Shi Y Q, Furuyoshi S, Hubacek I, Rando R R
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1993 Mar 30;32(12):3077-80. doi: 10.1021/bi00063a019.
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.
卵磷脂视黄醇酰基转移酶（LRAT）将酰基从卵磷脂的sn-1位区域特异性地转移至全反式视黄醇（维生素A）及类似的类视黄醇。LRAT对于视觉色素发色团11-顺式视黄醛的生物合成至关重要。LRAT也是维生素A一般饮食动员所必需的。该酶与膜结合，已被溶解并进行了部分但未完全纯化。本文证明全反式视黄醇α-溴乙酸酯（RBA）是LRAT的一种有效的不可逆亲和标记剂。测得的抑制常数KI = 12.1 μM，抑制的伪一级速率常数kinh = 8.2×10⁻⁴ s⁻¹。非LRAT底物的疏水醇的α-溴乙酸酯衍生物，如胆固醇和β-紫罗兰醇，不是该酶的抑制剂，这一观察结果进一步证明了抑制过程的特异性。用³H-RBA对部分纯化的酶进行标记，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示出一条分子量约为25,000的单一放射性标记带。
