Journal of Lipid Research, Vol 21, 100-109, Copyright © 1980 by Lipid Research, Inc.

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The binding protein for retinoic acid from rat testis cytosol: isolation and partial characterization

AC Ross, N Adachi and DS Goodman

This study reports the isolation and partial characterization of a soluble protein with binding specificity for retinoic acid from rat testis cytosol. Cytosol, labeled in vitro by incubation with [14C]retinoic acid, was fractionated by a series of procedures that included ion exchange chromatography on DEAE-Sepharose and on DEAE- cellulose, gel filtration on Sephadex (G-50, and preparative polyacrylamide gel electrophoresis. The resulting cytosol bindin; protein for retinoic acid had been purified approximately 16,000-fold, with recovery of 11% of the specifically bound [14C]retinoic acid. The purified binding protein was homogeneous on disc gel electrophoresis at pH 4.5 or in the presence of SDS, but displayed microheterogeneity on isoelectric focusing, where both a major and a minor band with apparent isoelectric points near 4.7 were observed. Purified retinoic acid binding protein had a molecular weight of approximately 14,600, and an ultraviolet absorption spectrum with two maxima near 277 and 346 nm. The fluorescence intensity of retinoic acid bound to the cytosol binding protein was approximately 30-times greater than that of retinoic acid in solution in any of five organic solvents, suggesting that retinoic acid is highly immobilized when bound to the cytosol binding protein. The properties of the rat testis cytosol binding protein for retinoic acid were compared directly with those of the testis cytosol binding protein for retinol and of serum retinol-binding protein. The two cytosol binding proteins were much more similar to each other than to the serum transport protein, from which they differed in a number of properties, including which they differed in a number of properties, including molecular size, affinity for prealbumin, immunological reactivity, and absorption and fluorescence spectral characteristics.
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