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Characterization and Significance of Carbamyl Phosphate Phosphatase¹

Department of Biochemistry, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27514

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 K_m 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° 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 < muscle < liver < kidney < brain. Ehrlich ascites carcinoma cell and muscle homogenate activities were comparable at 72 and 73 nmol/min/mg at pH 7.4 and 24°. 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. The CAP phosphatase activity was relatively constant at 60 to 70 nmol/min/mg even for mutants with variable N-(phosphonacetyl)-L-aspartate sensitivity but similar aspartate transcarbamylase levels; thus, the variable N-(phosphonacetyl)-L-aspartate sensitivity in these mutant cell lines was not due to a difference in CAP phosphatase levels. CAP phosphatase activity should have the greatest influence on flux through the pyrimidine pathway, when both aspartate transcarbamylase is inhibited and the ratio of CAP phosphatase activity to aspartate transcarbamylase is relatively high.

¹ This work was supported by NIH Grant HD12787 and American Cancer Society, Inc., Grant IN-15V. A preliminary report of this work was presented at the 74th Annual Meeting of the American Society of Biological Chemists in San Francisco, CA (7).

² Recipient of a Special Fellowship from the Leukemia Society of America, Inc.

³ To whom requests for reprints should be addressed.

Received 4/ 9/84. Accepted 7/ 2/84.