Albert Böni

Inhibition of human elastase from polymorphonuclear leucocytes by a glycosaminoglycan polysulfate (Arteparon

Abstract Human lysosomal elastase from polymorphonuclear leucocytes is inhibited by the glycosaminoglycan polysulfate Arteparon. The inhibition is of a mixed type: hyperbolic uncompetitive. The interaction between inhibitor and enzyme occurs via electrostatic forces, and the binding is very tight ( K i ranges from 10 −8 to 10 −7 M). Depending on the chain length of the polysulfated glycosaminoglycan, two, three or five enzyme molecules can be tightly bound by a single inhibitor molecule. These findings suggest a possible therapeutic role of Arteparon as an inhibitor of elastase, a potent mediator of connective tissue breakdown, since the enzyme is inhibited by a drug concentration as small as 1 μg/ml or less.

Inhibition of human elastase from polymorphonuclear leucocytes by gold sodium thiomalate and pentosan polysulfate (SP-54®)

Abstract Human lysosoma) elastase, the serine proteinase from the azurophil granules of polymorphonuclear leucocytes, is inhibited by gold thiomalate and pentosan polysulfate (SP-54®). The kinetic mechanism of the inhibition was studied using succinyl-alanyl-alanyl-prolyl-valyl-4-methyl-7-coumarylamide and (t-butyloxycarbonyl-alanyl-p-nitrophenylester as subsrates. The degree of inhibition was also tested using insoluble elastin as substrate. Independent of the substrate, the maximal inhibition of elastase by gold thiomalate and pentosan polysulfate was 40% and 60%, respectively. Pentosan polysulfate behaved as a simple intersecting, hyperbolic, non-competitive inhibitor and k′i. the dissociation constant of the E-S-I complex, was 1.8 × 10−7M. The interaction between inhibitor and enzyme is driven by electrostatic forces. Gold thiomalate showed a hyperbolic mixed type inhibition (intersecting, slope-hyperbolic, intercept-hyperbolic, non-competitive inhibition) with k′i = 5.4 × 10−5M. The overall kinetic mechanism of lysosomal elastase conforms to that of other serine proteinases. With both the ester and peptide substrates the rate limiting step of the reaction has been identified with the formation of the acyl-enzyme.

Kinetics of the Different Susceptibilities of the Four Human Immunoglobulin G Subclasses to Proteolysis by Human Lysosomal Elastase

Human lysosomal clastase cleaves human monoclonal IgG into components that closely resemble the fragments produced by papain digestion IgG1 produced Fab, Fe and Fab‐Fc fragments; cleavage of IgG2 produced Fab‐Fc, Fab and Fc fragments: IgG3 gave rise to almost pure Fab und Fch (Fc covalently Joined to the extended hinge region polypetide of IgG3). and from IgG4, F(ab)2, Fab and Fc fragments were recovered. The relative susceptibilities of the four human IgG subclasses to proteolytic attack by elastase were studied kinetically and showed the following decrease order of susceptibility: IgG3 >IgG1 >IgG2 >IgG4. The Fab fragment from papain digestion of IgG1 and the corresponding fragment from clastase digestion showed indistinguishable molecular weights and immunochemical identity

Degradation in vivo of articular cartilage in rheumatoid arthritis by leucocyte elastase from polymorphonuclear leucocytes

SummaryUsing a specific substrate, no leucocyte elastase activity could be detected in 55 synovial fluids, including 29 from patients with rheumatoid arthritis (RA). However, a high percentage of samples contained phagocytic inclusions of elastase, α1-proteinase inhibitor (α1-PI) and α2-macroglobulin (α2-MG) in both the polymorphonuclear (PMN) and mononuclear phagocytes. Immunofluorescence and indirect peroxidase-antiperoxidase staining of articular cartilage (ACA) from 52% of 21 patients with RA and one with juvenile RA (JRA) showed presence of elastase in the superficial layer of microscopically intact but proteoglycan depleted pannus-free ACA. In histologically altered pannus-free RA-ACA superficial elastase deposits were found in 24% of the cases. Adjacent ACA sections contained IgG, C 3, α1-PI and rarely α2-MG. RA-ACA below or surrounded by pannus showed close contact with intact and decaying PMN in 62% and 48% of the cases, respectively. ACA specimens from patients with degenerative disease and systemic lupus were negative. These findings strongly suggest that PMN leucocyte elastase is operative in the degradation of RA-ACA and JRA-ACA, and that this activity is largely dependent upon the presence of entrapped immune complexes in such cartilage.

Action of collagenase and elastase from human polymorphonuclear leukocytes on human articular cartilage

SummaryCollagenase from human polymorphonuclear leukocytes (neutrophil collagenase) attacks collagen type II in solution at a rate intermediate to those of type I and III collagens. This enzyme alone is not able to initiate degradation of native human articular cartilage. If the cartilage is first treated with leukocyte elastase, collagenase slowly degrades collagen.Confirming earlier findings by other investigators, elastase has a dual action on cartilage: The enzyme removes proteoglycans, thus demasking collagen fibers and giving collagenase access to them, and solubilizes collagen at a sizable rate.Although neutrophil collagenase cleaves collagen type II in solution at a high rate, the native, cross-linked status of collagen in cartilage makes it a relatively poor substrate for this enzyme. On a weight by weight scale, elastase and collagenase display about the same collagenolytic potential on human articular cartilage.The elastase/collagenase system from human polymorphonuclear leukocytes could represent a cooperative proteolytic complex in the destruction of cartilage in rheumatoid arthritis.

A handy assay for collagenase using reconstituted fluorescein-labeled collagen fibrils

Abstract This report describes an assay for measuring the activity of collagenases using reconstituted fibrils of fluorescein-labeled soluble collagen as substrate. The labeling of commercially available material is easy and not expensive. The assay is very sensitive, reproducible, and is linear with collagenase concentration up to 100% consumption of the reconstituted fibrils. Readings of collagenolytic activity can be determined directly by measuring the fluorescence of the released labeled peptides without further processing of the data.

Cleavage of human IgM with human lysosomal elastase

Abstract Human lysosomal elastase, a serine proteinase stored in the azurophil granules of polymorphonuclear leucocytes, cleaves human monoclonal IgM producing two fragments and dialyzable peptides. An F(ab) 2 μ-like fragment, called IgMe in this report, retains some reactivity with an anti-Fcμ-antiserum and is antigenically deficient with respect to both the subunit (IgMs) produced by reduction and alkylation of IgM and the similar fragment (IgMp) produced by papain digestion. The other fragment is very similar to Fabμ generated by papain digestion, as indicated by immunochemical identity and a similar molecular weight.

Partial Purification and Characterization of the High Molecular Weight Latent Collagenase from Human Leukocytes

Two latent collagenases whose apparent molecular weights were ca. 150 000 and 60 000 have been detected in human leukocytes and the partial purification of the high molecular weight component was accomplished by the following steps: acetone precipitation, ammonium sulphate fractionation, gel filtration on Sephacryl S-200, chromatography on DEAE-Sepharose, and a final gel filtration on Sephacryl S-200. After activation by an activator extracted from human rheumatoid synovial fluid, the enzyme was able to cleave collagen into the classical 1/4 and 3/4 fragments, and was inhibited by chelating agents and other typical collagenase inhibitors.

Experimental Production of Rheumatoid Factor-Like Antibodies and Antibodies Against the Cathepsin D Site of IgG Following the Injection of Autologous Fab2

In a very high proportion of rabbits, repeated intra- or extra-articular injections of autolous Fab2 produced by homologous cathepsin D induce the formation of Rf-like antibodies reacting with both homologous and human IgG. Moreover, intra-articular injections of this kind cause a significant rise in the titre of thm of all the animals so far tested. Rf-like antibodies against human IgG appear earlier and have higher serum titres than those reacting with homologous IgG. The reason for this latter observation seems to be the blocking of the anti-rabbit IgG antibodies by the animals own IgG. The anti-rabbit IgG antibodies can be absorbed only on aggregated rabbit IgG. The anti-human IgG antibodies cross-react to some extent with rabbit IgG. The results of inhibition studies suggest that the formation of anti-Fab2 homoreactants is directly stimulated by the injected Fab2, whereas the Rf-like antibodies owe their appearance to immune complexes formed in vivo by the injected Fab2 and the naturally occuring anti-Fab2 homoreactants. In respect of immunoglobulin class, the two kinds of Rf-like antibody are possibly of both IgM and IgG type.