Gollapalli Deviprasad R, Rando Robert R
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Biochim Biophys Acta. 2003 Sep 23;1651(1-2):93-101. doi: 10.1016/s1570-9639(03)00239-5.
The endergonic trans-->cis isomerization of retinoids is an essential element in rhodopsin regeneration in vertebrates. All-trans-retinyl esters, which are generated by lecithin retinol acyltransferase (LRAT), are on the isomerization pathway. The critical isomerohydrolase activity, which catalyzes the trans-->cis isomerization/hydrolysis reaction of all-trans-retinyl esters, remains to be identified. It is demonstrated here that 11-cis-retinyl bromoacetate (cRBA) is a potent and specific inactivator of the bovine retinyl pigment epithelial (RPE) isomerohydrolase activity, with a measured K(I)=0.19 microM and a pseudo-first-order rate of inactivation k(inh)=1.83 x 10(-3) s(-1). This demonstrates that the isomerization is indeed enzyme-mediated. This inactivator should facilitate the identification and study of isomerohydrolase, or at least an essential component of it. Labeling of crude RPE membranes with 3H-cRBA reveals the presence of several labeled bands that may be isomerohydrolase candidates.
类视黄醇的吸能反式→顺式异构化是脊椎动物视紫红质再生中的一个关键要素。由卵磷脂视黄醇酰基转移酶（LRAT）生成的全反式视黄酯处于异构化途径上。催化全反式视黄酯反式→顺式异构化/水解反应的关键异构水解酶活性仍有待确定。本文证明，11-顺式视黄醇溴乙酸酯（cRBA）是牛视网膜色素上皮（RPE）异构水解酶活性的一种强效且特异性的失活剂，测得的抑制常数K（I）=0.19微摩尔，假一级失活速率k（inh）=1.83×10⁻³ 秒⁻¹。这表明异构化确实是由酶介导的。这种失活剂应有助于异构水解酶或其至少一个必需组分的鉴定和研究。用³H-cRBA对粗制RPE膜进行标记，揭示出几条可能是异构水解酶候选物的标记条带。
