Bernstein P S, Law W C, Rando R R
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1987 Dec 15;262(35):16848-57.
We have previously shown that membranes from the retinal pigment epithelium can transform added all-trans-retinol into a mixture of 11-cis-retinoids, demonstrating the "missing reaction" in the visual cycle for the first time (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853). In this article, this isomerase activity is further characterized. Double-label experiments with [15-3H]- and [15-14C]all-trans-retinol as the substrate show that the tritium label is retained in the 11-cis-retinol and 11-cis-retinyl palmitate products. This requires that isomerization occur at the alcohol level of oxidation. All-trans-retinyl esters, such as the palmitate, acetate, butyrate, and hexanoate esters, are not directly transformed into their 11-cis counterparts by the membranes. The data are consistent with the presence of an all-trans-retinol isomerase enzyme system or enzyme complex, which produces 11-cis-retinol. Other isomeric retinols were tested for substrate activity. Neither 9-cis-retinol(al) nor 13-cis-retinol were processed by the isomerase. Since the membranes containing the isomerase possess other retinol metabolizing activities, such as retinyl ester synthetase and dehydrogenase activities, further purification was attempted. Appreciable quantities of all detergents tested led to the disappearance of isomerase activity, and high salt or EDTA did not dissociate isomerase activity from the membranes. However, extensive sonication of the membranes did produce a 100,000 x g supernatant fraction of light membranes depleted of other all-trans-retinol processing activities. The isomerase activity in these membranes was saturable with all-trans-retinol, as required for a biologically significant process, and showed a Vmax of 5 pmol/h/mg of protein, a KM of 0.8 microM, and a pH optimum of 8. The isomerase was destroyed by proteinase K, by phospholipase C, by heating, or by ethanol at concentrations greater than 1%. The addition of high energy compounds, such as MgATP, MgGTP, or palmitoyl-CoA, did not appear to stimulate isomerase activity in the 100,000 x g supernatant.
我们之前已经表明，视网膜色素上皮的膜能够将添加的全反式视黄醇转化为11-顺式视黄醇类的混合物，首次证明了视觉循环中“缺失的反应”（伯恩斯坦，P.S.，劳，W.C.，和兰多，R.R.（1987年）《美国国家科学院院刊》84，1849 - 1853）。在本文中，对这种异构酶活性进行了进一步表征。以[15 - 3H] - 和[15 - 14C]全反式视黄醇作为底物的双标记实验表明，氚标记保留在11-顺式视黄醇和11-顺式视黄基棕榈酸酯产物中。这要求异构化发生在氧化的醇水平。全反式视黄酯，如棕榈酸酯、乙酸酯、丁酸酯和己酸酯，不会被这些膜直接转化为它们的11-顺式对应物。这些数据与存在一种产生11-顺式视黄醇的全反式视黄醇异构酶酶系统或酶复合物一致。测试了其他异构视黄醇的底物活性。异构酶既不处理9-顺式视黄醇（醛）也不处理13-顺式视黄醇。由于含有异构酶的膜具有其他视黄醇代谢活性，如视黄酯合成酶和脱氢酶活性，因此尝试进一步纯化。测试的所有去污剂的可观量都会导致异构酶活性消失，高盐或乙二胺四乙酸（EDTA）不会使异构酶活性与膜解离。然而，对膜进行广泛的超声处理确实产生了一个100,000×g的上清液部分，其中轻膜不含其他全反式视黄醇处理活性。这些膜中的异构酶活性对全反式视黄醇是可饱和的，这是一个生物学上重要过程所必需的，其Vmax为5 pmol/小时/毫克蛋白质，KM为0.8 microM，最适pH为8。异构酶会被蛋白酶K、磷脂酶C、加热或浓度大于1%的乙醇破坏。添加高能化合物，如MgATP、MgGTP或棕榈酰辅酶A，似乎不会刺激100,000×g上清液中的异构酶活性。
