1. Intralysosomal hydrolysis of glycyl-L-phenylalanine 2-naphthylamide
M Jadot, C Colmant, S Wattiaux-De Coninck, R Wattiaux Biochem J. 1984 May 1;219(3):965-70. doi: 10.1042/bj2190965.
Glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap), a cathepsin C substrate, induces an increase of the free and unsedimentable activities of this enzyme when incubated with a total mitochondrial fraction of rat liver. 1 mM-ZnSO4 considerably inhibits the cathepsin C total activity, measured with Gly-L-Phe-2-NNap as the substrate, in the presence of Triton X-100. The inhibition is markedly less pronounced when the free activity is determined; a high activity remains that depends on the integrity of the lysosomes; it decreases as the free activity of N-acetylglucosaminidase increases when lysosomes are subjected to treatments able to disrupt their membrane. Cathepsin C activity is reduced when thioethylamine hydrochloride is omitted from the incubation medium. Under these conditions at 37 degrees C, the free activity equals the total activity, although the lysosomes are intact, as indicated by the low free activity of N-acetylglucosaminidase. 1 mM-ZnSO4 strikingly inhibits the total activity, whereas more than 80% of the free activity remains. These observations are presented as evidence that Gly-L-Phe-2-NNap can possibly cause a disruption of the lysosomes as a result of its hydrolysis inside these organelles. In the presence of ZnSO4, intralysosomal hydrolysis becomes apparent, owing to a preferential inhibition by Zn2+ of extralysosomal hydrolysis; in the absence of thioethylamine hydrochloride, it is measurable because the disruption of lysosomes by Gly-L-Phe-2-NNap is delayed as a result of a slow-down of the reaction. The usefulness of Gly-L-Phe-2-NNap and related dipeptidyl naphthylamides in lysosomal-membrane-permeability studies is emphasized.
2. Natural structural variation in enzymes as a tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state probe (2,2'-dipyridyl disulphide) and with a specific synthetic substrate (N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide)
F Willenbrock, K Brocklehurst Biochem J. 1984 Sep 15;222(3):805-14. doi: 10.1042/bj2220805.
Cathepsin B (EC 3.4.22.1) from bovine spleen and the analogous enzyme from rat liver were investigated at 25 degrees C at I0.1 in acidic media by kinetic study of (a) the reactions of their catalytic-site thiol groups towards the two-protonic-state reactivity probe 2,2'-dipyridyl disulphide and (b) their catalysis of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide. Reactivity-probe kinetics showed that nucleophilic character is generated in the sulphur atom of cathepsin B by protonic dissociation with pKa 3.4, presumably to form an S-/ImH+ ion-pair. Substrate-catalysis kinetics showed that ion-pair formation is not sufficient to generate catalytic competence in cathepsin B, because catalytic activity is not generated as the pH is raised across pKa 3.4 but rather as it is raised across pKa 5-6 (5.1 for kcat; 5.6 for kcat./Km for the bovine spleen enzyme and 5.8 for kcat./Km for the rat liver enzyme). The implications of these results and of known structural differences between the catalytic sites of the rat liver enzyme and papain (EC 3.4.22.2) for the mechanism of cysteine-proteinase-catalysed hydrolysis are discussed.
3. Factors affecting oral malodor in periodontitis and gingivitis patients
Thuy A V Pham, Masayuki Ueno, Kayoko Shinada, Yoko Kawaguchi J Investig Clin Dent. 2012 Nov;3(4):284-90. doi: 10.1111/j.2041-1626.2012.00155.x.
Aim: To examine the associations between oral health status, the presence of N-benzoyl-DL-arginine-2-naphthylamide-positive bacteria, and oral malodor in periodontal patients. Methods: A total of 137 periodontitis and 80 gingivitis patients were included in the study. Oral malodor was measured by an organoleptic test and the OralChroma. An oral examination was conducted, including the assessment of decayed teeth, periodontal status, and tongue coating. The presence of N-benzoyl-DL-arginine-2-naphthylamide-positive bacteria in the subgingiva, tongue coating, and saliva was evaluated by the N-benzoyl-DL-arginine-2-naphthylamide test. Results: In the periodontitis group, oral malodor was significantly correlated with decayed teeth, periodontal parameters, and tongue coating. Among the N-benzoyl-DL-arginine-2-naphthylamide test parameters, the highest correlation of oral malodor was found with N-benzoyl-DL-arginine-2-naphthylamide subgingiva, followed by N-benzoyl-DL-arginine-2-naphthylamide tongue coating and N-benzoyl-DL-arginine-2-naphthylamide saliva. In the gingivitis group, oral malodor was significantly correlated with the plaque index, bleeding on probing, and tongue coating. Among the N-benzoyl-DL-arginine-2-naphthylamide test parameters, the highest correlation of oral malodor was found with N-benzoyl-DL-arginine-2-naphthylamide tongue coating, followed by N-benzoyl-DL-arginine-2-naphthylamide saliva and N-benzoyl-DL-arginine-2-naphthylamide subgingiva. Conclusion: Dental plaque, bleeding on probing, tongue coating, and N-benzoyl-DL-arginine-2-naphthylamide-positive bacteria contribute to oral malodor, but with different degrees in periodontitis and gingivitis patients.
