Terjesen B F, Rønnestad I, Norberg B, Anderson P M
Department of Zoology, University of Bergen, Norway.
Comp Biochem Physiol B Biochem Mol Biol. 2000 Aug;126(4):521-35. doi: 10.1016/s0305-0491(00)00221-2.
The presence of carbamoyl phosphate synthetase III (CPSase III), catalyzing the first step of the urea cycle in fish, in Atlantic halibut (Hippoglossus hippoglossus L.) yolk-sac larvae and adult white muscle has been established using gel filtration chromatography to separate the CPSase III from the pyrimidine-pathway related CPSase II. The results are consistent with the hypothesis that teleostean fish express urea cycle enzymes during early development and with recent observations of low levels of CPSase III in muscle tissue. The presence of CPSase III in crude extracts could not be established using sensitive assay conditions to discriminate between CPSase III and CPSase II. However, kinetic characterization after chromatographic separation identified each as typical CPSase II and CPSase III activities, respectively. The CPSase III was less sensitive to activation by N-acetyl-L-glutamate and had a higher Km for ammonia than CPSase III found in other species. These results suggest that precise quantitation of low levels of CPSase III in the presence of CPSase II by assaying crude extracts may be difficult unless the enzymes are first separated and the kinetic properties of CPSase III are determined; the results indicate that assaying larval extracts of Atlantic halibut in the presence of uridine triphosphate results in CPSase activity that reflects mostly CPSase III and can, therefore, be used to measure changes in CPSase III activity.
利用凝胶过滤色谱法将氨基甲酰磷酸合成酶III（CPSase III）与嘧啶途径相关的CPSase II分离，已确定大西洋庸鲽（Hippoglossus hippoglossus L.）卵黄囊幼虫和成体白肌中存在催化鱼类尿素循环第一步的CPSase III。这些结果与硬骨鱼在早期发育过程中表达尿素循环酶的假说一致，也与近期在肌肉组织中发现低水平CPSase III的观察结果相符。在使用敏感的检测条件来区分CPSase III和CPSase II时，无法确定粗提物中是否存在CPSase III。然而，色谱分离后的动力学特征分别将二者鉴定为典型的CPSase II和CPSase III活性。与其他物种中的CPSase III相比，该CPSase III对N - 乙酰 - L - 谷氨酸激活的敏感性较低，对氨的Km值较高。这些结果表明，除非首先分离酶并确定CPSase III的动力学性质，否则在存在CPSase II的情况下通过检测粗提物来精确定量低水平的CPSase III可能很困难；结果表明，在三磷酸尿苷存在的情况下检测大西洋庸鲽幼虫提取物，所得到的CPSase活性主要反映CPSase III，因此可用于测量CPSase III活性的变化。
