Probing subunit interactions in glutathione S-transferases using affinity labeling

Affinity labeling, using reactive analogs of natural ligands, provides a powerful approach to probe for the existence of cooperative interactions between subunits as well as to locate specific xenobiotic binding sites in glutathione 5-transferases (GST). Thus, 3β-(iodoacetoxy)-dehydroisoandrosterone (3β-IDA) and 17-(iodoacetoxy)estradiol-3-sulfate (17β-IES) function as affinity labels of the nonsubstrate steroid binding site of rat liver GST, isozyme 1-1. Modification of Cys¹⁷ or Cys¹¹¹ on Subunit A prevents reaction of the same cysteine on Subunit B. Interactions between the subunits of both the 4-4 and 1-1 isozymes are also illustrated by photoaffinity labeling of the active sites of these homodimers by glutathionyl S[4-(succinimidyl)-benzophenone], an analog of the product of glutathione and xenobiotic substrate.