mersalyl and 4-hydroxymercuribenzoate

Topics: Alkylation; Animals; Arginine; Cell Nucleus; Cyclohexanones; Dose-Response Relationship, Drug; Hydroxymercuribenzoates; Kinetics; Lysine; Mersalyl; Molecular Weight; Polymers; Protein Conformation; Pyridoxal Phosphate; Receptors, Estrogen; Structure-Activity Relationship; Sulfhydryl Compounds

Recently developed "exchange assays" have been used to measure total cytosolic glucocorticoid receptor (GR) binding activity as compared to standard GR assays which measure unoccupied receptor. In the current study we modified these methods and extended the applications of such assays. Experiments defined the conditions whereby two sulfhydryl-binding agents, p-hydroxymercuribenzoate (PHMB) and mersalyl, completely inhibited binding of the glucocorticoid receptor to ligand in mouse renal cytosol. Reactivation of steroid-binding activity was restored by addition of dithiothreitol. The present study demonstrates 12% higher GR binding activity when this exchange assay is performed using saturated glucocorticoid-receptor complex, rather than standard cytosol. Combining the data from the standard and exchange mouse renal cytosolic GR assays, it was determined that, at physiologic tissue corticosterone levels, the respective mean concentrations of unoccupied, occupied, and total GR were 467, 89, and 556 fmol/mg cytosol protein. Measurement of receptor concentrations by the use of these methods permits precise experimental differentiation of factors which affect total, as well as unoccupied GR.

Topics: Animals; Binding, Competitive; Cytosol; Dexamethasone; Dithiothreitol; Hydroxymercuribenzoates; Kidney; Kinetics; Mersalyl; Mice; Organomercury Compounds; Receptors, Glucocorticoid

Pure human skin fibroblast procollagenase has been utilized in this study as a model system in which to examine the pathways of organomercurial and trypsin activation. Three organomercurials, p-(hydroxymercuri) benzoate, mersalyl, and p-aminophenylmercuric acetate, were able to fully activate human skin procollagenase with no accompanying loss of molecular weight. Lower molecular weight species were subsequently produced, particularly with a fourth organomercurial, phenylmercuric chloride. The activation process was dependent upon the concentration of the organomercurial compound and the time of incubation, but not on enzyme protein concentration. No evidence of a role for free sulfhydryls was found. Trypsin produced an initial cleavage product of procollagenase which was collagenolytically inactive yet underwent a concentration independent autocatalysis. Thus, procollagenase appeared to have an autocatalytic property which was enhanced by treatment with a variety of agents, all of which may function by perturbation of the zymogen conformation.

Topics: Cell Line; Collagenases; Enzyme Activation; Enzyme Precursors; Fibroblasts; Humans; Hydroxymercuribenzoates; Mersalyl; Microbial Collagenase; Molecular Weight; Organomercury Compounds; Phenylmercuric Acetate; Skin; Trypsin