Human placental sphingomyelinase. Purification to homogeneity, antigenic properties and partial amino-acid sequences of the enzyme.
Sphingomyelinase from human placenta was purified to homogeneity in five steps: concanavalin A Sepharose, butyl agarose. Blue Sepharose, sphingosylphosphocholine Sepharose chromatography and FPLC-Mono Q. This lysosomal enzyme has a pH optimum around pH 5.0-6.0. It is a glycoprotein with an approximate molecular mass of 70 kDa which is reduced to 60 kDa by enzymatic deglycosylation. Monospecific antibodies against sphingomyelinase were isolated using sphingomyelinase covalently linked to Sepharose as affinity matrix. These antibodies effectively inhibit the sphingomyelinase activity. Peptides were released from sphingomyelinase by cyanogen bromide or proteolytically by trypsin, proteinase V8 and Lys C for gas phase sequencing. Amino-acid sequences are reported which proved to be the prerequisite for antibody and oligonucleotide screening of the respective human placenta cDNA libraries for the determination of the complete amino acid sequence of human lysosomal sphingomyelinase. In situ hybridisation with a labelled antisense RNA synthesized in vitro using cloned sphingomyelinase-specific cDNA as template, which encodes the peptide sequences described here, revealed the strong expression of sphingomyelinase in human placental villi and normal fibroblasts. Fibroblasts of a Niemann-Pick patient, however, were free of mRNA expressing the sphingomyelinase described here.
Kurth
J
J
Institut für Biochemie, Medizinische Fakultät, Universität zu Köln.
Stoffel
W
W
D000595:Amino Acid Sequence; D001345:Autoradiography; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D012697:Serine Endopeptidases; D013108:Sphingomyelin Phosphodiesterase / Q000302:isolation & purification*; D014357:Trypsin
D013108:Sphingomyelin Phosphodiesterase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D003488:Cyanogen Bromide