Mata N L, Villazana E T, Tsin A T
Division of Life Sciences, The University of Texas at San Antonio, 78249-0662, USA.
Invest Ophthalmol Vis Sci. 1998 Jul;39(8):1312-9.
To identify the subcellular locale of 11-cis retinyl esters in bovine retinal pigment epithelium (RPE) and to characterize the enzymic mechanism responsible for liberation of 11-cis retinoids in this compartment.
Endoplasmic reticulum (ER)- enriched and plasma membrane (PM)-enriched protein fractions were prepared from bovine RPE microsomes using sequential discontinuous sucrose and Percoll gradient fractionation. Enzyme markers for ER (such as carboxylesterase), and PM (such as 5'-nucleotidase [5'-ND]; alkaline phosphatase [AP]; and ouabain-sensitive Na+,K+-ATPase [ATPase]) were used to identify the subfractions. Membrane-associated retinoids were quantified by high-performance liquid chromatography (HPLC) and retinyl ester hydrolase (REH) activities were determined by radiometric and chromatographic (HPLC) means.
Chromatographic analyses of membrane-associated retinoids showed that 11-cis retinyl esters are localized mainly in PM-enriched fractions, whereas all-trans retinyl esters are associated predominantly with ER-enriched membranes; profiles of the distribution of 11-cis- and all-trans REH activities were consistent with the retinyl ester distribution. Further purification of the crude PM fraction yielded a fraction (P2) that was significantly enriched with 5'-ND (fivefold), ATPase (15-fold), AP (10-fold), and 11-cis retinyl ester hydrolase (11-cis REH; threefold) activities, but was relatively devoid of carboxylesterase and all-trans REH activities. Apparent kinetic constants (Km(app) and Vm(app)) for 11-cis REH activity in P2 were 18 microM and 1800 picomoles/min per mg, respectively.
This is the first identification of an 11-cis-specific REH activity in RPE plasma membrane. Results from these studies demonstrate the capacity of RPE plasma membranes to accommodate and hydrolyze 11-cis retinyl esters. Plasma membrane storage and mobilization of 11-cis retinyl esters represents a novel compartmentalization of retinoid metabolism that is distinct from the sites where 11-cis retinoids are produced. The implication of these findings for present theories of visual chromophore biosynthesis are discussed.
确定11-顺式视黄酯在牛视网膜色素上皮（RPE）中的亚细胞定位，并描述该区域中负责释放11-顺式视黄醛的酶促机制。
使用连续不连续蔗糖和Percoll梯度分级分离法，从牛RPE微粒体中制备富含内质网（ER）和富含质膜（PM）的蛋白质组分。使用ER的酶标志物（如羧酸酯酶）和PM的酶标志物（如5'-核苷酸酶[5'-ND]；碱性磷酸酶[AP]；和哇巴因敏感的Na +，K + -ATP酶[ATP酶]）来鉴定亚组分。通过高效液相色谱（HPLC）对膜相关视黄醛进行定量，并通过放射性和色谱法（HPLC）测定视黄酯水解酶（REH）活性。
对膜相关视黄醛的色谱分析表明，11-顺式视黄酯主要定位于富含PM的组分中，而全反式视黄酯主要与富含ER的膜相关；11-顺式和全反式REH活性的分布曲线与视黄酯分布一致。对粗PM组分的进一步纯化产生了一个组分（P2），该组分富含5'-ND（5倍）、ATP酶（15倍）、AP（10倍）和11-顺式视黄酯水解酶（11-顺式REH；3倍）活性，但相对缺乏羧酸酯酶和全反式REH活性。P2中11-顺式REH活性的表观动力学常数（Km(app)和Vm(app)）分别为18 microM和1800皮摩尔/分钟/毫克。
这是首次在RPE质膜中鉴定出11-顺式特异性REH活性。这些研究结果表明RPE质膜能够容纳和水解11-顺式视黄酯。11-顺式视黄酯的质膜储存和动员代表了视黄醛代谢的一种新型区室化，这与11-顺式视黄醛产生的部位不同。讨论了这些发现对当前视觉发色团生物合成理论的意义。
