Connaris, Helen; (1992) Heterogeneity of proteinases from the hyperthermophilic archaeobacterium, Pyrococcus furiosus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In order to investigate the role of proteolysis in Pyrococcus furiosus, cultures were grown on a variety of complex and simple carbon substrates and assayed for proteolytic activity using azocasein as the protein substrate. Growth on complex protein substrates such as peptone or tryptone induced high levels of extracellular proteinase activity, whereas growth in the presence of the oligosaccharide maltose largely repressed proteinase activity. Gelatin SDS PAGE revealed thirteen proteolytically active bands in both cell extracts and culture supernatants, with apparent molecular weights ranging from 66kDa to 135kDa. It was concluded that these bands were not artefacts but probably discrete polypeptides. The band pattern was observed after a variety of treatments, which included different incubation times and temperature, SDS concentration, different proteolytic substrate (casein) and thiol-interchange mechanisms. Using a variety of chromatographic techniques, only the 66kDa proteinase could be resolved. Mono Q, Hydrogen Bond cellulose, sucrose density gradient centrifugation, and gel permeation under non-denaturing conditions all produced similar band patterns suggesting that the polypeptides existed as active aggregates or as a high molecular weight complex. The proteinase 'complex' from cell extracts was purified to near homogeneity with a yield of 35%. The molecular weight was estimated to be approximately 10⁶ Daltons. The pH optimum for activity was between 6 and 8, and the ''temperature optimum" was 100°C. At 95°C, the proteinase complex had a half-life of 69h. The proteinase complex was stable at 95°C in 6M urea, 10mM dithiothreitol and in 4.4M guanidinium chloride. Enhanced stability was observed in the presence of 0.1% and 1% Triton X-100, whereas the detergents SDS and CTAB produced a large destabilising effect. Organic solvents (ethanol, acetone and dimethylformamide) significantly reduced thermostability of the proteinase complex at 100°C. Enhanced stability of the complex was observed in the presence of 1M NaC1. Inhibition studies indicated that the activity of the complex was predominantly of a serine-type. Inhibition by EDTA of a PMSF-insensitive proteinase was observed in gelatin SDS gels, suggesting the presence of at least one metalloproteinase activity in the complex. A preliminary determination of substrate specificity indicated a preference for small basic amino acids and substrates containing phenylalanine adjacent to the leaving group.

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