Jolly S O, Tolbert N E
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824.
Plant Physiol. 1978 Aug;62(2):197-203. doi: 10.1104/pp.62.2.197.
A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO(3). The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in V(max).The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.
已从黑暗处理54小时的大豆（Glycine max L. Merr. Var. Amsoy）幼叶中分离出一种NADH-硝酸还原酶抑制剂。抑制剂的存在最初是通过匀浆中缺乏硝酸还原酶活性而推测出来的，直到通过二乙氨基乙基（DEAE）-纤维素色谱法去除抑制剂。该抑制剂可使光照下收获的叶片匀浆中的酶失活。通过蔗糖梯度分离的单个完整细胞中的硝酸还原酶，无论叶片是在光照还是黑暗条件下生长，其活性均相同，但加入活性抑制剂后会受到抑制。通过DEAE-纤维素色谱法、Sephadex G-100过滤、硫酸铵沉淀并透析的方法，将NADH-硝酸还原酶抑制剂纯化了2500倍，得到一种电泳纯的蛋白质。检测基于硝酸还原酶抑制作用。还开发了一种快速部分分离程序，通过DEAE-纤维素色谱法并用KNO₃洗脱，将硝酸还原酶与抑制剂分离。该抑制剂是一种热不稳定蛋白，分子量约为31,000，有两个相同的亚基。在聚丙烯酰胺凝胶上电泳后，出现两条相邻的蛋白带；一种活性形式和一种静置后形成的无活性形式。活性因子抑制叶片的NADH-硝酸还原酶，但不抑制NADPH-硝酸还原酶、细菌硝酸还原酶或其他所测试的酶。抑制位点可能在还原型黄素腺嘌呤二核苷酸-NR反应处，因为它不阻断NADH-细胞色素c还原酶的部分反应。该抑制剂似乎不是蛋白酶。在酶存在的情况下，通过Sephadex G-75观察到抑制剂迁移率的变化，表明活性抑制剂与硝酸还原酶存在某种形式的结合。硝酸还原酶的抑制作用对硝酸盐是非竞争性的，但导致V(max)降低。分离出的抑制剂在光照下失活，但在黑暗中放置24小时后，完全恢复抑制活性。无论叶片是在光照还是黑暗条件下收获，抑制剂的含量相同，只是从光照下的叶片中快速分离时，抑制剂最初无活性。黄色不纯抑制剂的光失活不需要额外成分，但纯化的无色抑制剂的失活需要加入黄素。给出了通过DEAE-纤维素色谱法部分分离一种刺激硝酸还原酶的成分的初步证据和程序。数据表明，硝酸还原酶活性的光暗变化受特定蛋白质抑制剂和刺激剂的调节。
