Characterization and Significance of Carbamyl Phosphate Phosphatase1

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 tis sues 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 activi ties 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 37Â°and was 8-fold greater than was the specific activity of the cytosolic fraction. The two frac tions 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 paniculate 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 ho mogenate activities were comparable at 72 and 73 nmol/min/mg at pH 7.4 and 24Â°. CAP phosphatase activity was measured in homogenates of SV40-transformed Syrian hamster mutant cell lines with various levels of aspartate transcarbamylase and var iable sensitivity to the aspartate transcarbamylase competitive inhibitor, A/-(phosphonacetyl)-L-aspartate. The CAP phosphatase activity was relatively constant at 60 to 70 nmol/min/mg even for mutants with variable A/-(phosphonacetyl)-L-aspartate sensi tivity but similar aspartate transcarbamylase levels; thus, the variable A/-(phosphonacetyl)-L-aspartate sensitivity in these mu tant cell lines was not due to a difference in CAP phosphatase levels. CAP phosphatase activity should have the greatest influ ence on flux through the pyrimidine pathway, when both aspar tate transcarbamylase is inhibited and the ratio of CAP phospha tase activity to aspartate transcarbamylase is relatively high. INTRODUCTION CAP4 is an important high-energy biological compound in volved in synthesis of pyrimidines and in synthesis of citrulline, arginine, and urea via the urea cycle (33, 34). Pyrimidine de novo biosynthesis occurs primarily in the cytoplasm, where CAP is synthesized by CAP synthetase II (glutamine requiring) and sub sequently is converted first to carbamyl aspartate by ATCase and then to dihydroorotate by dihydroorotase. CAP synthetase II, ATCase, and dihydroorotase are all part of the cytoplasmic trifunctional protein CAD. In contrast, the first 2 steps of mam malian urea biosynthesis occur in the mitochondria of liver and small intestine (35), where CAP is synthesized by CAP synthe tase I (ammonia requiring) and then is converted to citrulline by ornithine transcarbamylase. The only other enzymes that can catabolize CAP are phosphatases which hydrolyze CAP to carbamate and orthophosphate (16, 28, 56). This catabolic enzyme activity, referred to here as CAP phosphatase activity, has not been well characterized. Enzymatic hydrolysis of CAP was first reported in a preliminary manner between 1954 and 1958 by Grisolia and coworkers (22, 24,25) and Krebs ef al. (40). Since that time, a variety of enzymes from a number of tissues have been reported to hydrolyze CAP. CAP phosphatase activity has been found in normal tissues of humans and other mammals (21-23) and in tumor tissues (28). In normal animals, CAP phosphatase activity has been demon strated in most major organs (56). Enzymes characterized as acyl phosphatase (EC 3.6.1.7.) (15, 55), glucose-6-phosphatase (51), alkaline phosphatase (61),5 Na+-K+-ATPase (4, 73), and Caz+-ATPase (42) have all been shown to hydrolyze CAP. In addition, an acid phosphatase has been shown to hydrolyze the CAP analogue, acetyl phosphate (67). The above enzymes are located in both the soluble and paniculate fractions of the cell. Acyl phosphatase was first described using acetyl phosphate as substrate by Lipmann (43) in 1946. Purified acyl phosphatases are generally soluble proteins that hydrolyze acetyl phosphate, CAP, benzoyl phosphate, and 1,3-diphosphoglycerate (56). They are distinct by having a molecular weight ranging from 8,000 to 14,000 and are often heat and acid stable (56). Clearly, the efficiency of CAP hydrolysis for a given tissue will be a function of all the different enzymes present which can catabolize CAP. In all tissues other than liver and small intestine, the concen tration of CAP in the cytosol should be low, <0.2 fiM, as sug gested by Christopherson and Jones (12). With Kâ„¢values for CAP phosphatase activity reported in the mM range (21, 28, 56), it would be unlikely for a phosphatase to compete efficiently with '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. 3To whom requests for reprints should be addressed. Received April 9, 1984; accepted July 2, 1984. 'The abbreviations used are: CAP, carbamyl phosphate; ATCase, aspartate transcarbamylase; CAD, the multifunctional protein catalyzing the initial 3 steps in de novo pyrimidine biosynthesis; EAC, Ehrlich ascites carcinoma; HEPES, N-2hydroxyethylpiperazine-AT-2-ethanesulfonic acid; PALA, N-<phosphonacetyl)-L-aspartate; sucrose:EDTA, 0.25 M sucrose:2.5 mM EDTA, pH 7.2; N fraction, nuclear fraction; M fraction, mitochondrial fraction; L fraction, lysosomal fraction; P fraction, microsomal fraction; S fraction, soluble fraction. * Hydrolysis of CAP by calf intestine alkaline phosphatase has been confirmd in