Yamanaka T, Hanada E, Suzuki K
J Biol Chem. 1981 Apr 25;256(8):3884-9.
Two new purification procedures for human brain sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12), one without any detergent and the other with Triton X-100, are described. These procedures were more readily reproducible than the earlier one from this laboratory (Yamaguchi, S., and Suzuki, K. (1977) J. Biol. Chem. 252, 3805-3813) and yield preparations of higher purity with the final specific activity in the range of 50 to 100 mumol/h/mg of protein. Although activities of most lysosomal hydrolases tested were effectively eliminated, substantial activities of galactosylceramidase and alpha-mannosidase remained in the purified preparation. They could be separated on the analytical scale from the sphingomyelinase activity by sucrose density gradient centrifugation. "Sphingomyelinase A" as we reported earlier appears to be aggregates of the sphingomyelinase (earlier designated as "sphingomyelinase B"). In Sephadex G-200 gel filtration, the enzyme showed an apparent molecular weight of 17 to 21 X 10(4). However, in the sucrose density gradient centrifugation in the presence of Triton X-100, it co-sedimented with bovine serum albumin (Mr = 67,000). Contrary to the earlier procedure, the new procedures eliminated the magnesium-dependent neutral sphingomyelinase activity from the acid sphingomyelinase preparations.
本文描述了两种用于纯化人脑鞘磷脂酶（鞘磷脂磷酸二酯酶，EC 3.1.4.12）的新方法，一种不使用任何去污剂，另一种使用 Triton X-100。与本实验室早期的方法（Yamaguchi, S., and Suzuki, K. (1977) J. Biol. Chem. 252, 3805 - 3813）相比，这些方法更易于重复，并且能获得更高纯度的制剂，最终比活性在 50 至 100 μmol/h/mg 蛋白质范围内。尽管所测试的大多数溶酶体水解酶的活性被有效消除，但纯化制剂中仍保留了大量的半乳糖神经酰胺酶和α-甘露糖苷酶活性。通过蔗糖密度梯度离心可在分析规模上将它们与鞘磷脂酶活性分离。正如我们之前报道的，“鞘磷脂酶 A”似乎是鞘磷脂酶（早期称为“鞘磷脂酶 B”）的聚集体。在 Sephadex G - 200 凝胶过滤中，该酶的表观分子量为 17 至 21×10⁴。然而，在 Triton X - 100 存在下的蔗糖密度梯度离心中，它与牛血清白蛋白（Mr = 67,000）共同沉降。与早期方法相反，新方法从酸性鞘磷脂酶制剂中消除了镁依赖性中性鞘磷脂酶活性。
