PubMed · 14134335
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L M ISHIMOVA. 1963. [SOME CURRENT PROBLEMS IN ALLERGY].. https://pubmed.ncbi.nlm.nih.gov/14134335/
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A proteolytic enzyme, Php-B ( Photorhabdus protease B), was purified from the entomopathogenic bacterium, Photorhabdus luminescens. The enzyme is intracellular, and its molecular mass is 74 kDa. Tested on various peptide and oligopeptide substrates, Php-B hydrolysed only oligopeptides, with significant activity against bradykinin and a 2-furylacryloyl-blocked peptide, Fua-LGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat=3.6x10(2) s(-1), K(m)=5.8x10(-5) M(-1), pH optimum approx. 7.0). The p K(a1) and the p K(a2) values of the enzyme activity (6.1 and 7.9 respectively), as well as experiments with enzyme inhibitors and bivalent metal ions, suggest that the activity of Php-B is dependent on histidine and cysteine residues, but not on serine residues, and that it is a metalloprotease, which most probably uses Zn2+ as a catalytic ion. The enzyme's ability to cleave oligopeptides that contain a sequence similar to collagen repeat (-Pro-Xaa-Gly-), bradykinin and Fua-LGPA (a synthetic substrate for bacterial collagenases and collagen peptidases), but not native collagens (types I and IV) or denatured collagen (gelatin), indicates that Php-B is probably a collagen peptidase, the first enzyme of this type to be identified in an insect pathogen, that might have a role in the nutrition of P. luminescens by degrading small collagen fragments. For the determination of enzyme kinetic constants, we fitted a numerically integrated Michaelis-Menten model to the experimental progress curves. Since this approach has not been used before in the characterization of proteases that are specific for the P1'-P4' substrate sites (e.g. collagenolytic enzymes), we present a comparison of this method with more conventional ones. The results confirm the reliability of the numerical integration method in the kinetic analysis of collagen-peptide-hydrolysing enzymes.
Human neutrophils play a pivotal role in acute inflammation. However, their capacity to generate bioactive kinin peptides has not been established as yet. We have examined the ability of neutrophil enzymes to release biologically active kinins in vitro from purified human H- and L-kininogens. Neutrophils isolated from human blood were stimulated with f-Met-Leu-Phe, thrombin, or human immunoglobulin G adsorbed to silica particles. Supernatants were incubated with iodinated kininogens, and polyacrylamide gel electrophoresis analyzed aliquots taken after a range of incubation times. A time-course analysis demonstrated that supernatants from stimulated neutrophils caused a rapid hydrolysis of both substrates, resulting in an accumulation of fragments ranging from 20 to less than 10 kDa. Radioimmunoassay (RIA) revealed that all supernatants were able to generate kinins in vitro. High-performance liquid chromatography of the generated peptides indicated that they had a retention time similar to that of bradykinin and Met-Lys-bradykinin, clearly recognized as kinin peptides when the corresponding fractions were tested by RIA. The kinin-immunoreactive fractions produced lowering of blood pressure and a dramatic increase in venular permeability. Biological activity of the neutrophil-generated kinins was completely abolished by the B2 receptor antagonist HOE140, indicating that over the time-course of the experiments, only kinin B2 agonists appeared to have been generated and that cellular actions of these were mediated by kinin B2 receptors. Together, our results demonstrate that human neutrophil proteases can release kinins from both plasma kininogens, suggesting that these peptides may participate actively during acute inflammation.
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