Xiong X F, Anderson P M
Department of Biochemistry, School of Medicine, University of Minnesota, Duluth 55812.
Arch Biochem Biophys. 1989 Apr;270(1):198-207. doi: 10.1016/0003-9861(89)90021-0.
Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.
在板鳃亚纲动物中，肝脏线粒体利用氨合成瓜氨酸的过程涉及谷氨酰胺的中间形成，这是由于高水平的谷氨酰胺合成酶和一种独特的依赖谷氨酰胺和N - 乙酰谷氨酸的氨甲酰磷酸合成酶的存在，这两种酶都具有依赖谷氨酰胺合成尿素功能所特有的性质，尿素以高浓度保留在板鳃亚纲动物组织中用于渗透调节[P.M.安德森和C.A.凯西（1984年）《生物化学杂志》259卷，456 - 462页；R.A.尚卡尔和P.M.安德森（1985年）《生物化学与生物物理学报》239卷，248 - 259页]。本研究的目的是确定催化线粒体瓜氨酸合成最后一步且此前未从任何鱼类物种中分离出来的鸟氨酸氨甲酰转移酶是否也具有与该功能独特相关的性质。从代表板鳃亚纲动物的棘鲨分离的肝脏线粒体中高度纯化了鸟氨酸氨甲酰转移酶。纯化后的酶是一种三聚体，亚基分子量为38,000，天然分子量约为114,000。pH的影响受鸟氨酸浓度显著影响；当鸟氨酸饱和时，最佳活性在pH 7.8。在pH 7.8时，鸟氨酸和氨甲酰磷酸的表观Km值分别为0.71和0.05 mM。在pH 7.8以上，鸟氨酸表现出相当程度的底物抑制。该活性不受渗透剂尿素或三甲胺 - N - 氧化物的生理浓度或许多其他代谢物的显著影响。动力学研究结果与底物的稳态有序添加（氨甲酰磷酸先结合）和产物的快速平衡随机释放一致。除了异常低的比活性外，纯化的板鳃亚纲动物酶的性质与来自哺乳动物排尿素动物和其他物种的鸟氨酸氨甲酰转移酶的性质相似，并且似乎并非其在依赖谷氨酰胺合成尿素以进行渗透调节的作用中所特有。
