Neutral sphingomyelinase from human urine. Purification and preparation of monospecific antibodies.
A neutral sphingomyelinase which cleaves phosphorylcholine from sphingomyelin at a pH optima of 7.4 was purified 440-fold to apparent homogeneity from normal human urine concentrate employing Sephadex G-75 column chromatography, preparative isoelectric focusing, and sphingosylphospholcholine CH-Sepharose column chromatography. The enzyme is composed of a single polypeptide whose apparent molecular weight is 92,000. Analytical isoelectric focusing revealed that the pI of this enzyme is 6.5. Purified neutral sphingomyelinase was devoid of beta-galactosidase and beta-N-acetylglucosaminidase activity originally present in the urine concentrate. The purified neutral sphingomyelinase (N-SMase) had low levels of phospholipase A1 and A2 activity when phosphatidylcholine was used as a substrate and detergents were included in the assay mixture. However, it had no phospholipase activity toward phosphatidylglycerol and sphingomyelin at pH 4.5 irrespective of the presence or absence of detergents. Monospecific polyclonal antibodies raised against N-SMase immunoprecipitated approximately 70% of N-SMase activity from urine, human kidney proximal tubular cells, and partially purified membrane-bound N-SMase from these cells. Western immunoblot assays revealed that the monospecific polyclonal antibody against urinary N-SMase recognized both the urinary N-SMase and the membrane-bound N-SMase. Because this enzyme is distinct biochemically and immunologically as compared to acid sphingomyelinase (EC 3.1.4.12), we would like to assign it an enzyme catalog number of EC 3.1.4.13. The availability of N-SMase and corresponding antibody will be useful in studying various aspects of this enzyme in biological systems.
Chatterjee
S
S
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Ghosh
N
N
D000118:Acetylglucosaminidase / Q000652:urine; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006801:Humans; D007525:Isoelectric Focusing; D007687:Kidney Tubules, Proximal / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000652:urine*; D012867:Skin / Q000201:enzymology; D013108:Sphingomyelin Phosphodiesterase / Q000276:immunology / Q000302:isolation & purification / Q000652:urine*; D013379:Substrate Specificity; D014462:Ultrafiltration; D001616:beta-Galactosidase / Q000652:urine
D000911:Antibodies, Monoclonal; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase; D001616:beta-Galactosidase; D000118:Acetylglucosaminidase