Detection of cathepsin B- and L-, elastase-, tryptase-, trypsin-, and dipeptidyl peptidase IV-like activities in crevicular fluid from gingivitis and periodontitis patients with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin.
Crevicular fluid samples were collected from 20 gingivitis and periodontitis patients using filter paper strips; these were then eluted into buffer. Portions of each sample were combined and the activities of this pooled eluate against different peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin (AFC) were examined with respect to their pH profiles and effector responses. Ca-thepsin B- and L-like activity was detected with Bz-Val-Lys-Lys-Arg-AFC; elastase-like activity with MeOSuc-Ala-Ala-Pro-Val-AFC; tryptase-like activity with Z-Ala-Ala-Lys-AFC; trypsin-like activity with Z-Gly-Gly-Arg-AFC; and dipeptidyl peptidase (DPP) IV-like activity with Ala-Pro-AFC. The selectivity and sensitivity of these assays were improved by choice of appropriate conditions. The cathepsin B- and L-, elastase-, tryptase-, and trypsin-like activities all had properties consistent with those from host sources, whilst partial inactivation of the DPP IV-like activity by heat treatment (60 degrees C for 30 min) suggested that it may have represented a mixture of human and Bacteroides gingivalis enzymes. Individual patient eluates showed wide variations in enzyme concentrations, but generally elastase-like activity was by far the highest. The sensitivity of the assays with AFC-linked substrates was such that it should prove possible to measure all five different types of activity in crevicular fluid samples from local periodontal disease sites.
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D000595:Amino Acid Sequence; D002401:Cathepsin B / Q000032:analysis; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis; D003374:Coumarins; D003546:Cysteine Endopeptidases; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000032:analysis; D010450:Endopeptidases; D004789:Enzyme Activation; D005883:Gingival Crevicular Fluid / Q000201:enzymology*; D005891:Gingivitis / Q000201:enzymology*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000032:analysis; D010447:Peptide Hydrolases / Q000032:analysis*; D010518:Periodontitis / Q000201:enzymology*; D013379:Substrate Specificity; D014357:Trypsin / Q000032:analysis
D003374:Coumarins; C055467:7-amino-4-trifluoromethylcoumarin; D002403:Cathepsins; D010450:Endopeptidases; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4; D010196:Pancreatic Elastase; D014357:Trypsin; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L