RIBOSOMAL-ASSOCIATED PHOSPHATIDYLSERINE SYNTHETASE FROM ESCHERICHIA-COLI - PURIFICATION BY SUBSTRATE-SPECIFIC ELUTION FROM PHOSPHOCELLULOSE USING CYTIDINE "5'-DIPHOSPHO-1,2-DIACYL-SN-GLYCEROL
RIBOSOMAL-ASSOCIATED PHOSPHATIDYLSERINE SYNTHETASE FROM ESCHERICHIA-COLI - PURIFICATION BY SUBSTRATE-SPECIFIC ELUTION FROM PHOSPHOCELLULOSE USING CYTIDINE "5'-DIPHOSPHO-1,2-DIACYL-SN-GLYCEROL
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DOI：
10.1021/bi00669a003
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发表时间：
1976-01-01
期刊：
影响因子：
2.9
通讯作者：
DOWHAN, W
中科院分区：
文献类型：
--
作者：
LARSON, TJ;DOWHAN, W
胞苷5“-二磷酸-1，2-二酰基-sn-甘油（CDP-甘油二酯）：L-丝氨酸O-磷脂酰转移酶（EC 2.7.8.8，磷脂酰丝氨酸合成酶）与大肠杆菌粗提物中的核糖体紧密结合。杆菌从核糖体中分离酶后，将其纯化至97%的均一性。总体5500倍纯化的主要部分通过在洗涤剂存在下使用CDP-甘油二酯从磷酸纤维素的底物特异性洗脱来获得。当在含有十二烷基硫酸钠的聚丙烯酰胺圆盘凝胶上进行电泳时，纯化的酶作为具有54，000的表观最小MW的单一条带迁移。纯化的酶催化CMP和CDP-甘油二酯之间以及丝氨酸和磷脂酰丝氨酸之间的交换反应。该酶还催化胞二磷甘油二酯水解形成胞苷酸和磷脂酸。在酶催化的所有反应中，dCDP-甘油二酯与CDP-甘油二酯相当。纯化的酶催化磷脂酰甘油或磷脂酰甘油磷酸的形成在一个非常缓慢的速度时，丝氨酸被取代作为底物由甘油或sn-甘油-3-磷酸，分别。显然，通过形成磷脂酰-酶中间体，通过乒乓机制发生催化作用。
Cytidine 5''-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase) is bound tightly to the ribosomes in crude extracts of E. coli. After separation of the enzyme from the ribosomes it was purified to 97% homogeneity. The major portion of the overall 5500-fold purification was attained by substrate-specific elution from phosphocellulose using CDP-diglyceride in the presence of detergent. The purified enzyme migrated as a single band with an apparent minimum MW of 54,000 when subjected to electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. The purified enzyme catalyzed exchange reactions between CMP and CDP-diglyceride and between serine and phosphatidylserine. The enzyme also catalyzed the hydrolysis of CDP-diglyceride to form CMP and phosphatidic acid. dCDP-diglyceride was equivalent to CDP-diglyceride in all reactions catalyzed by the enzyme. The purified enzyme catalyzed the formation of phosphatidylglycerol or phosphatidylglycerophosphate at a very slow rate when serine was replaced as substrate by glycerol or sn-glycero-3-phosphate, respectively. Apparently, catalysis occurs via a ping-pong mechanism through the formation of a phosphatidyl-enzyme intermediate.