Human placental steroid-sulfatase solubilized with a cholic-acid derivative: molecular mass, kinetic properties and susceptibility to glycosidases.
Human placental steroid-sulfatase was extracted nearly quantitatively from microsomes as well as from acetone dry powder of placenta homogenates using CHAPS as detergent. The solubilized enzyme was enriched 10-fold by ammonium sulfate precipitation and gel chromatography. The sulfatase extracted from both microsomes and acetone dry powder eluted as a single fraction on Sepharose 6B, but with different apparent molecular masses (390 and 270 kDa, respectively). Kinetic experiments with the sulfate esters of dehydroepiandrosterone, 16 alpha-hydroxydehydroepiandrosterone, estrone, and estriol as substrates or inhibitors indicated that the solubilized sulfatase was fully active. Both the particulate and the extracted enzyme showed higher affinities for the 16-unsubstituted than for the 16 alpha-hydroxylated substrates. Whereas a competitive inhibition was observed in mixed substrate incubations with dehydroepiandrosterone sulfate/16 alpha-hydroxydehydroepiandrosterone sulfate and estrone sulfate/estriol sulfate, diverse patterns of inhibition were obtained with dehydroepiandrosterone sulfate/estrone sulfate, depending on the sulfatase preparation used. However, evidence for the distinct nature of the steroid-sulfatase and the estrogen-sulfatase was not obtained. The membrane-bound, but not the solubilized enzyme was to a certain degree sensitive to lipase and acetone. The solubilized sulfatase strongly bound to ConA-Sepharose. This observation together with the elution by alpha-methyl mannoside were indicative of the presence of carbohydrates on the sulfatase. Since its enzymatic activity was markedly decreased by the effects of alpha-mannosidase and N-acetylglucosaminidase, a possible involvement of the carbohydrate moiety in the catalytic activity of the sulfatase might be considered.
D002241:Carbohydrates / Q000032:analysis; D002793:Cholic Acids; D005260:Female; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D017830:Octoxynol; D010920:Placenta / Q000201:enzymology*; D011092:Polyethylene Glycols; D011247:Pregnancy; D011506:Proteins / Q000378:metabolism; D043266:Steryl-Sulfatase; D013429:Sulfatases / Q000302:isolation & purification*
D002241:Carbohydrates; D002793:Cholic Acids; D011506:Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D013429:Sulfatases; D043266:Steryl-Sulfatase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate