Affinity purification of retinoic acid-binding proteins using immobilized 4-(2-hydroxyethoxy)retinoic acid.
An affinity chromatographic matrix that purifies cellular retinoic acid-binding protein to near homogeneity from rat testes cytosol has been developed. The three-step procedure includes an acid precipitation, a batch treatment with CM Bio-Gel, and affinity chromatography on 4-(2-hydroxyethoxy)retinoic acid coupled to epoxy-activated Sepharose 6B. The binding protein was purified approximately 8500-fold based on total soluble testicular protein and with a recovery in excess of 80%. In addition, further enhancement of the purity of the protein can be attained by size-exclusion HPLC to increase purification to 21,000-fold. The recovered protein has an apparent M(r) 14,300 as determined by size-exclusion HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is isolated in the apo-form and retains its ability to bind retinoic acid as evidenced by the binding of [3H]retinoic acid. An apparent retinoic acid-binding protein of M(r) 18,000 has also been isolated from rat testes nuclei by the affinity chromatography step. The affinity phase has been used for 6 months without any detectable loss in its ability to purify cellular retinoic acid-binding protein.
Chapman
J M
JM
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus 43210.
Curley
R W
RW
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002467:Cell Nucleus / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D008297:Male; D008970:Molecular Weight; D051381:Rats; D018168:Receptors, Retinoic Acid; D013737:Testis / Q000737:chemistry; D014212:Tretinoin / Q000031:analogs & derivatives / Q000378:metabolism*
D002352:Carrier Proteins; D018168:Receptors, Retinoic Acid; C077923:4-(2-hydroxyethoxy)retinoic acid; D014212:Tretinoin