Anderson P M
Department of Biochemistry, University of Minnesota, Duluth 55812.
Biochem J. 1989 Jul 15;261(2):523-9. doi: 10.1042/bj2610523.
The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation.
海洋板鳃亚纲鱼类肝脏中尿素合成的前两个步骤分别涉及由氨形成谷氨酰胺以及由谷氨酰胺形成氨甲酰磷酸，这两个过程分别由谷氨酰胺合成酶和氨甲酰磷酸合成酶催化[安德森和凯西（1984年）《生物化学杂志》259卷，456 - 462页]；这两种酶都仅定位于线粒体基质中。本研究的目的是确定棘鲨（一种具有代表性的板鳃亚纲鱼类）中氨甲酰磷酸形成的酶学机制以及其在嘧啶核苷酸生物合成中的利用情况。在角鲨的肝脏中未检测到天冬氨酸氨甲酰转移酶。然而，脾脏提取物具有谷氨酰胺依赖性氨甲酰磷酸合成酶、天冬氨酸氨甲酰转移酶、二氢乳清酸酶和谷氨酰胺合成酶活性，所有这些酶都定位于胞质溶胶中；还存在二氢乳清酸脱氢酶、乳清酸磷酸核糖转移酶和乳清苷 - 5'-脱羧酶活性。除谷氨酰胺合成酶外，所有活性水平都非常低。氨甲酰磷酸合成酶活性受到UTP的抑制，并被5 - 磷酸核糖 - 1 - 焦磷酸激活。在多种不同条件下进行凝胶过滤层析时，嘧啶途径的前三种酶活性在明显不同的位置被洗脱；尽管不能排除在提取过程中多功能复合物的结构域间区域发生完全蛋白水解的情况，但证据表明，与哺乳动物不同，在角鲨中，嘧啶途径的这三种酶以单个多肽链的形式存在。这些结果：（1）确定角鲨表达两种不同的谷氨酰胺依赖性氨甲酰磷酸合成酶活性，（2）证实了[史密斯、里特和坎贝尔（1987年）《生物化学杂志》262卷，198 - 202页]关于角鲨表达两种不同谷氨酰胺合成酶的报道，（3）提供了间接证据表明在肝脏中谷氨酰胺可能无法用于除尿素形成之外的生物合成反应。
