Purification, primary structure characterization, and cellular distribution of two forms of cellular retinol-binding protein, type II from adult rat small intestine.
Cellular retinol-binding protein type II (CRBP(II)) is a major protein in the small intestine, accounting for more than 1% of the soluble protein recovered from rat jejunal mucosa. Two forms of the protein, called CRBP(II)A and CRBP(II)B, were purified from rat small intestine using a three-column procedure. The two forms were present in equal abundance. The primary structures of CRBP(II)A and CRBP(II)B were determined using a combination of techniques including amino acid composition and sequence analyses, and fast atom bombardment and gas chromatography-electron impact mass spectrometry. The primary structures of both proteins were found to be identical, but they differed in their NH2-terminal processing. CRBP(II)B was acetylated at its NH2 terminus, while CRBP(II)A was not. The results also confirmed the amino acid sequence of CRBP(II)A that was deduced from the cDNA sequence by Li et al. (Li, E., Demmer, L. A., Sweetser, D. A., Ong, D. E., and Gordon, J. I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5770-5783). Antibodies capable of distinguishing between the two forms of CRBP(II) were used for immunohistochemical studies which indicated that the organ and cellular distributions of the two forms were identical. The 50% acetylation observed here in vivo fits the pattern predicted by recent in vitro studies which described the effect of NH2-terminal sequence on cotranslational NH2-terminal processing of cytosolic proteins (Boissel, J. P., Kasper, T. J., and Bunn, H. F. (1988) J. Biol. Chem. 263, 8443-8449). Our results provide a basis for investigating the possibility of different roles of CRBP(II)A and CRBP(II)B within cells, as well as the importance of acetylation of the amino terminus for these biological functions.
Schaefer
W H
WH
Department of Pharmacology, Vanderbilt University Nashville, Tennessee.
Kakkad
B
B
Crow
J A
JA
Blair
I A
IA
Ong
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007124:Immunoenzyme Techniques; D007421:Intestine, Small / Q000032:analysis* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D012177:Retinol-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D054840:Retinol-Binding Proteins, Cellular
D010446:Peptide Fragments; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular