Detection of cathepsin B‐ and L‐, elstase‐, 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

Abstract

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.

Purification and Characterization of a Novel Cysteine Proteinase (Periodontain) from Porphyromonas gingivalis

“…Despite the importance of these bacterial proteinases, the abundance of host-derived metalloproteinases and serine proteinases, including active HNE, in the gingival crevicular fluid of individuals with severe periodontitis, is still believed to be the primary factor responsible for much of the extracellular matrix destruction that occurs in this disease (36). The concentration of HNE within the neutrophil is near 3.0 M, and it is likely to be as high as 268 M at sites of inflammation (37), such as those that occur during the development of another major connective tissue disease, pulmonary emphysema.…”

Cystatin F Is a Glycosylated Human Low Molecular Weight Cysteine Proteinase Inhibitor

“…They should therefore serve a protective function to regulate the activities of such endogenous proteinases, which otherwise may cause uncontrolled proteolysis and tissue damage. Cysteine proteinase activity can not normally be measured in body fluids, but can be detected extracellularly in conditions like endotoxin-induced sepsis (2) and metastasizing cancer (3) and at local inflammatory processes such as in rheumatoid arthritis (4), purulent bronchiectasis (5), and periodontitis (6), which indicates that a tight enzyme regulation by cystatins is a necessity in the normal state. A deficiency state in which the levels of the intracellular cystatin, cystatin B, are lowered due to mutations has recently been shown to segregate with a form of progressive myoclonus epilepsy (7), which points to additional specialized functions of cystatins.…”

Dipeptidyl Aminopeptidase IV fromStenotrophomonas maltophiliaExhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

“…The crystal structures of human, porcine, and rat DPIV enzymes have been determined (14,21,35,47). Although DPIV enzymes from bacteria and fungi, including Flavobacterium meningosepticum (56), Bacteroides fragilis (32), Prevotella intermedia (50), Porphyromonas gingivalis (11), Trichophyton rubrum (41), Lactobacillus helveticus (37), Aspergillus fumigatus (5), Aspergillus oryzae (52), etc., have been identified, no structural information has been reported regarding microbial DPIV. Some of these are known to be pathogenic bacteria, causing diseases such as athlete's foot (T. rubrum), periodontitis (P. intermedia and P. gingivalis), and a variety of opportunistic infectious diseases (F. meningosepticum, A. fumigatus, and B. fragilis).…”