Hope B T, Michael G J, Knigge K M, Vincent S R
Kinsmen Laboratory of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, Canada.
Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2811-4. doi: 10.1073/pnas.88.7.2811.
NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affinity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPH-dependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.
NADPH黄递酶组织化学可选择性地标记整个神经系统中许多离散的神经元群体。这种简单而可靠的技术已在大量实验和神经病理学研究中得到应用；然而，这种酶的功能一直是个推测的问题。因此，我们着手对这种酶进行生化特性分析。通过生化和免疫化学分析，利用亲和层析和阴离子交换高效液相色谱法从大鼠脑中纯化出了纯度明显较高的NADPH黄递酶。用针对NADPH黄递酶的特异性抗体进行蛋白质免疫印迹转移和免疫染色，标记出了一条150 kDa的单一蛋白质条带。最近研究表明，一氧化氮合酶是脑中一种150 kDa的NADPH依赖性酶。它负责在钙/钙调蛋白的作用下，从L-精氨酸合成鸟苷酸环化酶激活剂一氧化氮。我们发现，一氧化氮合酶活性与NADPH黄递酶共同纯化至同质状态，并且两种活性都能用识别神经元NADPH黄递酶的抗体进行免疫沉淀。此外，一氧化氮合酶受到NADPH黄递酶底物氮蓝四唑的竞争性抑制。因此，神经元NADPH黄递酶就是一种一氧化氮合酶，所以，NADPH黄递酶组织化学为产生一氧化氮的神经元提供了一种特异性组织化学标记。
