Modulation of partially purified rat liver mitochondrial carbamoyl phosphate synthetase I using two glutamic acid analogues

Abstract

Mitochondrial c arbamoyl ph osphate synthetase I (CPS I) i s the first enzyme involved in urea biosynthesis in ureotelic mammals and has an absolute requirement for N - acetyl - L - glutamate (NAG) or N - carbamyl - L - glutamate (NCLG) in absence of NAG as its allosteric modulator. To investigate effect of diet on CPS I acti vation , three male albino rats were maintained under normal laboratory diet (control) and another three on high protein egg white diet for 10 days . The percentage mean weight gain for the normal diet was 6.4 % while the percentage mean weight loss for the high protein diet group 18.6 %. The rats were sacrificed and CPS I isolated from the liver mitochondria through differential centrifugation and partially purified by ammonium sulphate precipitation, gel filtration on Sephadex G - 200 and native polyacrylamid e gel electrophoresis (PAGE). Lactate dehydrogenase - pyruvate kinase (LDH - PK) coupled assay system was devised to determine the effect of NAG and its structural analogue NCLG in the activation of CPS I from rats fed on the two diets. CPS I activity of 268.1 6 nmol/min/mg in the control group doubled to 553.86 nmol/min/mg in rats fed on high protein. An initial velocity of CPS I of 3.07 nmol/min/mg was observed when activated by 0.57 mM NAG and a lowered activity of 2.2 nmol/min/mg when replaced with 0.57 mM N CLG. Both NAG and NCLG activated CPS I at all concentrations tested in the assay system devised with improved activity seen when CPS I activity was measured in presence of NAG