Murine steroid sulfatase (mSTS): purification, characterization and measurement by ELISA.
The murine steroid sulfatase (mSTS) is a microsomal enzyme, important in steroid metabolism. In the mouse, the gene encoding mSTS is pseudoautosomal and thus escapes X-inactivation. We have purified steroid sulfatase approximately 30-fold from mouse liver microsomes and its properties have been investigated. The major steps in the purification procedure included solubilization with Triton X-100, gel filtration chromatography, DEAE-Sephadex chromatography and HPLC gel filtration chromatography. The purified sulfatase showed a relative molecular weight of 128 kDa on HPLC gel filtration, whereas the enzyme migrated as two bands of 60 and 68 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.2 by column chromatofocusing. Polyclonal antibodies to the purified protein were prepared. An Enzyme Linked Immunosorbent Assay (ELISA) was developed using purified monospecific anti-mSTS antibodies labelled with peroxidase. The standard criteria of precision and reproducibility were satisfied. The assay was applicable to routine determination of mSTS samples in research laboratories. Differences in mSTS liver concentrations were used to identify putative alleles for the mSTS gene (Sts). Results in ELISA confirmed the polymorphism previously demonstrated for an enzymatic mSTS activity assay in two inbred mouse strains.
Mortaud
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S
URA CNRS 1294 Génétique, Neurogénétique et Comportement, U.F.R. Biomédicale des Saints-Pères, Université Paris V-René Descartes, France.
Donsez-Darcel
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Roubertoux
P L
PL
Degrelle
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H
D000818:Animals; D001192:Arylsulfatases / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification*; D004303:Dosage Compensation, Genetic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004966:Estrogens, Conjugated (USP) / Q000378:metabolism; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008862:Microsomes, Liver / Q000201:enzymology*; D015203:Reproducibility of Results; D043266:Steryl-Sulfatase
D004966:Estrogens, Conjugated (USP); D004970:Estrone; D001192:Arylsulfatases; D043266:Steryl-Sulfatase; C017296:estrone sulfate