title: Affinity labeling of lecithin retinol acyltransferase.
abstract: Lecithin retinol acyltransferase (LRAT) transfers acyl groups regiospecifically from the sn-1 position of lecithins to all-trans-retinol (vitamin A) and similar retinoids. LRAT is essential for the biosynthesis of 11-cis-retinal, the visual pigment chromophore. LRAT is also required for the general dietary mobilization of vitamin A. The enzyme is membrane-bound and has been solubilized and partially, but not completely, purified. It is demonstrated here that all-trans-retinyl alpha-bromoacetate (RBA) is a potent irreversible affinity labeling agent of LRAT. The measured KI = 12.1 microM and the pseudo-first-order rate constant for inhibition is kinh = 8.2 x 10(-4) s-1. The specificity of the inhibition process is further evidenced by the observation that alpha-bromoacetate derivatives of hydrophobic alcohols which are not substrates for LRAT, such as cholesterol and beta-ionol, are not inhibitors of the enzyme. Labeling of the partially purified enzyme with 3H-RBA showed a single radiolabeled band of molecular weight approximately 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Shi
authors.forename: S
authors.initials: S
authors.lastname: Furuyoshi
authors.forename: I
authors.initials: I
authors.lastname: Hubacek
authors.forename: R R
authors.initials: RR
authors.lastname: Rando
mesh_terms: D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels; D000478:Alkylation; D000818:Animals; D002417:Cattle; D004224:Diterpenes / Q000378:metabolism / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004793:Enzyme Reactivators; D007700:Kinetics; D008970:Molecular Weight; D010777:Photochemistry; D000084562:Retinyl Esters
chemical_list: D000345:Affinity Labels; D004224:Diterpenes; D004793:Enzyme Reactivators; D000084562:Retinyl Esters; C034831:retinyl bromoacetate; D000217:Acyltransferases; C056942:lecithin-retinol acyltransferase