PARENT SESSION
Gonadal Steroids
362
DEVELOPMENT AND CHARACTERIZATION OF A STERYL SULFATASE ANTIBODY.
Selcer, Kyle1, Beyer, Amanda1, Parker, Sarah1, Li, Pui-Kai2, Clemens, Jeffrey1, 1 2
ABSTRACT- The enzyme steryl sulfatase (EC 3.1.6.2) hydrolyzes the sulfate group present on 3-hydroxysteroid sulfates. This enzyme is best known for its role in estrogen production in the fetal adrenal-placental pathway during human pregnancy; however, it also has important functions in other physiological and pathological steroid pathways. Recently, steryl sulfatase has been implicated in the conversion of inactive conjugated steroids to their active forms in various hormone-dependent cancers, particularly breast cancer. Deficiency of steryl sulfatase in humans results in a condition known as X-linked-ichthyosis, a genetic disorder caused by a deletion in the steryl sulfatase gene. Furthermore, steryl sulfatase may play a role in ovarian steroidogenesis. Despite the importance of steryl sulfatase in normal physiological and pathological processes, little is known about its regulation. Enzymatic activity of this microsomal enzyme can be easily quantified and RT-PCR assays have been developed to measure steady-state mRNA levels; however, no reagents are available to determine protein levels. To address this, we generated a rabbit polyclonal antiserum against peptide representing a conserved region of the steryl sulfatase protein, based on a multiple alignment of human, rat and mouse cDNA sequences. In Western blotting experiments using microsomal preparations from extracts of human breast cancer cells (MCF-7 and MDA-MB-231), the antiserum crossreacted with a 66 kDa protein, the reported size of steryl sulfatase. Crossreactivity was also shown for microsomal preparations from human placenta, rat liver, rat granulosa cells, and porcine and monkey ovaries. Each of these tissues has significant steryl sulfatase activity, as determined by tritiated estrone sulfate conversion assays. These data suggest that the antiserum recognizes authentic steryl sulfatase in various tissues from several species. This antibody may be useful in studies of steryl sulfatase regulation. (Supported in part by DAMD 17-00-0238).
KEY WORDS: steryl sulfatase, antibody, steroidogenesis