1. A selective colorimetric assay for cathepsin L using Z-Phe-Arg-4-methoxy-beta-naphthylamide
R C Kamboj, S Pal, N Raghav, H Singh Biochimie. 1993;75(10):873-8. doi: 10.1016/0300-9084(93)90042-q.
Among the intracellular proteinases, the thiol proteinases such as cathepsin B (EC 3.4.22.1), cathepsin H (EC 3.4.22.16) and cathepsin L (EC 3.4.22.15) which act at slightly acidic pHs are more likely to play an important role in lysosomal protein catabolism. Out of these, cathepsin L plays a major role primarily because it has high degradative activity on cellular and matrix proteins. However, the studies on cathepsin L in crude homogenates and subcellular fractions have always been hampered by the lack of a specific substrate to exclusively measure the activity of this proteinase. The only synthetic substrate alpha-N-benzyloxycarbonyl-L-Phe-L-Arg-4-methoxy-beta-naphthylamide (Z-Phe-Arg-NNapOMe) which is hydrolysed by cathepsin L is hydrolysed equally well by cathepsin B. This substrate was manipulated to act as a selective substrate for cathepsin L. In presence of 4 M urea at pH 5.0, cathepsin B (the only other cathepsin which also hydrolyses Z-Phe-Arg-NNapOMe) was inactivated and, therefore, under these conditions, the enzyme activity quantitated by using this substrate is only due to cathepsin L. Using this newly-developed colorimetric assay method specific for cathepsin L, the subcellular and regional distribution of this proteinase were established in goat brain tissue. About 80% cathepsin L activity was recovered in the lysosomal fraction thus establishing its lysosomal nature. Among the various brain parts, highest activity was found in cerebrum followed by cerebellum, pituitary body, pons-varolli, thalamus, medulla-oblongata and hypothalamus.
2. A comparative study of the chymotrypsin-like activity of the rat liver multicatalytic proteinase and the ClpP from Escherichia coli
J Arribas, J G Castaño J Biol Chem. 1993 Oct 5;268(28):21165-71.
A comparative study of the chymotrypsin-like activity of the purified recombinant ClpP protease and the multicatalytic proteinase from rat liver is presented. The peptidase activity of both enzymes has been analyzed with several synthetic fluorogenic peptides, containing either aromatic or nonpolar amino acids in their P1 position. The respective Vmax, Km, and Vmax/Km were calculated from kinetic experiments. The substrate specificity of the multicatalytic proteinase, as expressed by Vmax/Km values, indicate the following substrate preference: N-Suc-IIW-MCA > N-Suc-LY-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > N-Cbz-GGL-beta-NA > Glut-GGF-beta-NA > FPAM-4-MNA. In the case of the ClpP the order of preference is: N-Suc-LY-MCA > N-Suc-IIW-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > or = N-Cbz-GGL-beta-NA > FPAM-4-MNA (where: N-Suc, N-succinyl-; MCA, 7-amido-4-methyl coumarin; beta-NA, beta-naphthylamide; N-Cbz, N-benzyloxycarbonyl-; 4-MNA, 4-methoxy-beta-naphthylamide; Glut, glutaryl. This similar substrate specificity is further supported by the lack of activity of both enzymes against SY-MCA and N-Suc-AAPF-MCA (known substrates of chymotrypsin), by very reduced activity against N-Suc-AAA-MCA and by no significant activity against LG-beta-NA. The results of mixed substrate experiments have shown that all the peptides that are substrates seem to be hydrolyzed by a single class of chymotrypsin-like site in both enzymes. The substrate specificity studies suggest a possible evolutionary relationship between the catalytic component of the ClpP of Escherichia coli and the multicatalytic proteinase chymotrypsin-like catalytic component. This conclusion is further supported by other circumstantial evidence: the fact that affinity-purified anti-ClpP antibodies cross-react with two polypeptide components of the rat liver multicatalytic proteinase complex, presented here and also shown previously; the known resemblance of both structures at the electron microscope level; and their reported role in the degradation of NH2-end rule substrates.
3. Correlations between gingival crevicular fluid enzymes and the subgingival microflora
H Suido, J J Zambon, P A Mashimo, R Dunford, R J Genco J Dent Res. 1988 Aug;67(8):1070-4. doi: 10.1177/00220345880670080201.
Bacteroides gingivalis is a Gram-negative micro-organism implicated in the pathogenesis of adult periodontitis and producing relatively large amounts of specific enzymes. In the present study, subgingival samples taken from adults with moderate periodontitis were examined for the presence and relative amounts of enzymatic activity toward certain substrates. Enzyme levels were then correlated with clinical periodontal indices and microbiological analysis of subgingival plaque, including darkfield microscopy for bacterial morphotypes and immunofluorescence microscopy for B. gingivalis and Bacteroides intermedius. The results of this study indicate a significant positive correlation between levels of enzyme capable of degrading N-benzoyl-D,L-arginine-beta-naphthylamide hydrochloride, and subgingival B. gingivalis (r = 0.55). There was a much lower correlation coefficient between this enzyme activity and subgingival B. intermedius (r = 0.26). Statistically significant (p less than 0.01) positive correlations were also demonstrated between total bacterial cell counts and levels of enzymatic activity against N-benzoyl-D,L-arginine-beta-naphthylamide hydrochloride (r = 0.76), N-carbobenzoxy-glycyl-glycyl-L-arginine-beta-naphthylamide hydrochloride (r = 0.72), and glycyl-L-proline-4-methoxy-beta-naphthylamide hydrochloride (r = 0.72), and glycyl-L-proline-4-methoxy-beta-naphthylamide hydrochloride (r = 0.69). There were significant differences in the levels of these three enzymatic activities between sites exhibiting various degrees of clinical severity of gingival inflammation and harboring various proportions of B. gingivalis. The data from this study indicate that measurement of specific enzymatic activities in subgingival samples can be useful in the diagnosis of B. gingivalis-associated periodontitis.
