Murine steroid sulfatase (mSTS): Purification, characterization and measurement by ELISA

Autor: Hervé Degrelle, E. Donsez-Darcel, S. Mortaud, Pierre L. Roubertoux
Přispěvatelé: Immunologie et Neurogénétique Expérimentales et Moléculaires (INEM), Centre National de la Recherche Scientifique (CNRS)-Université d'Orléans (UO)
Rok vydání: 1995
Předmět:
Male
Estrone
Endocrinology
Diabetes and Metabolism
Clinical Biochemistry
Size-exclusion chromatography
Enzyme-Linked Immunosorbent Assay
Mice
Inbred Strains
Biochemistry
High-performance liquid chromatography
Mice
03 medical and health sciences
0302 clinical medicine
Endocrinology
Dosage Compensation
Genetic
Steroid sulfatase
Animals
Molecular Biology
ComputingMilieux_MISCELLANEOUS
Arylsulfatases
030304 developmental biology
Gel electrophoresis
0303 health sciences
Estrogens
Conjugated (USP)
Chromatography
biology
Chemistry
Chromatofocusing
Sulfatase
Reproducibility of Results
Cell Biology
Isoelectric point
Polyclonal antibodies
Microsomes
Liver
biology.protein
Molecular Medicine
Steryl-Sulfatase
[SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]
030217 neurology & neurosurgery
Zdroj: Journal of Steroid Biochemistry and Molecular Biology
Journal of Steroid Biochemistry and Molecular Biology, Elsevier, 1995, 52 (1), pp.91-96. ⟨10.1016/0960-0760(94)00143-a⟩
ISSN: 0960-0760
DOI: 10.1016/0960-0760(94)00143-a
Popis: 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.
Databáze: OpenAIRE
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