Acid sphingomyelinase from human urine: purification and characterization.
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).
Quintern
L E
LE
Institut für Organische Chemie und Biochemie der Universität Bonn, F.R.G.
Weitz
G
G
Nehrkorn
H
H
Tager
J M
JM
Schram
A W
AW
Sandhoff
K
K
D006801:Humans; D008823:Micelles; D009711:Nucleotides / Q000494:pharmacology; D017830:Octoxynol; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000652:urine*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000652:urine*; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism
D008823:Micelles; D009711:Nucleotides; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase