U.S. Department of Agriculture-Agricultural Research Service, Beltsville, Maryland 20705.
Plant Physiol. 1989 Nov;91(3):1014-9. doi: 10.1104/pp.91.3.1014.
Plasma membranes isolated from three-day-old maize (Zea mays L.) roots by aqueous two-phase partitioning were used as starting material for the purification of a novel electron transport enzyme. The detergent-solubilized enzyme was purified by dyeligand affinity chromatography on Cibacron blue 3G-A-agarose. Elution was achieved with a gradient of 0 to 30 micromolar NADH. The purified protein fraction exhibited a single 27 kilodalton silver nitrate-stained band on sodium dodecyl sulfate polyacrylamide gel electrophoretograms. Staining intensity correlated with the enzyme activity profile when analyzed in affinity chromatography column fractions. The enzyme was capable of accepting electrons from NADPH or NADH to reduce either ferricyanide, juglone, duroquinone, or cytochrome c, but did not transfer electrons to ascorbate free-radical or nitrate. The high degree of purity of plasma membranes used as starting material as well as the demonstrated insensitivity to mitochondrial electron transport inhibitors confirmed the plasma membrane origin of this enzyme. The purified reductase was stimulated upon prolonged incubation with flavin mononucleotide suggesting that the enzyme may be a flavoprotein. Established effectors of plasma membrane electron transport systems had little effect on the purified enzyme, with the exception of the sulfhydryl inhibitor p-chloromercuriphenyl-sulfonate, which was a strong inhibitor of ferricyanide reducing activity.
用双水相分配法从 3 天龄玉米（Zea mays L.）根中分离的质膜作为纯化新型电子传递酶的起始材料。去污剂溶解的酶通过 Cibacron blue 3G-A-琼脂糖的亲和配体色谱进行纯化。用 0 至 30 微摩尔 NADH 的梯度洗脱。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上，纯化的蛋白质部分显示出单一的 27 千道尔顿硝酸银染色带。在亲和色谱柱馏分中分析时，染色强度与酶活性谱相关。该酶能够接受 NADPH 或 NADH 的电子，将铁氰化物、胡桃醌、二氢醌或细胞色素 c 还原，但不能将电子转移到抗坏血酸自由基或硝酸盐。用作起始材料的质膜的高度纯度以及对线粒体电子传递抑制剂的不敏感性证实了该酶的质膜起源。用黄素单核苷酸长时间孵育可刺激纯化的还原酶，表明该酶可能是一种黄素蛋白。除了巯基抑制剂对氯汞苯磺酸盐外，已建立的质膜电子传递系统的效应物对纯化酶的影响很小，对氯汞苯磺酸盐是铁氰化物还原活性的强烈抑制剂。
