Affinity purification of cellular retinoic acid-binding protein on 14-carboxy-13-cis-retinamide-sepharose 4B.
A biologically active bifunctional retinoid, ethyl 14-carboxyretinoate, has been synthesized and shown to bind cellular retinoic acid (RA)-binding protein (CRABP) via its free carboxy group. We describe herein the synthesis of 14-carboxy-13-cis-retinamide-Sepharose 4B, which is an affinity matrix bearing an all-trans-RA moiety, and thus was used to purify and characterize CRABP from chick-embryo skin. An amide bond was first formed between the free carboxy group of the retinoid and a primary amino group of aminohexyl-Sepharose 4B, by reaction with carbodi-imide, and the ester group of the resin-bound retinoid was then hydrolysed in an alkaline medium. Polyacrylamide-gel electrophoresis and f.p.l.c. Superose column-chromatographic analysis demonstrated that the affinity-purified CRABP (Mr 15,000) was close to electrophoretic homogeneity (greater than 90%) and specifically interacts with RA. By using affinity gel chromatography, conversion of holo-CRABP into apo-CRABP by treatment with p-hydroxymercuribenzoate and a possible involvement of a thiol group in RA binding to CRABP were established. This affinity procedure provides several advantages: (i) 14-carboxy-13-cis-retinamide-Sepharose exhibited high efficiency and selectivity for RA-binding protein (i.e. retinol- or fatty-acid-binding proteins did not bind); (ii) the presence of the amide linkage between the ligand and the matrix makes this affinity resin relatively stable to cytosolic enzymes; and (iii) other RA-binding proteins, e.g. nuclear receptor(s), may be purified.
Singh
R K
RK
Kettering-Meyer Laboratories, Southern Research Institute, Birmingham, AL 35255.
Sani
B P
BP
Dawson
M I
MI
Shealy
Y F
YF
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification*; D002642:Chick Embryo; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008722:Methods; D018168:Receptors, Retinoic Acid; D012685:Sepharose / Q000138:chemical synthesis; D014212:Tretinoin / Q000031:analogs & derivatives / Q000138:chemical synthesis
D002352:Carrier Proteins; D018168:Receptors, Retinoic Acid; C061933:14-carboxy-13-cis-retinamide-Sepharose 4B; D014212:Tretinoin; D012685:Sepharose