Purification and partial characterization of a novel binding protein for retinoic acid from neonatal rat.
This report describes the purification and partial characterization of a novel retinoic acid-binding protein (CRABP-II) from neonatal rat pups. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-cellulose, and high performance liquid chromatography (HPLC) on a DEAE 5PW column. Two retinoic acid-binding peaks were resolved at the DEAE-cellulose step, with CRABP-I in the major peak and CRABP-II in the minor peak. Apparent homogeneity was achieved for both binding proteins after the HPLC step. CRABP-II consists of a single polypeptide, migrating with an apparent Mr of 15,000 in sodium dodecyl sulfate-polyacrylamide gel. It has an isoelectric point of 5.0. The dissociation constant for CRABP-II of retinoic acid was estimated to be 65 nM by fluorescence titration. Amino-terminal sequence analysis showed that CRABP-II has a distinct sequence, while the CRABP-I sequence is exactly identical to that of the rat testis CRABP. Despite the extensive sequence homology between CRABP-I and CRABP-II, antibodies directed against CRABP-I did not cross-react with CRABP-II.
Bailey
J S
JS
Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Canada.
Siu
C H
CH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000831:Animals, Newborn; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D018168:Receptors, Retinoic Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014212:Tretinoin / Q000378:metabolism*
D000596:Amino Acids; D002352:Carrier Proteins; D018168:Receptors, Retinoic Acid; D014212:Tretinoin