Characterization of cellular retinoic acid-binding protein I from chick embryo and its ligand binding properties.
In order to characterize cellular retinoic acid-binding protein (CRABP) from chick embryos, we resolved the [3H]RA-binding activity of embryo extracts by Mono Q anion-exchange chromatography. Fractions containing [3H]RA-binding proteins eluted in two peaks at 7 and 10 min with the latter showing 10-fold higher activity than the former. When the [3H]RA-binding activity in the major peak was resolved on a Superose-12 size-exclusion column, a protein of about 15,000 Da, similar in size to CRABP I or II, was eluted. The identity of this RA-binding component as CRABP I was confirmed by its immunopositive reaction with a CRABP I-specific monoclonal antibody. The chick embryo CRABP I, upon electrophoresis on native gel, however, showed slower migration than the mouse CRABP I, although both exhibited similar isoelectric pH (pI) of about 4.5. Equilibrium binding studies performed under saturating levels of RA indicated that the retinoid bound to the chick CRABP I with a Kd of 27 nM, a value similar to that reported for the native form of this protein from other species. Moreover, as indicated by their IC50 values, the relative binding affinities of various RA analogs for chick CRABP I are consistent with those obtained with human and mouse CRABP I. These results demonstrate that the major RA-binding protein expressed in chick embryo, while having a different charge as judged by electrophoretic mobility, is similar to mouse CRABP I in its size, pI, antigenic specificity, and ligand binding properties.
Venepally
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P
Kettering-Meyer Laboratories, Southern Research Institute, Birmingham, Alabama, 35255, USA.
Reddy
L G
LG
Sani
B P
BP
D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002642:Chick Embryo; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D008024:Ligands; D051379:Mice; D011485:Protein Binding; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism
D008024:Ligands; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; C082927:retinoic acid binding protein I, cellular; D014212:Tretinoin