Institute of Biology, Leipzig University, Leipzig, Germany.
Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Leipzig University, Leipzig, Germany.
IUBMB Life. 2023 Jan;75(1):66-76. doi: 10.1002/iub.2650. Epub 2022 May 26.
In the present study, low concentrations of the very mild detergent n-dodecyl-α-d-maltoside in conjunction with sucrose gradient ultracentrifugation were used to prepare fucoxanthin chlorophyll protein (FCP) complexes of the centric diatom Thalassiosira pseudonana. Two main FCP fractions were observed in the sucrose gradients, one in the upper part and one at high sucrose concentrations in the lower part of the gradient. The first fraction was dominated by the 18 kDa FCP protein band in SDS-gels. Since this fraction also contained other protein bands, it was designated as fraction enriched in FCP-A complex. The second fraction contained mainly the 21 kDa FCP band, which is typical for the FCP-B complex. Determination of the lipid composition showed that both FCP fractions contained monogalactosyl diacylglycerol as the main lipid followed by the second galactolipid of the thylakoid membrane, namely digalactosyl diacylglycerol. The negatively charged lipids sulfoquinovosyl diacylglycerol and phosphatidyl glycerol were also present in both fractions in pronounced concentrations. With respect to the pigment composition, the fraction enriched in FCP-A contained a higher amount of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt), whereas the FCP-B fraction was characterized by a lower ratio of xanthophyll cycle pigments to the light-harvesting pigment fucoxanthin. Protein analysis by mass spectrometry revealed that in both FCP fractions the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (DDE) was present. In addition, the analysis showed an enrichment of DDE in the fraction enriched in FCP-A but only a very low amount of DDE in the FCP-B fraction. In-vitro de-epoxidation assays, employing the isolated FCP complexes, were characterized by an inefficient conversion of DD to Dt. However, in line with the heterogeneous DDE distribution, the fraction enriched in FCP-A showed a more pronounced DD de-epoxidation compared with the FCP-B.
在本研究中，使用非常低浓度的温和去污剂 n-十二烷基-α-d-麦芽糖苷与蔗糖梯度超速离心相结合，制备了中心硅藻塔玛多藻的岩藻黄素叶绿素蛋白（FCP）复合物。在蔗糖梯度中观察到两种主要的 FCP 级分，一种在上层，一种在梯度的下部高蔗糖浓度处。第一级分在 SDS-凝胶中以 18 kDa 的 FCP 蛋白带为主。由于该级分还包含其他蛋白带，因此将其指定为富含 FCP-A 复合物的级分。第二级分主要包含 21 kDa 的 FCP 带，这是 FCP-B 复合物的典型特征。脂质组成的测定表明，两个 FCP 级分都含有单半乳糖基二酰基甘油作为主要脂质，其次是类囊体膜的第二种类脂，即双半乳糖基二酰基甘油。带负电荷的脂质磺基奎诺糖基二酰甘油和磷脂酰甘油在两个级分中也以明显的浓度存在。就色素组成而言，富含 FCP-A 的级分含有较高量的叶黄素循环色素二氢玉米黄质（DD）和二氢玉米黄质（Dt），而 FCP-B 级分的叶黄素循环色素与捕光色素岩藻黄素的比例较低。通过质谱分析蛋白质，发现两个 FCP 级分都存在叶黄素循环酶二氢玉米黄质去环氧化酶（DDE）。此外，分析表明，富含 FCP-A 的级分中 DDE 富集，但 FCP-B 级分中 DDE 含量非常低。采用分离的 FCP 复合物进行的体外去环氧化测定表明，DD 向 Dt 的转化率较低。然而，与 DDE 分布不均一致，富含 FCP-A 的级分与 FCP-B 相比，DD 的去环氧化作用更为明显。
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