mersalyl and 4-aminophenylmercuriacetate

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

1. Trypsin has been reacted with dithiothreitol and with a naturally occurring thiol-containing trypsin inhibitor to form enzyme-inhibitor complexes. This complex formation is known to be via a reversible intermolecular disulphide linkage. 2. These latent forms of trypsin have been re-activated with mersalyl [N-(O-carboxymethylsalicyloyl)-3-hydroxymercuric-2-methoxypropylamine], 4-aminophenylmercuric acetate and with cystine. 3. Active-site titration analysis of trypsin in the presence of incremental additions of dithiothreitol demonstrated the simultaneous inhibition and modification of the enzyme active site, demonstrating a direct involvement of a significant disulphide controlling the conformation of the active site of the enzyme. 4. Mersalyl addition to the dithiothreitol-reduced trypsin resulted in a regain of enzymic activity and a corresponding regain of availability of the active sites for titration. 5. Mersalyl and 4-aminophenylmercuric acetate were shown to re-activate the trypsin-inhibitor complex. 6. A molecular mechanism for the organomercurial re-activation of latent enzymes of this particular type (involving disulphide exchange) has been proposed.

Topics: Binding Sites; Cystine; Dithiothreitol; Enzyme Activation; Kinetics; Mersalyl; Organomercury Compounds; Phenylmercuric Acetate; Phenylmercury Compounds; Protein Binding; Trypsin