Purification of acid sphingomyelinase from human placenta: characterization and N-terminal sequence.
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 approximately 75 kDa, which was reduced to approximately 60 kDa after complete deglycosylation. Microsequencing of the purified ASM revealed the N-terminal amino acid sequence of the mature placental enzyme.
Lansmann
S
S
Institut für Organische Chemie und Biochemie, Universität Bonn, Germany.
Ferlinz
K
K
Hurwitz
R
R
Bartelsen
O
O
Glombitza
G
G
Sandhoff
K
K
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010920:Placenta / Q000201:enzymology*; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000302:isolation & purification*
D013108:Sphingomyelin Phosphodiesterase