Action of surface-active agents on arylsulfatase-C of human cultured fibroblasts.
Arylsulfatase-C is a microsomal membrane-bound enzyme with unusual biochemical and genetic properties. Whether it is a single enzyme hydrolyzing different sterol sulfates or a complex of enzymes, with each enzyme hydrolyzing a specific substrate, has not been resolved. Its locus has been mapped to the human X chromosome but appears to escape inactivation. As a first step to clarify its biochemical properties, a systematic search was undertaken for a suitable detergent that can release this enzyme from human cultured fibroblast membranes in a form that is biologically active and electrophoretically mobile. Four non ionic (Triton X-100, Nonidet P-40, Digitonin, and saponin) and four amphoteric (lysolecithin, Zwittergent, Miranol, and Chaps) detergents were studied. At 1% concentration, they released more than 80% of the activity into a low-speed supernatant fraction, except for Saponin which had no effect. With Triton X-100 and Miranol representing the two groups of detergents, significant release occurred only when the detergent concentrations exceeded their respective critical micelle concentrations, thus indicating that arylsulfatase-C is an integral membrane protein. The apparent molecular weight of the detergent-enzyme complex, ascertained by gel filtration, was 85,000 in the presence of Triton X-100 and 335,000 in the presence of Miranol. However, only the preparation solubilized by Miranol (and Chaps, to a lesser degree) permitted migration of the enzyme in nitrocellulose acetate during electrophoresis at pH 7.0, while the enzyme extracted with all other detergents remained at the origin. Therefore, the amphoteric detergent, Miranol, appears to fulfill the requirements for further characterization of the membrane-bound arylsulfatase-C in human cultured fibroblasts.
D001192:Arylsulfatases / Q000302:isolation & purification*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis; D012995:Solubility; D043266:Steryl-Sulfatase; D013429:Sulfatases / Q000302:isolation & purification*; D013501:Surface-Active Agents
D011506:Proteins; D013501:Surface-Active Agents; D013429:Sulfatases; D001192:Arylsulfatases; D043266:Steryl-Sulfatase