Neutral sphingomyelinase from human urine. Purification and preparation of monospecific antibodies

Research output: Contribution to journalArticle

Abstract

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 sphingosylphosphocholine 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 β-galactosidase and β-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 form 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.

Original languageEnglish (US)
Pages (from-to)12554-12561
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number21
StatePublished - 1989

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Sphingomyelin Phosphodiesterase
Purification
Urine
Antibodies
Enzymes
Column chromatography
serine-ethanolaminephosphate phosphodiesterase
Sphingomyelins
Isoelectric Focusing
Detergents
Assays
Phospholipases A1
Galactosidases
Membranes
Acetylglucosaminidase
Agarose Chromatography
Phosphatidylglycerols
Phosphorylcholine
Phospholipases
Phospholipases A2

ASJC Scopus subject areas

  • Biochemistry

Cite this

Neutral sphingomyelinase from human urine. Purification and preparation of monospecific antibodies. / Chatterjee, Subroto B; Ghosh, N.

In: Journal of Biological Chemistry, Vol. 264, No. 21, 1989, p. 12554-12561.

Research output: Contribution to journalArticle

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