S-THIOLATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE INDUCED BY THE PHAGOCYTOSIS-ASSOCIATED RESPIRATORY BURST IN BLOOD MONOCYTES

Chemical oxidants can induce the covalent binding of low molecular wei ght thiols to reactive sulfhydryls on proteins (S-thiolation), We foun d that stimulation of the respiratory burst of human blood monocytes r esulted in S-thiolation of several proteins, most prominently one of 3 8 kDa. This purified protein was identified as glyceraldehyde-3-phosph ate dehydrogenase (GAPDH) by enzyme activity, immunoblotting, and amin o acid analysis. After stimulation of the respiratory burst, S-thiolat ion of GAPDH gradually increased, and cytosol GAPDH activity decreased ; so that at 60 min, GAPDH activity was reduced by similar to 40%, Act ivity was restored by the addition of the sulfhydryl-reducing agent di thioerythritol. H2O2 appeared to be particularly important in mediatin g S-thiolation during the respiratory burst. Exposure of monocytes to H2O2 induced concentration-dependent S-thiolation of GAPDH and a conco mitant decrease in enzyme activity. The addition of respiratory burst stimuli to lymphocytes, which lack a full respiratory burst, had no ef fect on GAPDH S-thiolation or activity; but H2O2 induced S-thiolation of lymphocyte GAPDH and inhibition of enzyme activity. Stimulation of monocytes from three patients with chronic granulomatous disease resul ted in no respiratory burst, S-thiolation of GAPDH, or inactivation of GAPDH activity. The thiols covalently bound to purified S-thiolated G APDH were removed by dithioerythritol and were identified as glutathio ne and cysteine; glutathione was predominant. These results indicate t hat during the respiratory burst in monocytes, low molecular weight th iols can bind to specific cytosolic proteins, including GAPDH. It is p ossible that S-thiolation of cytosolic proteins serves to modulate cel lular metabolic events during phagocytosis.