Acid Sphingomyelinase from Human Urine: Purification and Characterisation

Abstract

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 up to 30 000 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 2.5–10 mmol sphingomyelin cleaved/h per mg protein and with a yield of about 3–16 %. Purified sphingomyelinase appeared to be homogeneous in sodium dodecyl sulphate Polyacrylamide gel electrophoresis with a mass of about 70 kDa. In the presence of 0.08 % sodium taurodeoxycholate the preparation showed phosphodiesterase activity towards several phospholipids. Sphingomyelin, phosphatidylcholine, phosphatidylglycerol and at a slow rate phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine were hydrolysed. The phospholipase C activity towards phosphatidylcholine and phosphatidylglycerol and acid sphingomyelinase activity copurified during the entire purification procedure, indicating that acid sphingomyelinase has phospholipase C activity towards these lipids.

Addition of 100 µM tripalmitoylglycerol to the assay system (which contains 100 µM sphingomyelin) instead of detergent, stimulated the reaction about 20-fold, thus offering a sensitive system for the assay of acid sphingomyelinase in a system free of detergents. Sphingomyelin degradation was strongly inhibited by phosphatidylinositol 4′, 5′-bis phosphate, adenosine 3′, 5′- diphosphate and adenine-9-β-D arabinofuranoside 5′-monophosphate (50 % inhibition at inhibitor concentrations of 1–5 µM and a substrate concentration of 100 µM sphingomyelin).