Igamberdiev Abir U, Seregélyes Csaba, Manac'h Nathalie, Hill Robert D
Department of Plant Science, University of Manitoba, R3T 2N2, Winnipeg, Manitoba, Canada.
Planta. 2004 May;219(1):95-102. doi: 10.1007/s00425-003-1192-3. Epub 2004 Jan 22.
Transgenic alfalfa ( Medicago sativa L.) root cultures expressing sense and antisense barley ( Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions. Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions. The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate. Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000- g membrane fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7-8 and a strong affinity to NADH and NADPH ( K(m) 3 microM for both). It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates. There was a correspondence between NO degradation and nitrate formation. The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35+/-4 kDa, which corresponds to the size of the hemoglobin dimer. The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor.
对表达正义和反义大麦（Hordeum vulgare L.）血红蛋白的转基因苜蓿（Medicago sativa L.）根培养物进行了代谢一氧化氮（NO）能力的检测。在常氧条件下，过表达血红蛋白的株系提取物的NO转化率约为对照株系或反义株系的两倍。只有对照株系在厌氧条件下NO降解速率显著增加。NO的下降依赖于还原型吡啶核苷酸的存在，NADH依赖的速率比NADPH依赖的速率快约2.5倍。大部分活性存在于提取物的胞质部分，而在细胞壁、线粒体和105,000×g膜部分中仅发现少量活性。NADH依赖的NO转化在pH 7 - 8范围内表现出较宽的最佳值，对NADH和NADPH具有较强的亲和力（两者的K(m)均为3 microM）。它对黄素蛋白抑制剂二苯基碘鎓敏感。应用针对重组大麦血红蛋白产生的抗体可使活性显著降低。与未转化细胞相比，过表达大麦血红蛋白的大肠杆菌提取物的NO代谢速率高4倍。血红蛋白表达不足的株系中NADH/NAD和NADPH/NADP比值较高，表明血红蛋白的存在对这些比值有影响。在缺氧和抗霉素A处理下，这些比值会升高。苜蓿根提取物以细胞色素c或重组大麦血红蛋白为底物时表现出高铁血红蛋白还原酶活性。NO降解与硝酸盐形成之间存在对应关系。该活性从Superose 12柱上以单一峰形式洗脱下来，分子量为35±4 kDa，这与血红蛋白二聚体的大小相对应。结果与一种类似NO双加氧酶的活性一致，即血红蛋白与黄素蛋白协同作用，利用NADH作为电子供体将NO代谢为硝酸盐。
