The current research of the Section on adrenocortical steroid-binding proteins can be divided into two areas: 1) cloning and expression of the cDNA for Mr 34000 pregnenolone-binding protein (PBP); and 2) purification and structural analysis of the pregnenolone-binding regulatory factor. Based on internal amino acid sequences for tryptic fragments of purified PBP, oligonucleotides were synthesized and using PCR techniques a full- length PBP cDNA was cloned. PBP cDNA expressed in a mammalian cell system produced a Mr 34000 protein that crossreacts with a highly specific PBP polyclonal antibody. The expressed protein, however, did not bind pregnenolone. A search of the data banks revealed that the PBP cDNA codes for an estrogen sulfotransferase (EST). When the expressed protein was re- examined it was found to have high EST activity. PBP had previously been shown to exist in 4 change isoforms (pI 6.4, 5.5, 5.4, 5.2); the pregnenolone binding activity is associated with the pI 5.5 isoform. The expressed protein has a pI of 6.4, and an antibody generated against the pI 5.5 isoform inhibits pregnenolone binding activity and strongly crossreacts with the expressed protein. In fact, as previously shown, antibodies raised against each specific PBP charge isoform crossreacts with the other isoforms. Thus, EST and PBP are immunologically related Mr 34000 change isoforms. The precise relationship between EST and PBP, as well as the other charge isoforms, remains to be determined. Steroid sulfotransferases (ST) require as a sulfate donor the cofactor, 3'-phosphoadenosine-5'- phosphosulfate (PAPS). A product of the ST reaction, in addition to the steroid sulfate, is 3'-phosphoadenosine-5'-phosphate (PAP). Since EST and PBP appear to be related and EST requires as a cofactor, PAPS, could PAP function to modulate steroid binding activity? This was indeed found to be the case. That is, PAP could substitute for the endogenous binding factor, and alkaline phosphatase-treated PAP, just as with the endogenous factor, was completely inactive. When partially purified PBP was examined for estradiol binding, high affinity binding was found; as is the case with pregnenolone binding, alkaline phosphatase treatment completely inhibited estradiol binding. When the binding of estradiol and pregnenolone was examined by isoelectric focusing using dual ligand labels, pregnenolone binding was associated with the pI 5.5 isoform and estradiol binding with the 5.4 isoform. The current hypothesis is that PBP is probably a pregnenolone sulfotransferase (PST), but the PST has not yet been identified on a focusing gel. The hypothesis has been further expanded to suggest that EST=EBP and PBP=PST. The fact that a product of the ST reaction, i. e., PAP, alters the affinity of the enzyme for substrate reveals a novel and exciting aspect of the steroid sulfotransferase systems.
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