Charge isoforms of the adrenocortical pregnenolone-binding protein: influence of phosphorylation on isoformation and binding activity.

Abstract

Isoelectric focusing of the Mr 34,000 pregnenolone-binding protein (PBP) isolated from the guinea pig adrenal cortex has revealed multiple charge isoforms. Alkaline phosphatase treatment resulted in the disappearance of the pI 5.4 isoform associated with the appearance of pI 5.9 and pI 6.1 isoforms; this alteration in the charge-isoform pattern of the PBP correlated with a loss in pregnenolone-binding activity. This finding appears to be novel for intracellular steroidbinding proteins and has not been demonstrated for steroid receptors, a well-studied group of phosphoproteins. Resolution of the PBP by nondenaturing polyacrylamide gel electrophoresis produced two radioactive peaks of [3Hlpregnenolone in an equilibrium system, while only one peak was present in a nonequilibrium system, suggesting high-and low-binding affinity forms of PBP. Isoelectric focusing of highly purified PBP resolved multiple forms of Mr 34,000 protein with pI values ranging from 6.4 to 5.2. Two of the Mr 34,000 charge isoforms were isolated, and each was used to generate polyclonal antibodies; both antisera were crossreactive against all forms ofMr 34,000 PBP. Western blot analysis revealed that the PBP was present in both the fasciculata and reticularis of the adrenal cortex, though the isoform patterns were not identical for the two zones. Additionally, the pregnenolone-binding activity was =l0-fold greater in the zona reticularis. In vitro alkaline phosphatase treatment of the PBP abolished pregnenolonebinding activity and caused an alteration in the charge-isoform pattern for PBP in the zona reticularis, where pregnenolone binding is high, to resemble the pattern found for the zona fasciculata, where pregnenolone binding is low. The results indicate that phosphorylation/dephosphorylation regulates pregnenolone-binding activity and influences the pattern of the PBP isoformation. The data further suggest that the pI 5.4 isoform may be the active steroid-binding molecule.The rate-limiting step in adrenocortical steroidogenesis is the conversion of cholesterol to pregnenolone (1, 2); however, the precise mechanism by which corticotropin (ACTH) regulates the steroidogenic process is not well understood. There has been an extensive search in recent years for a noncatalytic protein that might play an important regulatory role, presumably by interacting with cholesterol. Three distinct proteins thought to function in such a manner have been described (3-5). The aforementioned proteins are not unique to the adrenal cortex, however, having been found in gonadal as well as nonsteroidogenic tissue (6-8); thus, the cholesterol-interacting proteins appear to play a more universal role in cholesterol metabolism and steroidogenesis. But what about pregnenolone, the product of the rate-limiting step? A specific adrenocortical pregnenolone-binding protein (PBP) has been described and partially purified, albeit its precise function is not understood (9,10 MATERIALS AND METHODSPreparation of Adrenocortical Cytosol. Male guinea pigs (NIH inbred s...

Steroid Sulfotransferases

“…An interesting feature of EST, as noted above, is that it can exist as multiple charge isoforms: at least two-charge isoforms for bovine placental EST (78) and four-charge isoforms for guinea pig adrenal EST (77) have been demonstrated. The four-charge isoforms for gpEST have pi values of 6.5, 5.5,5.4, and 5.2 and manifest a high degree of relatedness as evidenced by complete immunocross-reactivity and essentially identical HPLC elution profiles during peptide mapping analysis (80).…”

“…Adams (76) speculated that the wave-like kinetics might be related to the presence of multiple isoforms. In this regard, it is noteworthy that native guinea pig adrenocortical EST exists as multiple charge isoforms (77). Likewise, gpEST expressed by CHO-K1 cells consists of multiple charge isoforms analogous to the native protein (69).…”