Phosphorylation and Dephosphorylation of Carbamoyl-Phosphate [Synthetase II Complex of Rat Ascites Hepatoma Cells<xref ref-type="fn" rid="fn1"><sup>1</sup></xref><xref ref-type="fn" rid="fn2"><sup>2</sup></xref>

Abstract

Carbamoyl-phosphate synthetase II [EC 6.3.5.5] of rat ascites hepatoma cells (AH 13), the first and regulatory enzyme of de novo pyrimidine nucleotide biosynthesis, exists as a multienzyme complex (molecular weight, 870, 000) with aspartate car-bamoyltransferase [EC 2.1.3.2] and dihydroorotase [EC 3.5.2.3] (Mori, M. & Tati-bana, M. (1975) J. Biochem. 78, 239–242). The purified complex was phospho-rylated by the catalytic subunit of cAMP-dependent protein kinase [EC 2.7.1.37] of rabbit skeletal muscle. The incorporation of 32P1 was 2.2 mol/mol of the complex. The phosphorylation was completely inhibited by the inhibitor protein of the cAMP-dependent protein kinase. Among the substrates and effectors of the enzyme complex tested, only MgUTP, an allosteric inhibitor of carbamoyl-phosphate synthetase II, strongly inhibited the phosphorylation; this inhibition was due probably to the competition of MgUTP with the substrate MgATP for the protein kinase. The complex that was phosphorylated by cAMP-dependent protein kinase was dephos-phorylated by phosphoprotein phosphatase [EC 3.1.3.16] of rat skeletal muscle. The complex was also phosphorylated by cAMP-independent protein kinase activity present in the extract of AH 13 cells and dephosphorylated by phosphoprotein phosphatase activity of the same origin. These results suggest that the complex is subject to phosphorylation and dephosphorylation in the living cells. Phosphorylation of the complex by cAMP-dependent protein kinase was associated only with a slight change, albeit definite, in the activity of carbamoyl-phosphate synthetase II under the assay conditions. Thus, the physiological significance of phosphorylation-dephosphorylation remains to be further studied.