Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins

• Published in: Biochemistry (Easton) Vol. 31; no. 29; pp. 6748 - 6755
• Main Authors: Herr, Fiona M, Ong, David E
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 28-07-1992
• Subjects: 4-Chloromercuribenzenesulfonate - pharmacology; Acyltransferases - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; cellular retinol-binding protein; Chromatography, Gel; Chromatography, Ion Exchange; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; interaction; intestine; Intestine, Small - enzymology; Intestine, Small - metabolism; Kinetics; lecithin-retinol acyltransferase; liver; Microsomes - enzymology; Microsomes - metabolism; Microsomes, Liver - enzymology; Protein Binding; Rats; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Retinol-Binding Proteins - genetics; Retinol-Binding Proteins - isolation & purification; Retinol-Binding Proteins - metabolism; Retinol-Binding Proteins, Cellular; Transferases; Vitamin A - metabolism; Vitamin A - pharmacology; Ligand binding; Rat; Enzyme; Liver; Rodentia; Molecular interaction; Retinol fatty-acyltransferase; Microsome; Esterification; Retinol; Binding protein; Vertebrata; Mammalia; Inhibition
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00144a014

Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.