STEROID SULFATASE - EXPRESSION, ISOLATION AND INHIBITION FOR ACTIVE-SITE IDENTIFICATION STUDIES

Steroid sulphatase, which can hydrolyse 3-hydroxysteroid sulphates. ha s important roles in several physiological and pathological processes. A number of steroid sulphatase inhibitors have now been developed, of which the most potent to date is oestrone-3-O-sulphamate (EMATE). Thi s inhibitor inactivates steroid sulphatase in an irreversible, time- a nd concentration-dependent manner. In order to be able to use a. radio labelled derivative of EMATE to study the active site, it will be esse ntial to prepare the steroid sulphatase in a pure form. For this, atte mpts have been made to express the protein, using the steroid sulphata se cDNA, in the pGEX2T expression system and also to express a mutant form of the protein, in which the putative membrane-spanning domain wa s deleted, in CHO cells. In addition, a soluble steroid sulphatase has been identified from the snail Helix pomatia. This steroid sulphatase is inhibited by EMATE in an irreversible manner, similar to the human steroid sulphatase and appears to possess a histidine residue at its active site. The expression and/or isolation of a steroid sulphatase, in conjunction with tile use of a radiolabelled derivative of EMATE sh ould allow important new information about the active site of this enz yme and the mechanism of its inactivation to be obtained. (C) 1998 Els evier Science Ireland Ltd. All rights reserved.