Formation of a polymethylene bis(disulfide) intersubunit crosslink between cysteine-281 residues in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase using octamethylene bis(methane[35S]thiosulfonate)

Abstract

The synthesis of a radioactive cross-linking agent, S,S''-octamethylene bis(methane[35S]thiosulfonate) (OBMTS), is described. The route of synthesis can be generally used in the synthesis of 35S-labeled thiosulfonates for the selective modification of thiols in proteins. Glyceraldehyde-3-phosphate dehydrogenase (G3PD) reacts asymmetrically with the bifunctional inhibitor. Initially 2 molecules of OBMTS react rapidly with the active-site thiol, Cys-149, on 2 of the 4 subunits to inhibit the enzyme completely without cross-linking. This is followed by the modification of 4 Cys-281 residues to incorporate 2 cross-links into the tetramer. Reduction of modified G3PD with 5 mM dithioerythritol under nondenaturing conditions released the inhibitor blocking the active-site and completely restored enzyme activity while leaving the cross-link intact. Sodium dodecyl sulfate [SDS] gel electrophoresis of the cross-linked enzyme under nonreducing conditions showed a dimer (MW 72,000) as the major species which was only cleaved by reduction in SDS containing .beta.-mercaptoethanol. The monomer formed was stiil radioactive, showing that the first disulfide in teh cross-link was reduced at a much faster rate than the second disulfide. The latter was only reduced by using vigorous conditions. The location of the intersubunit cross-linked residues was established by isolation of the cyanogen bromide and tryptic subdigest peptides containing modified Cys-281. These were identified by MW, amino terminal sequence, and amino acid composition.