Human placental steroid sulfatase: purification and properties.
Steroid sulfatase is recovered quantitatively from the 105,000 g h supernatant of human placental microsomes extracted with Triton X-100. The solubilized enzyme has been purified using conventional techniques. Throughout the purification procedure, steroid sulfatase appears to be heterogeneous as evidenced by certain, but not all, criteria. Following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the final preparation exhibits a major component and varying amounts of two minor ones. Antibodies raised in rabbits with the heterogeneous immunogen give rise to a single precipitation line when the native enzyme is analyzed by double immunodiffusion or by immunoelectrophoresis. In addition, using aged preparations of microsomes and immunoaffinity techniques, steroid sulfatase activity was found to be associated with the fastest migrating minor component. This finding would suggest that the apparent heterogeneity of purified steroid sulfatase is linked to degradation processes occurring within the microsomal preparations. Steroid sulfatase has a Stokes radius of 56 A, a sedimentation coefficient of 4.85 +/- 0.15S (in Triton-containing buffers) and binds 1.3 g of Triton X-100-per g of protein. The molecular weight of the Triton-protein complex was calculated to be 166,000 in which the glycoprotein portion contribution is about 43% (72,000). In contrast, the apparent molecular weight of the major polypeptide determined on calibrated SDS-gels is 62,000. The purified enzyme exhibits two pH optima with cholesterol sulfate as substrate, an acidic one at pH 5.0 and a second one at pH 7.5. The Km values for cholesterol sulfate, dehydroandrosterone sulfate and p-nitrophenylsulfate were 5.26, 14 and 1,320 microM, respectively.
D003902:Detergents; D005260:Female; D006801:Humans; D005779:Immunodiffusion; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D017830:Octoxynol; D010920:Placenta / Q000201:enzymology*; D011092:Polyethylene Glycols; D011247:Pregnancy; D043266:Steryl-Sulfatase; D013429:Sulfatases / Q000302:isolation & purification* / Q000378:metabolism
D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D013429:Sulfatases; D043266:Steryl-Sulfatase