| | Bioinfo Publications | | 29 A RADIOCHEMICAL METHOD FOR CARBAMOYL-PHOSPHATE SYNTHETASE-I: APPLICATION TO RATS FED A HYPERPROTEIC DIET

Abstract- A method for the measurement of carbamoyl-phosphate synthetase I activity in animal tissues has been developed using the livers of rats under normal and hyperproteic diets. The method is based on the incorporation of 14C-ammonium bicarbonate to carbamoyl-phosphate in the presence of ATP-Mg and N-acetyl-glutamate. The reaction is stopped by chilling, lowering the pH and adding ethanol. Excess bicarbonate is flushed out under a gentle stream of cold CO2. The only label remaining in the medium was that incorporated into carbamoyl-phosphate, since all 14C-CO2 from bicarbonate was eliminated. The method is rapid and requires only a low pressure supply of CO2 to remove the excess substrate. The reaction is linear up to 10 min using homogenate dilutions of 1:20 to 1:200 (w/v). Rat liver activity was in the range of 89±8 nkat/g. Hyperproteic diet resulted in a significant 1.4-fold increase. The design of the method allows for the pro-cessing of multiple samples at the same time, and incubation medium manipulation is unnecessary, since the plastic incubation vial and its contents are finally counted together. Keywords- carbamoyl-phosphate synthetase I, urea cycle, hyperproteic diet