NADPH diaphorase detects S-nitrosylated proteins in aldehyde-treated biological tissues.
NADPH diaphorase detects S-nitrosylated proteins in aldehyde-treated biological tissues.
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DOI：
10.1038/s41598-020-78107-6
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发表时间：
2020-12-03
影响因子：
4.6
通讯作者：
Lewis SJ
中科院分区：
文献类型：
--
作者：
Seckler JM;Shen J;Lewis THJ;Abdulameer MA;Zaman K;Palmer LA;Bates JN;Jenkins MW;Lewis SJ
NADPH黄递酶被用作一氧化氮合酶（NOS）的组织化学标记物，在去甲肾上腺素处理的组织。据认为，NOS的催化活性促进NADPH依赖性还原硝基四氮唑蓝（NBT）的二甲。然而，有人认为，一氧化氮合酶以外的蛋白质因子是负责生产二甲肼治疗的组织。我们建议这是一个NO-含有的因素，如S-亚硝基硫醇和/或二亚硝基铁（II）半胱氨酸复合物或亚硝化蛋白质，包括NOS。我们现在报告：（1）S-亚硝基硫醇共价修饰NBT和TNBT，但仅改变修饰后NBT的还原电位，（2）向NBT溶液中加入S-亚硝基硫醇或β-或α-NADPH不会引发二甲瓒，（3）在不存在或存在多聚甲醛的情况下，向NBT + β-或α-NADPH溶液中加入S-亚硝基硫醇会引发二甲瓒的快速形成，（4）在NBT + β-或α-NADP溶液中加入S-亚硝基硫醇不产生二甲，（5）S-亚硝基硫醇不促进NADPH依赖的四硝基蓝四唑（TNBT）还原，TNBT中所有四个酚环都被硝化，（6）已知用于NADPH黄递酶染色的血管内皮细胞的胞质囊泡富含S-亚硝基硫醇，和（7）加速S-亚硝基硫醇分解的方法，显著减少随后进行多聚甲醛固定的组织切片中的NADPH黄递酶染色。我们的研究结果表明，NADPH黄递酶在去磷酸化固定的组织中不是酶促的，而是由于存在含NO的因子（游离的SNOs或亚硝化蛋白质，如NOS），其促进NBT还原为二甲瓒的NADPH依赖性还原。
NADPH diaphorase is used as a histochemical marker of nitric oxide synthase (NOS) in aldehyde-treated tissues. It is thought that the catalytic activity of NOS promotes NADPH-dependent reduction of nitro-blue tetrazolium (NBT) to diformazan. However, it has been argued that a proteinaceous factor other than NOS is responsible for producing diformazan in aldehyde-treated tissues. We propose this is a NO-containing factor such as an S-nitrosothiol and/or a dinitrosyl-iron (II) cysteine complex or nitrosated proteins including NOS. We now report that (1) S-nitrosothiols covalently modify both NBT and TNBT, but only change the reduction potential of NBT after modification, (2) addition of S-nitrosothiols or β- or α-NADPH to solutions of NBT did not elicit diformazan, (3) addition of S-nitrosothiols to solutions of NBT plus β- or α-NADPH elicited rapid formation of diformazan in the absence or presence of paraformaldehyde, (4) addition of S-nitrosothiols to solutions of NBT plus β- or α-NADP did not produce diformazan, (5) S-nitrosothiols did not promote NADPH-dependent reduction of tetra-nitro-blue tetrazolium (TNBT) in which all four phenolic rings are nitrated, (6) cytoplasmic vesicles in vascular endothelial cells known to stain for NADPH diaphorase were rich in S-nitrosothiols, and (7) procedures that accelerate decomposition of S-nitrosothiols, markedly reduced NADPH diaphorase staining in tissue sections subsequently subjected to paraformaldehyde fixation. Our results suggest that NADPH diaphorase in aldehyde-fixed tissues is not enzymatic but is due to the presence of NO-containing factors (free SNOs or nitrosated proteins such as NOS), which promote NADPH-dependent reduction of NBT to diformazan.
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DOI：
10.1152/ajpheart.1998.274.4.h1066
发表时间：
1998-04-01
影响因子：
4.8
作者：
Colombari, E;Davisson, RL;Lewis, SJ
通讯作者：
Lewis, SJ
影响因子：
3.9
作者：
Freitas, I;Griffini, P;Vairetti, M
通讯作者：
Vairetti, M
影响因子：
3
作者：
Hashmi-Hill, Maleka P.;Sandock, Kevin;Lewis, Stephen J.
通讯作者：
Lewis, Stephen J.
影响因子：
11.4
作者：
Chvanov, Michael;Gerasimenko, Oleg V.;Tepikin, Alexei V.
通讯作者：
Tepikin, Alexei V.
影响因子：
8.3
作者：
Davisson, RL;Bates, JN;Lewis, SJ
通讯作者：
Lewis, SJ