Purification and properties of steroid sulfatase from human placenta.
Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.
Suzuki
T
T
Department of Obstetrics and Gynecology, Showa University, School of Medicine, Tokyo, Japan.
Hirato
K
K
Yanaihara
T
T
Kadofuku
T
T
Sato
T
T
Hoshino
M
M
Yanaihara
N
N
D000596:Amino Acids / Q000032:analysis; D001192:Arylsulfatases / Q000737:chemistry / Q000172:deficiency / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004589:Electrophoresis, Disc; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D012995:Solubility; D043266:Steryl-Sulfatase
D000596:Amino Acids; D001192:Arylsulfatases; D043266:Steryl-Sulfatase