Abstract
Carbamoyl-phosphate synthetase II (CPSase II), aspartate transcarbamoylase (ATCase), and dihydroorotase (DHOase) catalyze the first three steps of de novo pyrimidine nucleotide biosynthesis, respectively. In mammalian species, these three enzyme activities exist in the cytosol in liver and other tissues as a multifunctional complex on a single polypeptide called carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) in the order of NH2-CPSase II-DHOase-ATCase-COOH. Previous studies provided evidence that in Squalus acanthias (spiny dogfish) these enzymes are not expressed in liver and that they exist as separate entities in the cytosol of extra-hepatic tissues such as testes and spleen (Anderson, P. M. (1989) Biochem. J. 261, 523-529). Here we report that the genes for these three enzymes are expressed in testes as a single transcript analogous to CAD in mammalian species and that these genes are not expressed in liver at levels that can be detected by Northern blots or by the polymerase chain reaction. The absence of the pyrimidine pathway in the liver may be related to the exclusive localization of glutamine synthetase in the mitochondrial matrix which provides for efficient assimilation of ammonia as glutamine for urea synthesis in these ureoosmotic species; thus glutamine may not be available for CPSase II or other amidotransferase activities in the cytosol. The amino acid sequence deduced from the nucleotide sequence of the shark CAD cDNA reported here is very similar to CAD from other species; alignment with the hamster CAD sequence shows 77% identical residues.
Highlights
We have recently reported that the predicted amino acid sequence based on the nucleotide sequence of the CPSase III eDNA from spiny dogfish (Squatus acanthias), a representative elasmobranch, has a high degree of identity to the amino acid sequences of rat, human, and frog CPSase I (Hong et al, 1994)
In addition to the unique difference from the CPSases related to the urea cycle in higher vertebrates, our previous studies with spiny dogfish have indicated that the CPSase activity related to pyrimidine biosynthesis in elasmobranchs may be different from the corresponding CPSase II activity in higher vertebrates (Anderson, 1989)
The results show that 1) the first three enzymes of the pyrimidine pathway are encoded as a single mRNA, which has a predicted amino acid sequence analogous to carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD); 2) the spiny dogfish CAD transcript is expressed in testes but not in liver; and 3) the transcript for CPSase III, as expected, is expressed in liver but not in testes
Summary
In ureoosmotic elasmobranch fishes (sharks, skates, and rays), carbamoyl phosphate formation in the urea cycle is catalyzed by a different type of CPSase, CPSase III (Anderson, 1980, 1991, 1995a). The apparent absence of ATCase activity in liver extracts suggests that CPSase II activity and the pyrimidine biosynthetic pathway may be expressed only in extra-hepatic tissues (Anderson, 1989). The results show that 1) the first three enzymes of the pyrimidine pathway are encoded as a single mRNA, which has a predicted amino acid sequence analogous to CAD; 2) the spiny dogfish CAD transcript is expressed in testes but not in liver; and 3) the transcript for CPSase III, as expected, is expressed in liver but not in testes
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