Photoaffinity labeling of human IRBP with all-trans-retinoic acid.
Interphotoreceptor retinoid-binding protein (IRBP), found only in photosensitive tissues, is a large approximately 135-kDa glycoprotein that contains a fourfold repeat structure. IRBP may function as a buffer and prevent retinoid toxicity and retinoid degeneration. Here we asked (i) whether each repeat of IRBP possesses the capability of photo-crosslinking all-trans-retinoic acid (RA), (ii) within Repeat 1 whether a single retinoic acid-binding domain exists, and (iii) whether protease and CNBr digestion of Repeat 1 bound RA indicate the exact location of the binding site. 3H-RA cross-linked to all four repeats, consistent with the current model of multiple binding sites in IRBP. Acetone precipitation was effective in removing unbound 3H-RA. LysC and tryptic digestion of the RA-Repeat 1 detected 18- and 5-kDa bands, respectively. CNBr digestion showed two bands about 9 and 11 kDa in size. Our data suggests a single binding site near positions 151-160 in the center of Repeat 1.
Rengarajan
K
K
Department of Ophthalmology, Emory Eye Center, Atlanta, Georgia, USA.
Pohl
J
J
Nickerson
J
J
D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry; D005136:Eye Proteins; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis; D019920:Photoaffinity Labels / Q000737:chemistry*; D010777:Photochemistry; D020449:Repetitive Sequences, Amino Acid; D012177:Retinol-Binding Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014212:Tretinoin / Q000737:chemistry*; D014316:Tritium; D000099214:Retinol-Binding Proteins, Interstitial
D003488:Cyanogen Bromide; D010450:Endopeptidases; D005136:Eye Proteins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D012177:Retinol-Binding Proteins; D014212:Tretinoin; D014316:Tritium; D000099214:Retinol-Binding Proteins, Interstitial