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.

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).