Purification of acid sphingomyelinase from human placenta: Characterization and N‐terminal sequence

FEBS Letters

Glombitza, Gereon¹  Sandhoff, Konrad¹  Lansmann, Stephanie¹  Hurwitz, Robert²  Ferlinz, Klaus¹  Bartelsen, Oliver¹ 

[1] Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany;Institut für Physiologische Chemie, Universität Bonn, Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany

关键词: Mature acid sphingomyelinase;    Human placenta;    Polyclonal antiserum;    Immunoaffinity purification;    N-terminal sequence;    ASM;    acid sphingomyelinase;    BCIP;    5-bromo-4-chloro-3-indolyl phosphate;    Con A;    concanavalin A;    COS;    COS-1;    CV1;    origin simian virus 40;    Endo H;    endo-β-N-acetylglucosaminidase H;    NBT;    nitroblue tetrazolium;    NP-40;    Nonidet P-40;    OG;    β-d-octylglucopyranoside;    PBS;    phosphate-buffered saline;    PVDF;    polyvinylidene difluoride;    SDS-PAGE;    sodium dodecyl sulfate-polyacrylamide gel electrophoresis;   

DOI  :  10.1016/S0014-5793(96)01331-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Human placental acid sphingomyelinase (ASM) was purified by sequential chromatography on Con A-Sepharose, octyl-Sepharose and Matrex gel red A. Final purification to apparent homogeneity was achieved by immunoaffinity chromatography employing polyclonal anti-ASM antibodies. The antibodies also allowed specific detection of ASM by Western blotting at various stages of purification. The ASM activity was enriched about 110 000-fold over that of the crude extract, yielding an enzyme preparation with a specific activity of about 1 mmol/h per mg protein in a detergent-containing assay system. Analysis of the final preparation by SDS-PAGE resulted in a single protein band with a molecular mass of ∼75 kDa, which was reduced to ∼60 kDa after complete deglycosylation. Microsequencing of the purified ASM revealed the N-terminal amino acid sequence of the mature placental enzyme.

【 授权许可】

Unknown