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Department of Physiology and Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, Texas, USA
¹ Maternal-Fetal Neonatal Care Center, Hamamatsu University School of Medicine, Hamamatsu, Japan

(Requests for offprints should be addressed to P J Hornsby, University of Texas Health Science Center, 15355 Lambda Drive STCBM 3.100, San Antonio, TX 78245, USA; Email: hornsby{at}uthscsa.edu)

^* (W Wang and L Wang contributed equally to this work)

In order to establish whether there are differences in DNA-binding proteins between zona fasciculata (ZF) and zona reticularis (ZR) cells of the human adrenal cortex, we prepared nuclear extracts from separated ZF and ZR cells. The formation of DNA–protein complexes was studied using an element in the first intron of the type I and type II 3ß-hydroxysteroid dehydrogenase genes (HSD3B1 and HSD3B2). Using the element in the HSD3B2 gene as a probe, a complex (C1) was formed with extracts from ZF cells but was formed only at a low level with ZR cell extracts. Another pair of complexes (C2/C3) was formed with both ZF and ZR cell extracts. The ZF-specific protein forming C1 was enriched by column chromatography on DEAE-Sepharose and carboxymethyl-Sepharose. Oligonucleotide competition analysis on the enriched fraction gave results consistent with those obtained on the unfractionated material. A further enrichment was brought about by passing the protein over an oligonucleotide affinity column based on the HSD3B2 element. The protein bound to the column was identified as -enolase by mass spectrometry. Although -enolase is a glycolytic enzyme, it binds to specific DNA sequences and has been found to be present in nuclei of various cell types. We performed immunohistochemistry on sections of adult human adrenal cortex and found -enolase to be located in nuclei of ZF cells but to be predominantly cytoplasmic in ZR cells. Transfection of an -enolase expression vector into NCI-H295R human adrenocortical cells increased HSD3B2 promoter activity, suggesting a possible functional role for this protein in regulation of HSD3B2 expression.

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