S-THIOLATION OF INDIVIDUAL HUMAN NEUTROPHIL PROTEINS INCLUDING ACTIN BY STIMULATION OF THE RESPIRATORY BURST - EVIDENCE AGAINST A ROLE FOR GLUTATHIONE DISULFIDE

Protein S-thiolation, a reversible modification of protein sulfhydryls resulting in formation of mixed-disulfides, was studied in human neut rophils stimulated with phorbol diester to produce superoxide anion. R apid S-thiolation of several proteins was examined by sodium dodecyl s ulfate-polyacrylamide gel electrophoresis. Glutathione was identified as the primary protein-bound thiol by HPLC chromatography, contributin g considerably more than 85% of the total. Minor amounts of homocystei ne and/or cysteine were also detected as protein-bound thiols. During the first 30 min after stimulation, 10% of the cellular glutathione be came protein bound (2 nmol/mg of protein). There was no increase in gl utathione disulfide suggesting that S-thiolation of the proteins did n ot occur by thiol/disulfide exchange. Approximately 10 mol% of one hea vily modified band (29 kDa) was S-thiolated after 30 min. A second maj or band of 42 kDa was identified as actin. It contained 1/10th of the total protein-bound glutathione and approximately 5 mol% was S-thiolat ed after 30 min. These experiments identify a subset of S-thiolated ne utrophil proteins, including actin, whose modification is related to t he phorbol diester stimulation of superoxide anion production in human neutrophils. Ten percent of the total glutathione pool became protein -bound without an appreciable change in non-bound concentration of glu tathione or glutathione disulfide. These results suggest that glutathi one was synthesized during initial phases of the respiratory burst, co mpensating for the amount of glutathione that became protein-bound. Si nce there was no significant increase in glutathione disulfide, it was probably not important in the observed protein S-thiolation. (C) 1994 Academic Press, Inc.