EXPRESSION ANALYSIS OF 2 MUTANT HUMAN ORNITHINE TRANSCARBAMYLASES IN COS-7 CELLS

Ornithine transcarbamylase (OTC) is located in the mitochondrial matri x of the liver and small intestine and catalyzes the second step of th e urea cycle. OTC deficiency (OTCD) is an X-linked inborn error of met abolism and causes hyperammonemia. We reported in 1992 the A152V and G 195R mutations in patients with OTCD. These mutant OTC cDNAs were prep ared by site-directed mutagenesis using the polymerase chain reaction (PCR). The wild-type and mutant cDNAs were transiently expressed in CO S-7 cells. The wild-type cDNA gave an OTC activity of 1180+/-47 nmol/m in per mg protein. The OTC activities of the A152V and the G195R mutan ts were 3.7% and 2.5% of that of wild-type, respectively. Immunoblot a nalysis showed that the quantities of OTC proteins in the A152V and G1 95R mutants were 29% and 12% of that of wildtype, respectively. In pul se-labeling and pulse-chase experiments, the precursor form of OTC was synthesized and processed to the mature form. The A152V mutant OTC wa s processed to the mature form as rapidly as the wild-type precursor. However, the processed, mature form of the mutant OTC was rapidly degr aded, presumably in the mitochondrial matrix. These results indicate t hat OTCD with the A152V mutation is due both to rapid degradation of t he processed, mature form, and to a lower specific activity of the rem aining protein.