Formation of a polymethylene bis(disulfide) intersubunit cross-link between cysteine-281 residues in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase using octamethylene bis(methane[35S]thiosulfonate).
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 two molecules of OBMTS react rapidly with the active-site thiol, Cys-149, on two of the four subunits to inhibit the enzyme completely without cross-linking. This is followed by the modification of four Cys-281 residues to incorporate two cross-links into the tetramer. Reduction of modified G3PD with 5 mM dithioerythritol under nondenaturing conditions released the inhibitor blocking the active-site thiol and completely restored enzyme activity while leaving the cross-link intact. Sodium dodecyl sulfate (Na-DodSO4) gel electrophoresis of the cross-linked enzyme under nonreducing conditions showed a dimer (Mr 72000) as the major species which was only cleaved by reduction in Na-DodSO4 containing beta-mercaptoethanol. The monomer formed was still radioactive, showing that the first disulfide in the 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. There were identified by molecular weight, amino terminal sequence, and amino acid composition.
D000476:Alkanesulfonates / Q000138:chemical synthesis* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001345:Autoradiography; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D003488:Cyanogen Bromide; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D008698:Mesylates; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits
D000476:Alkanesulfonates; D000596:Amino Acids; D003432:Cross-Linking Reagents; D008698:Mesylates; C034827:octamethylene bis(methanethiosulfonate); D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine; D003488:Cyanogen Bromide