Gueli M C, Nicotra C M, Pintaudi A M, Paganini A, Pandolfo L, De Leo G, Di Bella M A
Istituto di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università di Palermo, Italia.
Arch Biochem Biophys. 1991 Aug 1;288(2):572-7. doi: 10.1016/0003-9861(91)90238-e.
In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.
在牛视网膜色素上皮细胞膜中，我们发现了三种对反式视黄醇棕榈酸酯具有活性的水解酶。通过检测不同亚细胞组分在3至9的pH范围内的活性，得以实现这一发现。在酶分析中使用 Triton X - 100有利于这些活性的检测，在去污剂与蛋白质比例约为1.5 - 3.0时，其浓度高达0.03%时具有激活作用。在研究分析条件优化后，确定了视黄酯水解酶的表观动力学参数。在pH 4.5、6.0和7.0下起作用的水解酶的Vmax值分别为156、55和70 nmol/h/mg。为了确定这些水解活性的亚细胞位点，对每个亚细胞制剂中各种细胞器的标记酶进行了分析，结果表明pH 4.5视黄酯水解酶起源于溶酶体，pH 6.0视黄酯水解酶起源于微粒体，并提示pH 7.0视黄酯水解酶起源于高尔基体复合体。
