Release of Sphingomyelin Phosphodiesterase (Acid Sphingomyelinase) by Ammonium Chloride from CL 1D Mouse L-Cells and Human Fibroblasts. Partial Purification and Characterization of the Exported Enzymes

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Abstract
In cultured human fibroblasts and mouse [neoplastic fibroblast] 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 concanavalin A-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 .times. mg) and 1434 nmol/(h .times. 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 .times. 10-5 M and 2.4 .times. 10-5 M, respectively, and an apparent molecular mass of 68 kDa [kilodaltons]. In isoelectric focusing the enzymes peaked at pH 4.78 (mouse enzyme) and pH 4.75 (human enzyme).