Weitz G, Lindl T, Hinrichs U, Sandhoff K
Hoppe Seylers Z Physiol Chem. 1983 Jul;364(7):863-71. doi: 10.1515/bchm2.1983.364.2.863.
In cultured human fibroblasts and mouse L-cells the lysosomotropic agent, ammonium chloride, caused release of acid sphingomyelinase into the culture medium. The water-soluble enzymes were partially purified by sequential chromatography on ConA-Sepharose, octyl-Sepharose and Sepharose CL-4B. Mouse sphingomyelinase was purified up to 64-fold and human sphingomyelinase 134-fold from the culture medium. Specific activities were 925 nmol/(h X mg) and 1 434 nmol/(h X mg), respectively. The final enzyme preparations obtained were free of other lysosomal enzyme activities tested and had very similar properties: optimal activity at pH 4.8 (mouse enzyme) and pH 4.4 (human enzyme), Km values of 6.2 X 10(-5)M and 2.4 X 10(-5)M, respectively, and an apparent molecular mass of 68 kDa. In isoelectric focusing the enzymes peaked at pH 4.78 (mouse enzyme) and pH 4.75 (human enzyme).
在培养的人成纤维细胞和小鼠L细胞中，溶酶体亲和剂氯化铵可导致酸性鞘磷脂酶释放到培养基中。通过先后在伴刀豆球蛋白A-琼脂糖、辛基琼脂糖和琼脂糖CL-4B上进行层析，对水溶性酶进行了部分纯化。从小鼠培养基中纯化的鞘磷脂酶达到64倍，从人培养基中纯化的鞘磷脂酶达到134倍。比活性分别为925 nmol/(h×mg)和1434 nmol/(h×mg)。最终获得的酶制剂不含所检测的其他溶酶体酶活性，且具有非常相似的特性：小鼠酶在pH 4.8时活性最佳，人酶在pH 4.4时活性最佳，Km值分别为6.2×10(-5)M和2.4×10(-5)M，表观分子量为68 kDa。在等电聚焦中，这些酶在pH 4.78（小鼠酶）和pH 4.75（人酶）时达到峰值。
