Glutamine-dependent Carbamyl Phosphate Synthetase

A quantitative assay for the glutamine-dependent carbamyl phosphate synthetase (Synthetase II) that depends upon the conversion of bicarbonate-14C to citrulline-14C in the presence of excess ornithine carbamyltransferase has been used to investigate the properties of the enzyme and its distribution in relation to growth rates in normal tissues and transplanted tumors of the rat that lack the hepatic type enzyme, ammonia-dependent carbamyl phosphate synthetase (Synthetase I). The rate of the enzyme reaction in the tissues studied was a small fraction of the rate of Synthetase I found in liver. Synthetase II is distinguished from Synthetase I by its utilization of glutamine as substrate, its inhibition by 6-diazo-5-oxo-l-norleucine, its lack of requirement for N-acetylglutamate, and its presence in the soluble fraction. It can also utilize NH3 as substrate but the affinity for glutamine is 600 times greater. Under optimal conditions the rate of reaction is proportional to tissue concentration and constant with time. Rapidly growing normal tissues (fetal liver, spleen, and testis) had much more Synthetase II activity than other tissues tested (lung, uterus, heart, brain, and muscle). Tumors were more active than most normal tissues and did not contain detectable Synthetase I. The Synthetase II activities in tumors were proportional to the measured rates of growth in volume of seven tumors, and significantly so when the enzyme was expressed per tumor cell.