liver P-enolpyruvate carboxykinase was 0.89 microM (micromoles/kg of tissue) and the kidney enzyme was 1.90 microM. Following a 48-h fast, the enzyme in liver increased to 2.83 microM and that in kidney to 6.83 microM. Although liver enzyme concentration increased 3-fold, total liver enzymic activity was increased only about 30%. The discrepancy was due to the decrease in liver weight which occurred during fasting. The kidneys, which do not lose weight during fasting, had a 65% elevation of total organ activity. Total organ enzyme activity was predominantly dependent on changes in enzyme mass. In the fed state, the kidney/liver enzyme mass ratio was 0.44, in the fasted state it was 0.68. Variation in enzyme concentration, enzyme half-life, and response to tryptophan and chronic triamcinolone administration all gave evidence for organ-specific regulation.
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001667:Binding, Competitive; D003600:Cytosol / Q000201:enzymology; D005215:Fasting; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000032:analysis* / Q000302:isolation & purification; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D011919:Rats, Inbred Strains; D014221:Triamcinolone / Q000031:analogs & derivatives / Q000494:pharmacology
D000906:Antibodies; D000936:Antigen-Antibody Complex; D014221:Triamcinolone; C030262:triamcinolone diacetate; D010729:Phosphoenolpyruvate Carboxykinase (GTP)