Rockholm D C, Yamamoto H Y
Department of Plant Molecular Physiology, University of Hawaii at Manoa, Honolulu 96822, USA.
Plant Physiol. 1996 Feb;110(2):697-703. doi: 10.1104/pp.110.2.697.
Violaxanthin de-epoxidase catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Its activity is optimal at approximately pH 5.2 and requires ascorbate. In conjunction with the transthylakoid pH gradient, the formation of antheraxanthin and zeaxanthin reduces the photochemical efficiency of photosystem II by increasing the nonradiative (heat) dissipation of energy in the antennae. Previously, violaxanthin de-epoxidase had been partially purified. Here we report its purification from lettuce (Lactuca sativa var Romaine) to one major polypeptide fraction, detectable by two-dimensional isoelectic focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using anion-exchange chromatography on Mono Q and a novel lipid-affinity precipitation step with monogalactosyldiacylglyceride. The association of violaxanthin de-epoxidase and monogalactosyldiacyglyceride at pH 5.2 is apparently specific, since little enzyme was precipitated by eight other lipids tested. Violaxanthin de-epoxidase has an isoelectric point of 5.4 and an apparent molecular mass of 43 kD. Partial amino acid sequences of the N terminus and tryptic fragments are reported. The peptide sequences are unique in the GenBank data base and suggest that violaxanthin de-epoxidase is nuclear encoded, similar to other chloroplast proteins localized in the lumen.
紫黄质脱环氧化酶在叶黄素循环中催化紫黄质脱环氧化生成花药黄质和玉米黄质。其活性在pH约5.2时最佳，且需要抗坏血酸盐。与类囊体膜内外pH梯度协同作用，花药黄质和玉米黄质的形成通过增加天线中能量的非辐射（热）耗散来降低光系统II的光化学效率。此前，紫黄质脱环氧化酶已得到部分纯化。在此我们报告了从生菜（长叶莴苣罗马生菜变种）中纯化该酶至一个主要多肽组分的方法，该方法通过二维等电聚焦/十二烷基硫酸钠-聚丙烯酰胺凝胶电泳可检测到，使用Mono Q阴离子交换色谱和一个用单半乳糖基二酰基甘油进行的新型脂质亲和沉淀步骤。在pH 5.2时，紫黄质脱环氧化酶与单半乳糖基二酰基甘油的结合显然具有特异性，因为测试的其他八种脂质几乎没有沉淀出该酶。紫黄质脱环氧化酶的等电点为5.4，表观分子量为43 kD。报告了其N端的部分氨基酸序列和胰蛋白酶片段。这些肽序列在GenBank数据库中是独特的，表明紫黄质脱环氧化酶是核编码的，类似于其他定位在类囊体腔中的叶绿体蛋白。
