Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.37 (neutrophil elastase)
 4,078 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM.
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PMID:High pressure gel-permeation assay for the proteolysis of human aggrecan by human stromelysin-1: kinetic constants for aggrecan hydrolysis. 141 2

Bovine nasal septum aggrecan and selected proteinase-digested products of aggrecan were evaluated in an inhibition ELISA using the anti-keratan sulfate (KS) monoclonal antibody 5-D-4 (5D4). Undegraded aggrecan was recognized with an IC50 of 0.27 microgram/ml. When aggrecan was treated with human stromelysin (SLN), human leukocyte elastase (HLE), or papain, the degradation fragments had different hydrodynamic sizes. Treatment with SLN produced the largest fragments, HLE generated intermediate fragments, and papain the smallest fragments. Whereas degradation of aggrecan by SLN had little effect on recognition of proteoglycan in the ELISA (IC50-0.5 microgram/ml), degradation by both HLE and papain significantly decreased the sensitivity for detection of KS epitope (IC50-700 and 215 micrograms/ml, respectively). In addition, 5D4 detected single chain costal and corneal KS with much less sensitivity (IC50-21 and 469 micrograms/ml, respectively) than undegraded aggrecan (IC50-0.27 microgram/ml).
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PMID:Sensitivity of monoclonal antibody, 5-D-4, for the detection of aggrecan, aggrecan fragments, and keratan sulfate. 750 1

In this study, we evaluated the in vitro and in vivo potency of human leukocyte elastase (HLE) and human cathepsin G (HCG) as proteoglycanases. In vitro evaluation was done using bovine nasal septum aggrecan and aggrecan/hyaluronan aggregate as substrates. Enzyme activity was assessed by the ability of the proteinases to abrogate the ability of aggrecan to aggregate with hyaluronan. In vivo activity of the proteinases was tested by injecting purified HLE and HCG intra-articularly into rabbit stifle joints and quantifying the levels of proteoglycan released into synovial fluids. On a molar basis, HCG was at least tenfold more potent than HLE as a proteoglycanase in vitro. Moreover, HCG was twofold more potent as a proteoglycanase in vivo. In contrast, HLE hydrolyzed elastin approximately 22-fold faster than HCG, but was only slightly more rapid than HCG when [3H]-transferrin was used as substrate. These data indicate that HCG is more potent than HLE as a proteoglycanase both in vitro and in vivo. Thus, HCG could be more important in the pathogenesis of rheumatoid arthritis than previously suspected.
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PMID:Comparison of the proteoglycanolytic activities of human leukocyte elastase and human cathepsin G in vitro and in vivo. 814 41

The objective of this study was to compare the specificity and potency of recombinant human SLN-1 (rhSLN) and human leukocyte elastase (HLE) as proteoglycan (PG)-degrading enzymes after intraarticular injection into rabbits. Another objective was to evaluate the elicitation of a rhSLN-induced hyaluronan-binding region (HABR) fragment from rabbit aggrecan in joints using a polyclonal antiserum (anti-FVDIPEN) against the synthetic peptide, Phe-Val-Asp-Ile-Pro-Glu-Asn (FVDIPEN). The intraarticular injection of either activated rhSLN or HLE resulted in enzyme-specific quantitative release of PG fragments into synovial fluid. Based on the criteria used herein, HLE appears to be a more potent PG-degrading enzyme than SLN. Intraarticular injection of rhSLN also resulted in time- and dose-dependent release of a new HABR fragment of aggrecan (HABR-FMDIPEN) into both articular cartilage and synovial fluid. HABR-FVDIPEN is likely to be a good marker of matrix metalloproteinase (MMP)-induced degradation of aggrecan.
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PMID:Proteoglycan-degrading activity of human stromelysin-1 and leukocyte elastase in rabbit joints. Quantitation of proteoglycan and a stromelysin-induced HABR fragment of aggrecan in synovial fluid and cartilage. 883 47

The present studies deal with polymorphonuclear neutrophil (PMN) adhesion inhibitory properties of cartilage surface proteoglycans. Normal human PMN were used in adhesion experiments with bovine cartilage surfaces exposed to neutrophil elastase and reconstituted with fibronectin (Fn) or on plastic-bound Fn. An extract of cartilage surface small proteoglycans (SE) and purified fibromodulin (FM), decorin (DCN), biglycan (BGN), and aggrecan (AGN) on the surface of normal cartilage were used to test for inhibition of Fn-dependent cell adhesion. The PMN did not adhere to intact articular cartilage surfaces, whereas significant adhesion was measured using cartilage explants digested with elastase and reconstituted with Fn. Incubation of elastase-treated, Fn-reconstituted cartilage with 45 microg/ml SE inhibited PMN adhesion by 50.7 +/- 5.8% (P < 0.0001). Addition of 50 microg/ml purified FM to the reconstituted articular surfaces inhibited cell adhesion by 71.2 +/- 13.9% (P < 0.0001). Inhibition of PMN adhesion to plastic-bound Fn was seen with 1.7 microg/ml SE (20.4 +/- 8.0%). Maximal inhibition of 67.4 +/- 14.8% (P < 0.01) was obtained with 17.0 microg/ml SE. With FM, concentrations of 4.3 microg/ml resulted in 34.7 25.2 inhibition (P < 0.001), and maximal inhibition of 66.3 16.2% (P < 0.01) was obtained with 43.0 microg/ml. Similar results were obtained with purified bovine DCN and BGN. The main component of cartilage matrix, AGN, failed to inhibit cell adhesion significantly. The results indicate that macromolecules normally present on articular cartilage surfaces act as a barrier to PMN adhesion. Since cartilage surface proteins are susceptible to breakdown by proteases from synovial fluid inflammatory cells, we postulate that the degradation of this barrier may be responsible for increasing PMN adhesion and subsequent cartilage damage in inflammatory arthritis.
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PMID:Polymorphonuclear leukocyte adhesion to articular cartilage is inhibited by cartilage surface macromolecules. 1151 37

Pentosan polysulfate (NaPPS) and chondroitin sulfates (ChSs) have recently been shown to exhibit both symptom and disease modifying activities in osteoarthritis (OA), but their respective mechanisms of action are still the subject of conjecture. Excessive catabolism of joint articular cartilage is considered to be responsible for the initiation and progression of OA but the abilities of these drugs to mitigate this process has received only limited attention. Human neutrophil elastase (HNE) is a proteinase, which can degrade the collagens and proteoglycans (PGs) of the cartilage directly or indirectly by activating latent matrix metalloproteinases. Hyaluronidase (HAase) is an endoglycosidase, which degrades glycosaminoglycans including hyaluronan, which provides the aggregating component of the PG aggrecan complex. In the present study the molecular interactions between the NaPPS, ChSs and some other sulfated polysaccharides with immobilized HNE, HAase or lysozyme (a cationic protein implicated in PG metabolism) were studied using a SPR biosensor device-BIAcore2000. The above three enzymes were covalently immobilized to a biosensor chip CM5 separately using amine coupling. The binding affinity of each sulfated polysaccharide and the kinetics of NaPPS over the concentration range of 0.3-5.0 microg/ml were determined. The inhibition of HNE by the sulfated polysaccharides as determined using the synthetic substrate succinyl-Ala-Ala-Val-nitroanilide (SAAVNA) in a functional assay was compared with their respective binding affinities for this proteinase using the BIAcore system. The results obtained with the two independent techniques showed good correlation and indicated that the degree and ring positions of oligosaccharide sulfation were major determinants of enzyme inhibitory activity. The observed difference in order of binding affinities of the drugs to the immobilized HNE, HAase and lysozyme suggests a conformational relationship, in addition to the charge interactions between the sulfate esters of the polysaccharides and the cationic amino acids of the enzymes. Significantly, the SPR biosensor technology demonstrated that small differences among sulfated polysaccharides, even subtle variations among different NaPPS batches, could be readily detected. The SPR technology therefore offers not only a sensitive and reproducible method for ranking noncompetitive enzyme inhibitors for drug discovery but a rapid and quantitative bioassay for monitoring batch consistency of manufacture.
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PMID:Biosensor analysis of the molecular interactions of pentosan polysulfate and of sulfated glycosaminoglycans with immobilized elastase, hyaluronidase and lysozyme using surface plasmon resonance (SPR) technology. 1256 52

In articular cartilage, the extracellular matrix (ECM) and chondrocyte-associated pericellular matrix (PCM) are characterized by a high concentration of proteoglycans (PGs) and their associated glycosaminoglycans (GAGs). These molecules serve important biochemical, structural, and biomechanical roles in the tissue and differences in their regional distributions suggest that different GAG/PG species contribute to the specific biomechanical properties of the ECM and PCM. The objective of this study was to investigate region-specific contributions of aggrecan, chondroitin and dermatan sulfate, and hyaluronan to the micromechanical properties of articular cartilage PCM and ECM in situ. Cryosections of porcine cartilage underwent digestion with ADAMTS-4, chondroitinase ABC, bacterial hyaluronidase or human leukocyte elastase. Guided by immunofluorescence for type VI collagen, AFM stiffness mapping was used to evaluate the elastic properties of matched PCM and ECM regions in paired control and digested cartilage sections. These methods were used to test the hypotheses that specific enzymatic digestion of GAGs or PGs would reduce both PCM and ECM elastic moduli. Elastase, which digests a number of PGs, some types of collagen, and non-collagenous proteins, was used as a positive control. ECM elastic moduli were significantly reduced by all enzyme treatments. However, PCM micromechanical properties were unaffected by enzymatic digestion of aggrecan, chondroitin/dermatan sulfate, and hyaluronan but were significantly reduced by 24% following elastase digestion. Our results provide new evidence for high resistance of PCM micromechanical properties to PG digestion and suggest a potential role for elastase in the degradation of the ECM and PCM.
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PMID:High resistance of the mechanical properties of the chondrocyte pericellular matrix to proteoglycan digestion by chondroitinase, aggrecanase, or hyaluronidase. 2415 81