Brczi A, Mller IM
Plant Physiol. 1998 Mar;116(3):1029-36. doi: 10.1104/pp.116.3.1029.
Amino acid analysis of internal sequences of purified NADH-hexacyanoferrate(III) oxidoreductase (NFORase), obtained from highly purified plasma membranes (PM) of spinach (Spinacia oleracea L.) leaves, showed 90 to 100% homology to internal amino acid sequences of monodehydroascorbate (MDA) reductases (EC 1.6.5.4) from three different plant species. Specificity, kinetics, inhibitor sensitivity, and cross-reactivity with anti-MDA reductase antibodies were all consistent with this identification. The right-side-out PM vesicles were subjected to consecutive salt washing and detergent (polyoxyethylene 20 dodecylether and 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane sulfonate [CHAPS]) treatments, and the fractions were analyzed for NFORase and MDA reductase activities. Similar results were obtained when the 300 mm sucrose in the homogenization buffer and in all steps of the salt-washing and detergent treatments had been replaced by 150 mm KCl to mimic the conditions in the cytoplasm. We conclude that (a) MDA reductase is strongly associated with the inner (cytoplasmic) surface of the PM under in vivo conditions and requires washing with 1.0 m KCl or CHAPS treatment for removal, (b) the PM-bound MDA reductase activity is responsible for the majority of PM NFORase activity, and (c) there is another redox enzyme(s) in the spinach leaf PM that cannot be released from the PM by salt-washing and/or CHAPS treatment. The PM-associated MDA reductase may have a role in reduction of ascorbate in both the cytosol and the apoplast.
对从菠菜（Spinacia oleracea L.）叶片高度纯化的质膜（PM）中获得的纯化的NADH-高铁氰化物（III）氧化还原酶（NFORase）内部序列进行氨基酸分析，结果显示其与来自三种不同植物物种的单脱氢抗坏血酸（MDA）还原酶（EC 1.6.5.4）的内部氨基酸序列具有90%至100%的同源性。特异性、动力学、抑制剂敏感性以及与抗MDA还原酶抗体的交叉反应均与该鉴定结果一致。对右侧外翻的PM囊泡进行连续的盐洗和去污剂（聚氧乙烯20十二烷基醚和3-[(3-胆酰胺丙基)-二甲基铵]-1-丙烷磺酸盐[CHAPS]）处理，并分析各组分的NFORase和MDA还原酶活性。当用150 mM KCl替代匀浆缓冲液以及盐洗和去污剂处理所有步骤中的300 mM蔗糖以模拟细胞质中的条件时，获得了相似的结果。我们得出以下结论：（a）在体内条件下，MDA还原酶与PM的内（细胞质）表面紧密结合，需要用1.0 M KCl洗涤或CHAPS处理才能去除；（b）与PM结合的MDA还原酶活性是PM中大部分NFORase活性的原因；（c）菠菜叶PM中存在另一种氧化还原酶，其不能通过盐洗和/或CHAPS处理从PM中释放出来。与PM相关的MDA还原酶可能在细胞质溶胶和质外体中抗坏血酸的还原过程中发挥作用。
