Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Mar;87(5):2003–2007. doi: 10.1073/pnas.87.5.2003

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

Y C Lee 1, W J Driscoll 1, C A Strott 1
PMCID: PMC53613  PMID: 2308958

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 steroid-binding 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 [3H]pregnenolone 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 proteins 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 of Mr 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 approximately 10-fold greater in the zona reticularis. In vitro alkaline phosphatase treatment of the PBP abolished pregnenolone-binding 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.

Full text

PDF
2003

Images in this article

2005Image
on p.2005
2005Image
on p.2005
2005Image
on p.2005
2005Image
on p.2005
2006Image
on p.2006
2006Image
on p.2006
2006Image
on p.2006

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Auricchio F. Phosphorylation of steroid receptors. J Steroid Biochem. 1989 Apr;32(4):613–622. doi: 10.1016/0022-4731(89)90397-x. [DOI] [PubMed] [Google Scholar]
  2. Besman M. J., Yanagibashi K., Lee T. D., Kawamura M., Hall P. F., Shively J. E. Identification of des-(Gly-Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4897–4901. doi: 10.1073/pnas.86.13.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Kubo M., Strott C. A. Calcium-dependent protein kinase activity and protein phosphorylation in zones of the adrenal cortex. J Steroid Biochem. 1988 Apr;29(4):407–413. doi: 10.1016/0022-4731(88)90250-6. [DOI] [PubMed] [Google Scholar]
  4. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  5. Lee Y. J., Strott C. A. Adrenocortical pregnenolone-binding protein: identification and antibody development. Biochem Biophys Res Commun. 1988 Jan 15;150(1):456–462. doi: 10.1016/0006-291x(88)90542-6. [DOI] [PubMed] [Google Scholar]
  6. Mertz L. M., Pedersen R. C. The kinetics of steroidogenesis activator polypeptide in the rat adrenal cortex. Effects of adrenocorticotropin, cyclic adenosine 3':5'-monophosphate, cycloheximide, and circadian rhythm. J Biol Chem. 1989 Sep 15;264(26):15274–15279. [PubMed] [Google Scholar]
  7. Mikami K., Nishikawa T., Strott C. A. Adenylate cyclase activity and cyclic AMP production in the outer and inner zones of the adrenal cortex. Biochem Biophys Res Commun. 1985 Jun 28;129(3):664–670. doi: 10.1016/0006-291x(85)91943-6. [DOI] [PubMed] [Google Scholar]
  8. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  9. Nishikawa T., Strott C. A. Cortisol production by cells isolated from the outer and inner zones of the adrenal cortex of the guinea pig. Endocrinology. 1984 Feb;114(2):486–491. doi: 10.1210/endo-114-2-486. [DOI] [PubMed] [Google Scholar]
  10. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  11. Pastuszyn A., Noland B. J., Bazan J. F., Fletterick R. J., Scallen T. J. Primary sequence and structural analysis of sterol carrier protein 2 from rat liver: homology with immunoglobulins. J Biol Chem. 1987 Sep 25;262(27):13219–13227. [PubMed] [Google Scholar]
  12. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pedersen R. C., Brownie A. C. Steroidogenesis-activator polypeptide isolated from a rat Leydig cell tumor. Science. 1987 Apr 10;236(4798):188–190. doi: 10.1126/science.3563495. [DOI] [PubMed] [Google Scholar]
  14. STONE D., HECHTER O. Studies on ACTH action in perfused bovine adrenals: the site of action of ACTH in corticosteroidogenesis. Arch Biochem Biophys. 1954 Aug;51(2):457–469. doi: 10.1016/0003-9861(54)90501-9. [DOI] [PubMed] [Google Scholar]
  15. Sigel M. B., Sinha Y. N., VanderLaan W. P. Production of antibodies by inoculation into lymph nodes. Methods Enzymol. 1983;93:3–12. doi: 10.1016/s0076-6879(83)93031-8. [DOI] [PubMed] [Google Scholar]
  16. Smith A. C., Elsasser M. S., Harmon J. M. Analysis of glucocorticoid receptor activation by high resolution two-dimensional electrophoresis of affinity-labeled receptor. J Biol Chem. 1986 Oct 5;261(28):13285–13292. [PubMed] [Google Scholar]
  17. Strott C. A., Goff A. K., Lyons C. D. Functional differences between the outer and inner zones of the guinea pig adrenal cortex. Endocrinology. 1981 Dec;109(6):2249–2251. doi: 10.1210/endo-109-6-2249. [DOI] [PubMed] [Google Scholar]
  18. Strott C. A., Goff A. K., Lyons C. D. Purification of a pregnenolone-binding protein in the soluble fraction of the guinea-pig adrenal cortex: differentiation from pregnenolone sulfotransferase. J Steroid Biochem. 1983 Apr;18(4):489–498. doi: 10.1016/0022-4731(83)90070-5. [DOI] [PubMed] [Google Scholar]
  19. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Vahouny G. V., Chanderbhan R., Noland B. J., Irwin D., Dennis P., Lambeth J. D., Scallen T. J. Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization. J Biol Chem. 1983 Oct 10;258(19):11731–11737. [PubMed] [Google Scholar]
  21. Yanagibashi K., Ohno Y., Kawamura M., Hall P. F. The regulation of intracellular transport of cholesterol in bovine adrenal cells: purification of a novel protein. Endocrinology. 1988 Oct;123(4):2075–2082. doi: 10.1210/endo-123-4-2075. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES