Quintern L E, Weitz G, Nehrkorn H, Tager J M, Schram A W, Sandhoff K
Institut für Organische Chemie und Biochemie der Universität Bonn, F.R.G.
Biochim Biophys Acta. 1987 Dec 14;922(3):323-36. doi: 10.1016/0005-2760(87)90055-5.
Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine in the presence of 0.1% Nonidet P-40. The activity could be enriched 23,000-fold by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue Sepharose and DEAE-cellulose. The last purification step yielded an enzyme preparation with a specific activity of about 2.5 mmol sphingomyelin cleaved/h per mg protein and with a yield of about 3%. Purified sphingomyelinase appeared to be homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 70 kDa. In the presence of 0.08% (w/v) sodium taurodeoxycholate the preparation showed phosphodiesterase activity toward sphingomyelin, phosphatidylcholine and phosphatidylglycerol. These activities co-purified during the entire purification procedure, indicating that the acid sphingomyelinase hydrolyses not only sphingomyelin but also the other two phospholipids, phosphatidylcholine and phosphatidylglycerol. Addition of 100 microM tripalmitoylglycerol to the assay system (which contains 100 microM sphingomyelin) instead of detergent, stimulated the reaction about 20-fold compared to an assay which did not contain detergents, thus offering a very sensitive and efficient system for the assay of sphingomyelinase in a system free of detergents. Sphingomyelin degradation was strongly inhibited by phosphatidylinositol 4',5'-bisphosphate, adenosine 3',5'-diphosphate and adenine-9-beta-D-arabinofuranoside 5'-monophosphate (50% inhibition at inhibitor concentrations of 1-5 microM).
酸性鞘磷脂酶（鞘磷脂磷酸二酯酶，EC 3.1.4.12）是在0.1%的诺乃洗涤剂P-40存在的情况下从人尿中纯化得到的。通过依次在辛基琼脂糖凝胶、伴刀豆球蛋白A琼脂糖凝胶、蓝色琼脂糖凝胶和二乙氨基乙基纤维素上进行层析，该酶的活性可富集23000倍。最后一步纯化得到的酶制剂的比活性约为每毫克蛋白质每小时裂解2.5 mmol鞘磷脂，产率约为3%。纯化后的鞘磷脂酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中显示为均一性，分子量为70 kDa。在0.08%（w/v）的牛磺去氧胆酸钠存在下，该制剂对鞘磷脂、磷脂酰胆碱和磷脂酰甘油表现出磷酸二酯酶活性。这些活性在整个纯化过程中共同纯化，表明酸性鞘磷脂酶不仅水解鞘磷脂，还水解另外两种磷脂，即磷脂酰胆碱和磷脂酰甘油。在测定系统（含有100 μM鞘磷脂）中加入100 μM三棕榈酰甘油而不是去污剂，与不含去污剂的测定相比，反应刺激约20倍，从而为在无去污剂系统中测定鞘磷脂酶提供了一个非常灵敏和有效的系统。磷脂酰肌醇4',5'-二磷酸、腺苷3',5'-二磷酸和腺嘌呤-9-β-D-阿拉伯呋喃糖苷5'-单磷酸对鞘磷脂降解有强烈抑制作用（抑制剂浓度为1 - 5 μM时抑制50%）。
