Kitabatake K, Piran U, Kamio Y, Doi Y, Nishida T
Biochim Biophys Acta. 1979 Apr 27;573(1):145-54. doi: 10.1016/0005-2760(79)90181-4.
A simple and convenient method for the purification of human plasma lecithin-cholesterol acyltransferase was developed. The method involves the adsorption of the enzyme from diluted human plasma on DEAE-Sephadex, treatment with 1-butanol in the presence of (NH4)2SO4, DEAE-Sephadex chromatography, treatment with dextran sulfate in the presence of Ca2+, and hydroxyapatite chromatography. The enzyme purified showed a single main band by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In addition, the enzyme obtained was stable for more than four weeks, when it was kept at 4 degrees C under N2 in a buffer of low ionic strength. The purified enzyme was used to study its specificity toward the acyl acceptor. This specificity was found to be broad in that not only sterols but also long chain primary alcohols exhibited considerable acceptor activity. Furthermore, in agreement with our previous observations with crude enzyme (Piran, U. and Nishida, T. (1976) J. Biochem. (Tokyo) 80, 887-889), the purified enzyme was found to be capable of hydrolyzing the ester linkage at the carbon-2 position of phosphatidylcholine. The transesterification, as well as the hydrolytic reaction, required the presence of the cofactor polypeptide, apolipoprotein A-I.
开发了一种简单便捷的人血浆卵磷脂胆固醇酰基转移酶纯化方法。该方法包括从稀释的人血浆中吸附酶到DEAE-葡聚糖凝胶上，在硫酸铵存在下用正丁醇处理，进行DEAE-葡聚糖凝胶色谱，在钙离子存在下用硫酸葡聚糖处理，以及羟基磷灰石色谱。纯化后的酶在有和没有十二烷基硫酸钠的情况下通过聚丙烯酰胺凝胶电泳均显示出单一主带。此外，所获得的酶在低离子强度缓冲液中于4℃下氮气保存时，可稳定超过四周。纯化后的酶用于研究其对酰基受体的特异性。发现这种特异性很广泛，因为不仅甾醇而且长链伯醇都表现出相当的受体活性。此外，与我们之前对粗酶的观察结果一致（皮兰，U.和西田，T.（1976年）《生物化学杂志》（东京）80，887 - 889），发现纯化后的酶能够水解磷脂酰胆碱碳-2位的酯键。转酯反应以及水解反应都需要辅因子多肽载脂蛋白A-I的存在。
