The sterylsulfatase of human placenta: Kinetic properties of the membrane-bound and the isolated enzyme

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Sulfohydrolase activities of microsomal membranes from human term placenta and of a homogenous sterylsulfatase preparation isolated from these membranes were studied using a number of steryl sulfates and nonsteroidal aryl sulfates as substrates. The results of these experiments clearly indicate that in vitro both classes of substrates can be hydrolyzed by the sterylsulfatase. The enzyme catalyzed (in the order of decreasing specific activity) the hydrolysis of sulfuric acid esters of non-steroidal phenols, of a phenolic steroid, and of neutral 3β, 21-, and (though at a very low rate) 17β-hydroxysteroids. Triiodothyronine sulfate and indoxyl sulfate likewise were cleaved by the sulfatase. However, among all substrates tested only the 3-sulfates of phenolic and neutral steroids exhibited a considerably high affinity towards the sterylsulfatase. The products of the hydrolytic reaction only slightly inhibited the sulfatase activity. K I values derived for free steroids were ten- to hundred-fold higher than K M values obtained for the respective sulfoconjugates. Inorganic sulfate was a rather weak inhibitor of the enzyme's activity. Its inhibitory potency, however, increased significantly in a time-dependent manner when it was preincubated with the enzyme prior to assay. In contrast to sulfate, the hypothetical transition-state analogues sulfite and vanadate acted as strong inhibitors of the sterylsulfatase activity. Treatment of the sulfatase with amino acid side-chain modifying reagents directed against arginine, cysteine, cystine, serine, or tyrosine did not result in significant alteration of its activity. The enzyme, however, was severely inactivated by diethylpyrocarbonate which is known to react preferentially with histidyl groups on proteins. In the presence of substrate, this inactivation was significantly reduced. Histidine thus appears to be essential for the sulfatase activity. Taken together, our results reveal a considerable similarity between the reaction mechanism of human placental sterylsulfatase and the ones reported for other sulfatases. Salicylate, taurocholate, and 4,4′-substituted stilbene-2,2′-disulfonates, well known inhibitors of protein-mediated transport of anions across biological membranes, likewise interfered with the sulfatase activity. The dose dependency pattern of these interferences resembled the one reported for inhibition of anion transport by the same substances. Since the sterylsulfatase in vivo is strongly associated with cellular membranes including the plasma membrane of the syncytiotrophoblast, this finding supports the speculation that similar molecular structures may be involved in both placental transport and hydrolysis of the anionic steryl sulfates.