MICROSOMAL STEROID SULFATASE - INTERACTIONS WITH CYTOSOLIC STEROID SULFOTRANSFERASES

Net sulfation of 4-methylumbelliferone in intact hepatocytes is regula ted, in part, by substrate cycling between sulfotransferases (SULT) an d arylsulfatases (ARS. Thus, ARS have the potential to influence rates of net sulfate conjugation of a variety of compounds in intact cells via interaction with SULT. Unlike ARSA and ARSB, which are lysosomal, steroid sulfate sulfatase (ARSC, also known as STS) is localized exclu sively in the endoplasmic reticulum (ER). The present study was design ed to assess the existence and extent of substrate cycling between ste roids and their sulfate conjugates through ARSC and SULT, and also to initiate studies of the topology of the catalytic site of ARSC in the rat liver ER. Addition of rat liver microsomes to cytosol and 3'-phosp hoadenosine 5'-phosphosulfate (PAPS) reduced rates of sulfation of deh ydroepiandrosterone (DHEA) by SULT, and similarly hydrolysis of DHEA s ulfate (DHEAS) was reduced when recombinant human hydroxysteroid SULT was added to rat liver microsomes in the presence of PAPS. There was n o evidence for ARSC latency in the presence of detergent at either 4 o r 37 degrees C, indicating that facilitated transport of steroid sulfa tes across the ER membrane may not be required for ARSC activity. The effect of proteases on ARSC activity in intact and disrupted microsome s was determined and compared with effects on components of the glucos e-6-phosphatase system known to be localized on the lumenal and cytopl asmic surfaces of the ER. In contrast to the components of the glucose -6-phosphatase system, activity of ARSC in both intact and disrupted m icrosomes was substantially more resistant to protease inactivation. O ur results indicate that substrate cycling of steroids anti their sulf ates does occur, and suggest that the active site of ARSC may be locat ed within the ER membrane. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.