SULFHYDRYL-DISULFIDE MODULATION AND THE ROLE OF DISULFIDE OXIDOREDUCTASES IN REGULATION OF THE CATALYTIC ACTIVITY OF NITRIC-OXIDE SYNTHASE IN PULMONARY-ARTERY ENDOTHELIAL-CELLS

The role of sulfhydryl groups (SH) and disulfide bonds as well as disu lfide oxidoreductases in regulation of the catalytic activity of the m embrane-bound constitutive isoform of nitric oxide (NO) synthase from porcine pulmonary artery endothelial cells (PAEC) was examined. Treatm ent of intact PAEC or a total membrane preparation isolated from PAEC with the SH alkylating agent N-ethylmaleimide (NEM) (10 to 50 mu M) or with the intramolecular disulfide-forming agent diamide (20 to 100 mu M) resulted in the reduction of NO synthase activity in a dose-depend ent fashion, Similar loss of enzyme activity was observed when purifie d NO synthase from the membrane fraction of PAEC was incubated in the presence of NEM. The loss of membrane protein SH content from NEM- and diamide-treated preparations was associated with loss of NO synthase activity, In contrast, when intact PAEC or isolated total membranes de rived from PAEC were treated with increasing concentrations (1 to 5 mM ) of the disulfide-reducing agent dithiothreitol (DTT), but not oxidiz ed DTT, NO synthase activity was increased by 20 to 85 %. DTT reductio n of native disulfides from NEM-treated preparations or of disulfides formed after diamide treatment of membranes reversed the inhibition of NO synthase activity. Similarly, enzymatic reduction by thioredoxin/t hioredoxin reductase, but not by glutaredoxin, reversed the inhibition of membrane fraction and purified NO synthase isolated from diamide-t reated cells. This enzyme-catalyzed disulfide reduction was > 1,000-fo ld more efficient than the DTT-induced reduction. These results demons trate that modulation of the SH and/or the disulfide bonds of NO synth ase is associated with alterations in the catalytic activity of the me mbrane-bound constitutive isoform of NO synthase in PAEC. This modulat ion appears to be part of a regulatory mechanism that involves the oxi dation-reduction of at least two conformationally vicinal reactive SH located in or near the catalytic domain of NO synthase and indicates t hat thioredoxin/thioredoxin reductase may play a physiologically impor tant role in the regulation of NO synthase activity through a redox me chanism.