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Purification and Partial Characterization of Cellular Retinol-Binding Protein, Type Two, from Human Small Intestine¹

Department of Biochemistry, School of Medicine, Vanderbilt University, Nashville, TN 37232

Two forms of human cellular retinol-binding protein, type II [hCRBP(II)A and hCRBP(II)B], were purified from the small intestine in a three-step purification procedure. The N-terminal sequence of CRBP(II)A was determined to 36 residues and found to have only a single residue difference when compared with the N-terminal sequence in rat; the amino acid change maintained charge. hCRBP(II)B was blocked at the N-terminus, presumably because of N-acetylation of the initial threonine residue as previously established for CRBP(II)B from rat. This blockage did not affect the binding properties of the protein. The ability of pure hCRBP(II) to bind all-trans-retinol, retinal and retinoic acid was examined by competitive binding assay and compared with the binding specificity of pure human cellular retinol-binding protein (hCRBP). Retinoic acid did not compete with retinol for binding to either protein. Retinal competed with retinol for binding to hCRBP(II) but not to hCRBP, consistent with what was observed for the homologous proteins of rats. Polyclonal antiserum against hCRBP(II)B was produced that recognized both forms of hCRBP(II) in Western blotting and RIA but did not react with hCRBP. Distribution of hCRBP(II) was determined in the jejunum. Higher concentrations of hCRBP(II) were observed in the proximal portion compared with the distal portion. Although concentrations varied in individual intestinal samples, concentrations up to 1% of total mucosal protein were observed in some samples. These results provide tools for further investigation of the role of hCRBP(II) and suggest that previous results from rats, in this important aspect, are relevant to human metabolism of vitamin A.

KEY WORDS: • vitamin A • humans • cellular retinol-binding protein • cellular retinol-binding protein, type two • small intestine

¹ Supported by National Institute of Health grants HD 25286 and DK 32642. S. Inagami was supported by a fellowship from the American Heart Association.

² To whom correspondence should be addressed.

Manuscript received 26 June 1991. Revision accepted 27 August 1991.