Purification and cDNA cloning of lactosylceramide synthetase.

从小鼠肝脏中部分纯化了一种乳糖神经酰胺合成酶（udp -半乳糖：葡萄糖神经酰胺β -1,4-半乳糖基转移酶）。纯化过程包括差速离心制备微粒体部分，用Triton X-100提取酶，并对udp -己内酰胺- sepharose， udp -半乳糖醛酸己内酰胺- sepharose和溶糖葡萄糖神经酰胺-甲酰纤维素进行顺序层析。在溶糖神经酰胺-甲酰基纤维素步骤中，酶变得不稳定。虽然该酶用甘油和蔗糖部分稳定，但很难进一步纯化。现在正在测试其他各种化学物质来稳定这种酶。该酶经过数千倍的部分纯化，比活性为11 nmol/mg/protein。葡萄糖神经酰胺和udp -半乳糖的Km值分别为7muM和200muM。该酶不与α -乳糖蛋白- Sepharose柱结合，表明该酶与乳糖合成酶不同。

A lactosylceramide synthetase (UDP-galactose : glucosylceramide beta-1,4-galactosyltransferase) has been partially purified from mouse liver. The purification procedure involved differential centrifugation for preparation of microsomal fraction, extraction of the enzyme with Triton X-100, and sequential chromatography on UDP-hexanolamine-Sepharose, UDP-galacturonic acidhexanolamine-Sepharose, and lysoglucosylceramide-formylcellulofine. At the step of lysoglucosylceramide-formylcellulofine, the enzyme became labile. Although the enzyme was partially stabilized with glycerol and sucrose, it was hardly purified further. Various other chemicals are now tested for stabilizing the enzyme. The enzyme was partially purified several thousands-fold with a specific activity of 11 nmol/mg/protein. The Km values for glucosylceramide and UDP-galactose were 7muM and 200muM,respectively. The enzyme did not bind to alpha-lactalbumin Sepharose column, indicating that the enzyme is different from lactose synthetase.