Department of Horticulture and Michigan State University-Atomic Energy Commission Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48823.
Plant Physiol. 1971 Jun;47(6):790-4. doi: 10.1104/pp.47.6.790.
A method has been devised for the detection and measurement of nitrite reductase activity in intact barley (Hordeum vulgare L. cv. Himalaya) aleurone layers. The technique involves feeding aleurone layers nitrite and measuring nitrite disappearance after a given time period. The method also allows simultaneous determination of nitrite uptake by the tissue. Quantitative recovery of nitrite is obtained by rapid heating of tissue in the presence of dimethyl sulfoxide.Using the procedure described, nitrite reductase activity in intact barley aleurone layers was determined. Enzyme activity was increased by prior incubation of the tissue with nitrate, but considerable activity was present in tissue incubated without nitrate. Nitrate-induced activity was inhibited by cycloheximide but not by actinomycin D. Enzyme activity in induced layers was inhibited by 2,4-dinitrophenol, and partially by antimycin A and 2-n-heptyl-4-hydroxyquinoline N-oxide. Activity in noninduced tissue appeared to be less sensitive to these respiratory inhibitors. In contrast, both activities were inhibited more than 90% by anaerobiosis; but nitrate-induced and noninduced aleurone layers were able to reduce nitrite anaerobically when the concentration of substrate in the assay medium was reduced from 250 mum to 25 mum. Nitrite uptake was relatively insensitive to anaerobiosis and to the inhibitors tested.Nitrite depletion from the medium by aleurone layers was rapid at pH 4.5 and negligible at pH 7.5. Nitrite accumulated at pH 4.5 under anaerobic conditions was rapidly released when the tissue was transferred to medium at pH 7.5. Nitrite release at pH 7.5 occurred whether the tissue was maintained under anaerobic or aerobic conditions.
一种用于检测和测量完整大麦（Hordeum vulgare L. cv. Himalaya）糊粉层中亚硝酸盐还原酶活性的方法已经被设计出来。该技术包括向糊粉层中添加亚硝酸盐并在给定时间段后测量亚硝酸盐的消失。该方法还允许同时测定组织对亚硝酸盐的摄取量。组织在二甲基亚砜存在下快速加热可定量回收亚硝酸盐。使用所描述的程序，测定了完整大麦糊粉层中亚硝酸盐还原酶的活性。组织先用硝酸盐孵育可增加酶活性，但在没有硝酸盐孵育的组织中也存在相当大的活性。硝酸盐诱导的活性被环己酰亚胺抑制，但不被放线菌酮 D 抑制。诱导层中的酶活性被 2,4-二硝基苯酚部分抑制，被抗霉素 A 和 2-庚基-4-羟基喹啉 N-氧化物部分抑制。非诱导组织中的活性似乎对这些呼吸抑制剂的敏感性较低。相比之下，两种活性在缺氧条件下都被抑制超过 90%；但当测定介质中亚硝酸盐底物浓度从 250 µm 降低到 25 µm 时，非诱导和诱导的糊粉层仍能够在缺氧条件下还原亚硝酸盐。亚硝酸盐的摄取对缺氧和测试的抑制剂相对不敏感。糊粉层在 pH 4.5 时迅速从介质中耗尽亚硝酸盐，在 pH 7.5 时则几乎没有。在缺氧条件下，当组织转移到 pH 7.5 的介质中时，在 pH 4.5 下积累的亚硝酸盐迅速释放。无论组织在缺氧还是有氧条件下，在 pH 7.5 时都会发生亚硝酸盐释放。
