Herr F M, Ong D E
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232.
Biochemistry. 1992 Jul 28;31(29):6748-55. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
卵磷脂 - 视黄醇酰基转移酶（LRAT）催化视黄醇（维生素A醇）与长链脂肪酸的酯化反应，这是维生素A吸收和储存过程中的重要步骤。细胞中的视黄醇与细胞视黄醇结合蛋白（CRBP）结合，CRBP存在于包括肝脏在内的大多数组织中，或者与II型细胞视黄醇结合蛋白[CRBP（II）]结合，后者存在于小肠的吸收细胞中。在此，我们通过比较游离或结合形式的视黄醇酯化反应的米氏常数，研究了视黄醇是否必须从这些载体蛋白上解离才能作为LRAT的底物。肝脏和肠道LRAT对游离视黄醇的酯化反应产生的Km值（分别为0.63和0.44微摩尔），与视黄醇 - CRBP酯化反应（分别为0.20和0.78微摩尔）以及视黄醇 - CRBP（II）酯化反应（分别为0.24和0.32微摩尔）所获得的值相似。由于视黄醇 - CRBP和视黄醇 - CRBP（II）的解离常数（Kd）值为10^(-8)-10^(-10) M，这些相似的Km值表明不需要预先解离，并且必须发生直接的结合蛋白 - 酶相互作用。当脱辅基CRBP被证明是LRAT的有效竞争性抑制剂时，获得了这种相互作用的证据，其抑制常数（KI，0.21微摩尔）低于CRBP - 视黄醇的Km值（0.78微摩尔）。相比之下，脱辅基CRBP（II）对视黄醇与CRBP（II）结合后的酯化反应竞争力较弱。与4 mM对 - （氯汞基）苯磺酸反应的脱辅基CRBP失去了视黄醇结合能力，但保留了抑制LRAT的能力，证实这种抑制不能用游离视黄醇浓度的降低来解释。（摘要截短于250字）
