Characterization and significance of carbamyl phosphate phosphatase.

Carbamyl phosphate (CAP) is a key compound of the biosynthetic pathways for pyrimidines and urea. CAP can also be catabolyzed by phosphatases which hydrolyze CAP to carbamate and orthophosphate. This CAP phosphatase activity was studied from tissue preparations of Ehrlich ascites carcinoma cells, SV-40-transformed Syrian hamster cells, and normal tissues of the rat. A procedure for subcellular fractionation of Ehrlich ascites carcinoma cells was developed, so that cellular contents might be divided into nuclear, mitochondrial, lysosomal, microsomal, and cytosolic fractions. Plasma membranes were found primarily in the lysosomal fraction. There were at least 3 different CAP-hydrolyzing phosphatases, each occurring predominantly in different fractions, namely, the lysosomal, cytosolic, and microsomal fractions. These fractions contained 51, 20, and 14% of homogenate CAP phosphatase activity, respectively. The pH maximum for each of these phosphatases was 8, 5.7, and 7.5, respectively, and all fractions were stimulated by Mg2+. It was determined that activity in the lysosomal fraction resided with the plasma membrane fragments. The CAP phosphatase activities associated with the plasma membrane and cytosol were further studied. The Km for CAP of both fractions was 1.1 mM. The lysosomal fraction specific activity for CAP hydrolysis was 602 nmol/min/mg at pH 7.4 and 37 degrees and was 8-fold greater than was the specific activity of the cytosolic fraction. The two fractions differed in response to Mg2+, K+, and other ions and inhibitors. A crude subcellular fractionation of rat liver was also performed, and 92% of the activity was found in the particulate fractions. Levels of CAP phosphatase activity in homogenates of normal rat tissues varied: whole blood less than muscle less than liver less than kidney less than brain. Ehrlich ascites carcinoma cell and muscle homogenate activities were comparable at 72 and 73 nmol/min/mg at pH 7.4 and 24 degrees. CAP phosphatase activity was measured in homogenates of SV40-transformed Syrian hamster mutant cell lines with various levels of aspartate transcarbamylase and variable sensitivity to the aspartate transcarbamylase competitive inhibitor, N-(phosphonacetyl)-L-aspartate.(ABSTRACT TRUNCATED AT 400 WORDS)