介绍一种部分纯化CTP:磷酸胆碱胞苷酰转移酶(CT)的方法,并对部分纯化CT的性质进行了研究。经盐析、DEAE-纤维素、磷酸-纤维素及CTP-聚琼脂糖柱层析可将大鼠肝胞液中的CT纯化100倍以上,结果重复性好。CT在胞液中及经初步纯化性质稳定,但进一步纯化则其活性很易丧失。鼠肝总磷脂及油酸可激活CT、使其聚合,磷脂酰絲氨酸是其中起主要作用的CT激活物;CT聚合物可被辛基-葡萄糖苷解聚,且保留80％酶活性。用CT常规底物CTP的类似物观察CT的特异性,发现dCTP是比CTP更好的底物,CDP、dCDP能抑制CT对CTP或dCTP的作用,而CMP、dCMP对酶活性无影响。结果提示分子中糖的2′-OH并非CT底物所必需,而和胞苷相连的三个磷酸则不可缺少。
Abstract
The key enzyme of phosphatidylcholine synthesis, CTP : cholinephosphate cytidylyltransferase (CT) (EC 2.7.7.15), was partially purified from rat liver cytosol by conventional methods, including salting-out, DEAE-Cellulose, Phospho-Cellulose and CTP-Agarose column chromatography, to more than 100 folds.The properties of CT were studied. It was quite stable in cytosol and after purifying by ion-exchange column, but loss of enzymatic activity occurred rapidly during further purification.Partially purified CT can be stimulated to form aggregate by total rat liver phospholipids,oleate and some unknown heat-stable f actor(s) . Phosphatidylserine seemed to be the most effective stimulator to CT. Aggregated CT could be dissociated by octyl-glucoside, a mild detergent, and retained about 80% of activity. Specificity study of CT showed that dCTP was a better substrate for CT than CTP; but dCDP or CDP showed some inhibitory effect on CT activity. From these results, we suggested that the triphosphate group linked to cytidine was the essential group for CT catalysis, but the 2'-OH group on the sugar moiety was not.
关键词
CTP:磷酸胆碱胞苷酰转移酶 /
鼠肝胞浆
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参考文献
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