A new approach of the estimation of Km of carbamyl phosphate synthetase for ammonia in isolated rat hepatocytes.

1. The Km for ammonia of carbamyl phosphate synthetase was determined by preincubating isolated liver cells for 30 min in the absence of ammonia and bicarbonate and in the presence of ornithine, chloroquine, which blocks lysosomal proteolysis, and aminoxy acetic acid, which inhibits transaminases. 2. The reaction was started with the addition of varying concentrations of ammonia and 10 mM bicarbonate. 3. The rate of citrulline formation was measured as related to ammonia concentration. 4. The pre-incubation with ornithine permits an accumulation of intracellular and mitochondrial ornithine concentrations which in turn allow rapid citrulline formation in the carbamyl phosphate form. 5. This prevents any feedback inhibition on a carbamyl phosphate synthetase or decreases in activity due to accumulation of carbamyl phosphate and/or absence of ornithine. 6. Using these methods in combination with [14C]bicarbonate permitted an estimation of exogenous ammonia for carbamyl phosphate synthesis. 7. The Km for ammonia was 1.5 mM, using a pK of 8.88 the Km for free NH3 was 48 microM.