Gingivain; A Cysteine Proteinase Isolated from Porphyromonas gingivalis

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Shah, H. N. ; Gharbia, S. E. ; Kowlessur, D. ; Wilkie, E. ; Brocklehurst, K. (2011)
  • Publisher: Microbial Ecology in Health and Disease
  • Journal: Microbial Ecology in Health and Disease (issn: 1651-2235, eissn: 1651-2235)
  • Related identifiers: doi: 10.3402/mehd.v4i5.7715

The extracellular proteinase produced by Porphyromonas gingivalis, which has often been described as ‘trypsin-like’, was isolated by thiol-disulphide interchange covalent chromatography. Evidence from the method of isolation, thiolspecific inhibition reactivation cycle and thiol-specific reactivity probe kinetics, all involving 2-pyridyl disulphides, compels the view that this enzyme, for which the name gingivain is here proposed, is a cysteine proteinase. All of the hydrolytic activity towards α-N-benzoyl-L-arginine-4-nitroanilide(L-BAPNA) and azocasein in the P. gingivalis vesicle/supernatant mixture was shown to be thiol dependent by its complete inhibition by 2,2’-dipyridyl disulphide and complete reactivation by 2-mercaptoethanol. The vesicles and all of the hydrolytic activity were isolated by precipitation in 70 per cent saturated ammonium sulphate, centrifugation for 22 h at 150,000 g and 4°C. The cysteine proteinase was prepared in fully active form by sequential elution covalent chromatography on Sepharose-glutathione 2-pyridyl disulphide gel, which separated the enzyme from the vesicles and from other thiol-containing protein devoid of catalytic activity towards L-BAPNA. The fully active isolated enzyme was: (a) completely inhibited by reaction with 2,2’-dipyridyl disulphide with complete reactivation by 2-mercaptoethanol, and (b) shown to possess a key catalytic site characteristic, typical of many cysteine proteinases. Thus, stopped-flow kinetic analysis of the reaction of its catalytically essential thiol group with 2,2’-dipyridyl disulphide showed the reactivity to be minimum at ca. pH 6, behaviour characteristic of the existence of a catalytic site cysteine-histidine interactive system.Keywords: Gingivain; a cysteine proteinase; P. gingivalis proteinase.
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