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NADPH diaphorase detects S-nitrosylated proteins in aldehyde-treated biological tissues.
Seckler, James M; Shen, Jinshan; Lewis, Tristan H J; Abdulameer, Mohammed A; Zaman, Khalequz; Palmer, Lisa A; Bates, James N; Jenkins, Michael W; Lewis, Stephen J.
Afiliación
  • Seckler JM; Department of Pediatrics, Case Western Reserve University, Cleveland, OH, 44106, USA.
  • Shen J; Department of Pharmacology, University of Iowa, Iowa City, IA, 52242, USA.
  • Lewis THJ; Department of Pharmacology and Physiology, University of Georgia, Athens, GA, 30602, USA.
  • Abdulameer MA; Department of Pediatrics, Case Western Reserve University, Cleveland, OH, 44106, USA.
  • Zaman K; Department of Pediatrics, Case Western Reserve University, Cleveland, OH, 44106, USA.
  • Palmer LA; Department of Pediatrics, University of Virginia, Charlottesville, VA, 801366, USA.
  • Bates JN; Department of Anesthesia, University of Iowa, Iowa City, IA, 52242, USA.
  • Jenkins MW; Department of Pediatrics, Case Western Reserve University, Cleveland, OH, 44106, USA.
  • Lewis SJ; Department of Bioengineering, Case Western Reserve University, Cleveland, OH, 44106, USA.
Sci Rep ; 10(1): 21088, 2020 12 03.
Article en En | MEDLINE | ID: mdl-33273578
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 ß- or α-NADPH to solutions of NBT did not elicit diformazan, (3) addition of S-nitrosothiols to solutions of NBT plus ß- 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 ß- 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.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Óxido Nítrico Sintasa / S-Nitrosotioles / NADPH Deshidrogenasa Límite: Animals Idioma: En Revista: Sci Rep Año: 2020 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Óxido Nítrico Sintasa / S-Nitrosotioles / NADPH Deshidrogenasa Límite: Animals Idioma: En Revista: Sci Rep Año: 2020 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido