Department of Chemistry, State University of New York, College of Environmental Science and Forestry, Syracuse, New York 13210.
Plant Physiol. 1978 Apr;61(4):611-6. doi: 10.1104/pp.61.4.611.
Squash cotyledon (Cucurbita pepo L.) NADH:nitrate reductase (NR) was purified 150-fold with 50% recovery by a single step procedure based on the affinity of the NR for blue-Sepharose. Blue-Sepharose, which is prepared by direct coupling of Cibacron blue to Sepharose, appears to bind squash NR at the NADH site. The NR can be purified in 2 to 3 hours to a specific activity of 2 mumol of NADH oxidized/minute * milligram of protein. Corn (Zea mays L.) leaf NR was also purified to a specific activity of 6.9 mumol of NADH oxidized/minute * milligram of protein using a blue-Sepharose affinity step. The blue-Sepharose method offers the advantages of a rapid purification of plant NR to a high specific activity with reasonable recovery of total activity.The kinetic mechanism of higher plant NR was investigated using these highly purified squash and corn NR preparations. Based on initial velocity and product inhibition studies utilizing both enzymes, a two-site ping-pong mechanism is proposed for NR. This kinetic mechanism incorporates the concept of the reduced NR transferring electrons from the NADH site to a physically separated nitrate site.
南瓜子叶 NADH：硝酸盐还原酶（NR）可通过基于 NR 对蓝琼脂糖亲和力的单一步骤程序，以 50％的回收率进行 150 倍纯化。蓝琼脂糖是通过将 Cibacron 蓝直接偶联到琼脂糖上制备的，它似乎在 NADH 结合部位结合南瓜 NR。NR 可以在 2 至 3 小时内纯化至 2 微摩尔 NADH 氧化/分钟毫克蛋白质的比活度。玉米（Zea mays L.）叶片 NR 也通过蓝琼脂糖亲和步骤纯化至 6.9 微摩尔 NADH 氧化/分钟毫克蛋白质的比活度。蓝琼脂糖法具有快速纯化植物 NR 的优点，具有较高的比活度和总活性的合理回收率。使用这些高度纯化的南瓜和玉米 NR 制剂研究了高等植物 NR 的动力学机制。基于初始速度和产物抑制研究，利用两种酶，提出了 NR 的双位点乒乓机制。该动力学机制包含了还原型 NR 将电子从 NADH 结合部位转移到物理分离的硝酸盐结合部位的概念。
