Abstract
It has been generally accepted that the orotic acid pathway is the major source of pyrimidines in a mammalian liver and carbamyl-P is considered as the initial substrate of this pathway. However, in spite of many attempts by previous investigators, carbamyl-P synthetase was not found in animal tissues other than the liver and small intestine 1-3). These negative results promoted varying speculations concerning the source of pyrimidines in these other tissues; it remained to be clarified whether carbamyl-P synthetase may be actually present though its detection has not been possible for some technical difficulty, or the tissues maydepend on preformed pyrimidines or precursors supplied by the liver, or an alternative pathway may operate in these tissues.
As the first approach to solve this problem, incorporation in vivoof14C 02 into the pyrimidine ring of the hematopoietic mouse spleen was studied and compared with that into the pyrimidine ring of the liver 4) . The labeling of uracil of uridine nucleotides in acid soluble fraction of the spleen took place without any noticeable lag after 14CO2 administration, and the specific radioactivity of the spleen UMP was higher than that of the liver UMP (Fig. 1). These results suggested that the uracil molecules synthesized in the liver play only a minor role, if at all, as the source of pyrimidines in spleen. When Cdistribution in uracil as well as in carbamylaspartate was examined, there was a marked difference between the labeling patterns of these compounds isolated from the spleen and those from the liver (TABLE I). These observations provided evidence against the possibility that the products or intermediates of the orotic acid pathway were transferred from the liver to the spleen tissue. In addition, the spleen slices were shown to be able to incorporate 14CO2 into carbamylaspartate, other precursors and pyrimidine nucleotides. It was concluded from these results that the spleen tissue is provided with the complete orotic acid pathway for pyrimidine biosynthesis starting from bicarbonate.
After several negative attempts, a successful detection of carbamyl-P synthetase of the spleen was achieved by the use of glycerol as a stabilizing agent for the enzyme (Fig.2) 5). The enzyme was purified about twenty fold from soluble fraction of the spleen by ammonium sulfate fractionation, followed by chromatography on a hydroxylapatite column. Studies on this preparation revealed the distinction of spleen carbamyl-P synthetase from liver enzyme, which is the only mammalian carbamyl-P synthetase so far well characterized. The findings are summarized as follows: 1) the spleen enzyme is fully active in the absence of N-acetylglutamate, the essential activator for the liver enzyme; 2) the spleen enzyme utilizes L-glutamine as well as NH4+ as carbamyl nitrogen donor; 3) the greater part of the spleen enzyme is localized in the soluble fraction; 4) the spleen enzyme activity is so low (0.5μmole carbamyl-P formation/g/hr) as limit the biosynthetic rate; 5) the enzyme is subject to feedback inhibition by UTP, thus playing a key role in the control of pyrimidine biosynthesis (TABLE II). On the other hand, it was found that aspartate transcarbamylase of the spleen is not involved in the control mechanism (TABLE III).
Through the use of glycerol as a stabilizing agent of enzyme, some other non-hepatic tissues were shown to contain definite activity of carbamyl-P synthetase (TABLE IV). The activity was high in tissues which are supposed to be of a rapid rate of cellular proliferation, namely, stomach, thymus, testis, bone marrow, reticulocytes, Ehrlich ascites tumor cells and so on.
