PMID- 8912669
OWN - NLM
STAT- MEDLINE
DCOM- 19961226
LR  - 20190501
IS  - 0264-6021 (Print)
IS  - 1470-8728 (Electronic)
IS  - 0264-6021 (Linking)
VI  - 319 ( Pt 2)
IP  - Pt 2
DP  - 1996 Oct 15
TI  - Nitric oxide-dependent NAD linkage to glyceraldehyde-3-phosphate dehydrogenase: 
      possible involvement of a cysteine thiyl radical intermediate.
PG  - 369-75
AB  - Previous studies have demonstrated that glyceraldehyde-3-phosphate dehydrogenase 
      (GAPDH) undergoes NAD(H) linkage to an active site thiol when it comes into 
      contact with .NO-related oxidants. We found that a free-radical generator 
      2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), which does not release 
      either .NO or .NO-related species, was indeed able to induce the NAD(H) linkage 
      to GAPDH. We performed spin-trapping studies with purified apo-GAPDH to identify 
      a putative thiol intermediate produced by AAPH as well as by .NO-related 
      oxidants. As .NO sources we used .NO gas and two .NO-donors, 
      S-nitroso-N-acetyl-D,L-penicillamine and 3-morpholinosydno-nimine hydrochloride 
      (SIN-1). Because SIN-1 produces .NO and a superoxide radical simultaneously, we 
      also tested the effects of peroxynitrite. All the .NO-related oxidants were able 
      to induce the linkage of NAD(H) to GAPDH and the formation of a protein 
      free-radical identified as a thiyl radical (inhibited by N-ethylmaleimide). .NO 
      gas and the .NO-donors required molecular oxygen to induce the formation of the 
      GAPDH thiyl radical, suggesting the possible involvement of higher nitrogen 
      oxides. Thiyl radical formation was decreased by the reconstitution of GAPDH with 
      NAD+. Apo-GAPDH was a strong scavenger of AAPH radicals, but its scavenging 
      ability was decreased when its cysteine residues were alkylated or when it was 
      reconstituted with NAD+. In addition, after treatment with AAPH, a thiyl radical 
      of GAPDH was trapped at high enzyme concentrations. We suggest that the NAD(H) 
      linkage to GAPDH is mediated by a thiyl radical intermediate not specific to .NO 
      or .NO-related oxidants. The cysteine residue located at the active site of GAPDH 
      (Cys-149) is oxidized by free radicals to a thiyl radical, which reacts with the 
      neighbouring coenzyme to form Cys-NAD(H) linkages. Studies with the NAD+ molecule 
      radio-labelled in the nicotinamide or adenine portion revealed that both portions 
      of the NAD+ molecule are linked to GAPDH.
FAU - Minetti, M
AU  - Minetti M
AD  - Laboratorio di Biologia Cellulare, Istituto Superiore di Sanita, Roma, Italy.
FAU - Pietraforte, D
AU  - Pietraforte D
FAU - Di Stasi, A M
AU  - Di Stasi AM
FAU - Mallozzi, C
AU  - Mallozzi C
LA  - eng
PT  - Journal Article
PL  - England
TA  - Biochem J
JT  - The Biochemical journal
JID - 2984726R
RN  - 0 (Free Radicals)
RN  - 0U46U6E8UK (NAD)
RN  - 31C4KY9ESH (Nitric Oxide)
RN  - EC 1.2.1.- (Glyceraldehyde-3-Phosphate Dehydrogenases)
RN  - K848JZ4886 (Cysteine)
SB  - IM
MH  - Cysteine/metabolism
MH  - Free Radicals/metabolism
MH  - Glyceraldehyde-3-Phosphate Dehydrogenases/*metabolism
MH  - Humans
MH  - NAD/*metabolism
MH  - Nitric Oxide/*metabolism
PMC - PMC1217778
EDAT- 1996/10/15 00:00
MHDA- 1996/10/15 00:01
PMCR- 1997/04/15
CRDT- 1996/10/15 00:00
PHST- 1996/10/15 00:00 [pubmed]
PHST- 1996/10/15 00:01 [medline]
PHST- 1996/10/15 00:00 [entrez]
PHST- 1997/04/15 00:00 [pmc-release]
AID - 10.1042/bj3190369 [doi]
PST - ppublish
SO  - Biochem J. 1996 Oct 15;319 ( Pt 2)(Pt 2):369-75. doi: 10.1042/bj3190369.