Xu Q, Bricker T M
Department of Botany, Louisiana State University, Baton Rouge 70803-1705.
J Biol Chem. 1992 Dec 25;267(36):25816-21.
The 33-kDa manganese-stabilizing protein stabilizes the manganese cluster in the oxygen-evolving complex. There has been, however, a considerable amount of controversy concerning the stoichiometry of this photosystem II (PS II) component. In this paper, we have verified the extinction coefficient of the manganese-stabilizing protein by amino acid analysis, determined the manganese content of oxygen-evolving photosystem II membranes and reaction center complex using inductively coupled plasma spectrometry, and determined immunologically the amount of the manganese-stabilizing protein associated with photosystem II. Oxygen-evolving photosystem II membranes and reaction center complex preparations contained 258 +/- 11 and 67 +/- 3 chlorophyll, respectively, per tetranuclear manganese cluster. Immunoquantification of the manganese-stabilizing protein using mouse polyclonal antibodies on "Western blots" demonstrated the presence of 2.1 +/- 0.2 and 2.0 +/- 0.3 molecules of the manganese-stabilizing protein/tetranuclear manganese cluster in oxygen-evolving PS II membranes and highly purified PS II reaction center complex, respectively. Since the manganese-stabilizing protein co-migrated with the D2 protein in our electrophoretic system, accurate immunoquantification required the inclusion of CaCl2-washed PS II membrane proteins or reaction center complex proteins in the manganese-stabilizing protein standards to compensate for the possible masking effect of the D2 protein on the binding of the manganese-stabilizing protein to Immobilon-P membranes. Failure to include these additional protein components in the manganese-stabilizing protein standards leads to a marked underestimation of the amount of the manganese-stabilizing protein associated with these photosystem II preparations.
33 kDa的锰稳定蛋白可稳定放氧复合体中的锰簇。然而，关于这种光系统II（PS II）组分的化学计量存在相当多的争议。在本文中，我们通过氨基酸分析验证了锰稳定蛋白的消光系数，使用电感耦合等离子体质谱法测定了放氧光系统II膜和反应中心复合体中的锰含量，并通过免疫法测定了与光系统II相关的锰稳定蛋白的量。放氧光系统II膜和反应中心复合体制剂中，每个四核锰簇分别含有258±11和67±3个叶绿素。在“Western印迹”上使用小鼠多克隆抗体对锰稳定蛋白进行免疫定量分析表明，在放氧PS II膜和高度纯化的PS II反应中心复合体中，每个四核锰簇分别存在2.1±0.2和2.0±0.3个锰稳定蛋白分子。由于在我们的电泳系统中锰稳定蛋白与D2蛋白共迁移，准确的免疫定量需要在锰稳定蛋白标准品中加入用CaCl2洗涤过的PS II膜蛋白或反应中心复合体蛋白，以补偿D2蛋白对锰稳定蛋白与Immobilon-P膜结合可能产生的掩盖效应。如果在锰稳定蛋白标准品中不包含这些额外的蛋白质成分，会导致与这些光系统II制剂相关的锰稳定蛋白量被明显低估。
