Purification and properties of cellular retinoic acid-binding protein from neonatal rat skin.
Cellular retinoic acid-binding protein (CRABP) was detected in cytosolic extracts of dermis and epidermis of neonatal rat skin using high-performance size-exclusion liquid chromatography and was more abundant in dermal tissue. CRABP was purified 1000-fold from an acid-precipitated, 50,000 x g supernatant of neonatal rat skin by ion-exchange chromatography on DEAE-Sephacel, followed by chromatofocussing and hydrophobic-interaction chromatography. The protein had an apparent Mr of 14,800. In chromatofocussing experiments the apoprotein and holoprotein gave different elution profiles, indicating a charge difference between the two forms. The ability of various retinoids to compete with all-trans-retinoic acid for binding to CRABP was assayed: 4-oxoretinoic acid and two synthetic retinoids were effective competitors, but 13-cis-retinoic acid, 3,4-didehydroretinoic acid and the acid derivative of etretinate competed poorly. The binding protein had a Kd for all-trans-retinoic acid of 8 nM using a dextran-charcoal assay, but a higher value was obtained using high-performance size-exclusion liquid chromatography. The holoprotein dissociated rapidly at room temperature and had a half-life of 4.7 min. At 0 degrees C, the holoprotein had a half-life of 200 min.
Daly
A K
AK
Department of Dermatology, University of Newcastle upon Tyne, Royal Victoria Infirmary, U.K.
Redfern
C P
CP
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D001059:Apoproteins / Q000302:isolation & purification; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D003600:Cytosol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004817:Epidermis / Q000032:analysis; D006207:Half-Life; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D018168:Receptors, Retinoic Acid; D012867:Skin / Q000032:analysis*; D014018:Tissue Distribution; D014212:Tretinoin / Q000378:metabolism
D001059:Apoproteins; D002352:Carrier Proteins; D018168:Receptors, Retinoic Acid; D014212:Tretinoin