Expression, purification, and binding properties of human cellular retinoic acid-binding protein type I and type II.
Human cellular retinoic acid-binding protein (CRABP) type I and type II were expressed in Escherichia coli from cloned cDNAs. Expressed proteins were purified by gel filtration and ion-exchange chromatography, resulting in highly pure proteins. The yield after gel filtration was approximately 50 mg/liter bacterial culture. In binding studies the equilibrium dissociation constant, Kd, of retinoic acid (RA) for E. coli-derived CRABP-I and CRABP-II was 6.8 and 39 nM, respectively. The Kd of the synthetic retinoid analog CD 367 was 2.2 nM for CRABP-I and 3.0 nM for CRABP-II. RA competed with the binding of CD 367 to CRABP-I and CRABP-II with IC50 values of 20.0 and 90.0 nM, respectively. Retinoid analogs competed with the binding of CD 367 to CRABP-I and CRABP-II in the following order: (p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphtyl)-1-propenyl]-benzoic acid (TTNPB) > 4-oxo-RA > 4-OH-RA > 13-cis-RA = 9-cis-RA. m-carboxy-TTNPB and CD 271 were found not to compete with the binding of CD 367 to CRABP-I or CRABP-II even at 500-fold molar excess. These data demonstrate that E. coli-derived CRABP-I has a higher affinity for RA than CRABP-II and that retinoic acid metabolites have a lower affinity for these proteins. The observed difference in affinity for RA supports the idea that CRABP-I, which is constitutively expressed, and CRABP-II, which is induced by RA, have different functions in the cell. In addition, 9-cis-RA, a natural ligand for the retinoid X receptors, is not a physiological ligand for either CRABP-I or CRABP-II.
Department of Dermatology, University of Michigan, Ann Arbor 48109.
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Fogh
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Voorhees
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Aström
D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011869:Radioligand Assay; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism*; D014316:Tritium
D002352:Carrier Proteins; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D014316:Tritium; D014212:Tretinoin