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1.
Specific cleavage of human type III collagen by human polymorphonuclear leukocyte elastase 总被引:11,自引:0,他引:11
C L Mainardi D L Hasty J M Seyer A H Kang 《The Journal of biological chemistry》1980,255(24):12006-12010
Purified polymorphonuclear leukocyte elastase degraded native human liver type III collagen at 27 degrees C by making a cleavage through the triple helix. The enzyme had no effect on human type I collagen. The reaction was inhibited by phenylmethanesulfonyl fluoride (PhCH2SO2F) but not by EDTA. The collagen reaction products were identical with those generated by human rheumatoid synovial collagenase when analyzed by polyacrylamide gel electrophoresis and gel filtration. NH2-trminal sequence analysis indicated that the enzyme cleaved at an isoleucyl-threonyl bond located 4 residues on the carboxyl side of the established cleavage site for animal collagenases. Therefore, it is likely that in pathologic states, type III collagen can be selectively depleted from the matrix by this enzyme. 相似文献
2.
Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins. 总被引:2,自引:0,他引:2
Bac5 and Bac7, antibiotics of the bactenecin (proline/arginine-rich peptide) family, are stored as proforms in the large granules of bovine neutrophils [Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H. and Romeo, D. (1990) J. Cell Biol. 111, 1363-1371]. These proforms have been purified to homogeneity from granule extracts by immunoaffinity and reverse-phase chromatography. While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium with minimal inhibitory concentrations of 6-12 micrograms/ml, proBac5 and proBac7 do not affect the growth of the same microorganisms, even at 500 micrograms/ml. Previous investigations have suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a neutral serine protease stored in the azurophil granules. Purified proBac5 and proBac7 were thus treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass spectrometry, and N-terminal sequence analysis. Of the three proteases, only elastase is able to catalyze the stepwise cleavage of probactenecins into the corresponding mature peptides, which have the same mass, N-terminal sequence and antibiotic activity of authentic Bac5 and Bac7. These results point to the importance of cooperation between azurophils and large granules in mounting a defense reaction. 相似文献
3.
We studied the proteolytic action in vitro of free and alpha 2-macroglobulin-bound porcine pancreatic elastase [EC 3.4.21.11] on the apolipoproteins of plasma: very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Polyacrylamide gel electrophoresis, isoelectric focusing and immunodiffusion tests of elastase-treated plasma lipoproteins revealed that apolipoprotein C-II and C-III polypeptides were more susceptible to elastase in free form than plasma apolipoproteins (A-I, A-II, B, and E). Elastase bound to alpha 2-macroglobulin did not show any such activities. 相似文献
4.
5.
Ten dextran sulphates and six chitosan sulphates of variable Mr and extent of sulphate substitution have been examined for their ability to inhibit human leukocyte elastase (HLE). All were potent partial non-competitive inhibitors of this enzyme, highest activity being obtained with compounds of large molecular weight and maximum sulphate incorporation (Ki = 5.0 X 10(-10)M]. In all cases, the dextran sulphates were more effective inhibitors than chitosan sulphates of similar size and charge, but both classes were inactive against bovine trypsin, chymotrypsin and porcine pancreatic elastase at concentrations less than 10(-4)M. The data suggest that drug binding to HLE occurs by stereospecific electrostatic interactions at site(s) removed from the catalytic reaction centre. 相似文献
6.
《Biochemical medicine》1980,23(2):219-222
The activity of human leukocyte elastase is strongly enhanced by ionic strength. For instance, with 5 m NaCl, the rate of the elastase catalyzed hydrolysis of N-succinyl-trialanine-p-nitroanilide is increased by a factor of 20. Under these conditions elastase concentrations as low as 1 nm (35 ng/ml) may be assayed accurately. Pancreatic elastase is much less sensitive to ionic strength than the leukocyte enzyme. Between 0.2 and 0.75 m NaCl, the rate of solubilization of elastin by leukocyte elastase is also strongly enhanced. 相似文献
7.
The recently reported Ki values for human leukocyte elastase and a series of trifluoroacetylated peptides are erroneous because the enzyme preparation was contaminated by a small amount of porcine pancreatic elastase. The correct Ki values are much higher. However, trifluoroacetylated peptides are still much more potent inhibitors than the corresponding acetylated peptides. 相似文献
8.
9.
Background
Neuronal cell loss contributes to the pathology of acute and chronic neurodegenerative diseases, including Alzheimer’s disease (AD). It remains crucial to identify molecular mechanisms sensitizing neurons to various insults and cell death. To date, the multifunctional, autophagy-related protein Beclin 1 has been shown to be both necessary and sufficient for neuronal integrity in neurodegenerative models associated with protein aggregation. Interestingly, besides its role in cellular homeostasis, Beclin 1 has also been ascribed a role in apoptosis. This makes it critical to elucidate whether Beclin 1 regulates neuronal death and survival across neurodegenerative conditions independent of protein clearance. Here, we provide experimental evidence for a direct functional link between proteolytic cleavage of Beclin 1 and apoptotic neuronal cell loss in two independent models of neurodegeneration in vivo.Methods
Proteolytic cleavage of Beclin 1 was characterized in lysates of human AD brain samples. We developed viral tools allowing for the selective neuronal expression of the various Beclin 1 forms, including Beclin 1 cleavage products as well as a cleavage-resistant form. The effect of these Beclin 1 forms on survival and integrity of neurons was examined in models of acute and chronic neurodegeneration in vitro and in vivo. Markers of neuronal integrity, neurodegeneration and inflammation were further assessed in a Kainic acid-based mouse model of acute excitotoxic neurodegeneration and in a hAPP-transgenic mouse model of AD following perturbation of Beclin 1 in the susceptible CA1 region of the hippocampus.Results
We find a significant increase in caspase-mediated Beclin 1 cleavage fragments in brain lysates of human AD patients and mimic this phenotype in vivo using both an excitotoxic and hAPP-transgenic mouse model of neurodegeneration. Surprisingly, overexpression of the C-terminal cleavage-fragment exacerbated neurodegeneration in two distinct models of degeneration. Local inhibition of caspase activity ameliorated neurodegeneration after excitotoxic insult and prevented Beclin 1 cleavage. Furthermore, overexpression of a cleavage-resistant form of Beclin 1 in hippocampal neurons conferred neuroprotection against excitotoxic and Amyloid beta-associated insults in vivo.Conclusions
Together, these findings indicate that the cleavage state of Beclin 1 determines its functional involvement in both neurodegeneration and neuroprotection. Hence, manipulating the cleavage state of Beclin 1 may represent a therapeutic strategy for preventing neuronal cell loss across multiple forms of neurodegeneration.10.
Human leukocyte elastase (HLE), a serine protease involved in inflammation and tissue degradation, can be irreversibly inactivated in a time- and concentration-dependent manner by ynenol lactones. Ynenol lactones that are alpha-unsubstituted do not inactivate but are alternate substrate inhibitors that are hydrolyzed by the enzyme. Ynenol lactones that are both substituted alpha to to the lactone carbonyl and unsubstituted at the acetylene terminus are rapid inactivators of HLE and inactivate pancreatic elastase and trypsin more slowly. 3-Benzyl-5(E)-(prop-2-ynylidene)tetrahydro-2-furanone inactivates HLE with biphasic kinetics and an apparent second-order rate of up to 22,000 M-1 s-1 (pH 7.8, 25 degrees C). The rate of inactivation is pH-dependent and is slowed by a competitive inhibitor. The partition ratio is 1.6 +/- 0.1. Rapid removal of ynenol lactone during the course of inactivation yields a mixture of acyl and inactivated enzyme species, which then shows a partial recovery of activity that is time- and pH-dependent. Inactivation is not reversible with hydroxylamine. The enzyme is not inactivated if the untethered allenone is added exogenously. All of these results are consistent with a mechanism involving enzyme acylation at serine-195 by the ynenol lactone, isomerization of the acyl enzyme to give a tethered allenone, and capture of a nucleophile (probably histidine-57) to inactivate the enzyme. Substitution at the acetylene terminus of ynenol lactones severely reduces their ability to inactivate HLE, because allenone formation is slowed and/or nucleophile capture is hindered. Chemical competence of each of these steps has been demonstrated [Spencer, R.W., Tam, T.F., Thomas, E.M., Robinson, V.J.,& Krantz, A. (1986) J. Am. Chem. Soc. 108, 5589-5597]. 相似文献
11.
William C. Groutas Lee S. Chong Jeffrey B. Epp Radhika Venkataraman Michael J. Brubaker Michael A. Stanga Eun-Hong Kim Charles E. Keller 《Bioorganic & medicinal chemistry letters》1992,2(12):1571-1574
The interaction of a series of derivatives of cis-N-hydroxy-3-phenyl-2-isoxazoline-4,5-dicarboximide toward human leukocyte elastase and cathepsin G was investigated. Both enzymes were rapidly acylated and the corresponding acyl enzymes exhibited variable stability. 相似文献
12.
R L Stein A M Strimpler P D Edwards J J Lewis R C Mauger J A Schwartz M M Stein D A Trainor R A Wildonger M A Zottola 《Biochemistry》1987,26(10):2682-2689
Kinetics of inhibition have been determined for the interaction of human leukocyte elastase (HLE) with two series of peptide trifluoromethyl ketones (TFMKs): X-Val-CF3,X-Pro-Val-CF3,X-Val-Pro-Val-CF3, and X-Lys(Z)-Val-Pro-Val-CF3, where X is MeOSuc or Z. These compounds are "slow-binding" inhibitors of HLE and, thus, allow the determination of Ki, the dissociation constant for the stable complex of inhibitor and enzyme, as well as kon and koff, the rate constants for formation and decomposition of this complex. Maximal potency is reached with Z-Lys(Z)-Val-Pro-Val-CF3, which displays a Ki less than 0.1 nM. Upon binding to HLE, these compounds undergo addition by the hydroxyl of the active site serine to form a hemiketal. The evidence supporting a hemiketal intermediate includes Ki values of 1.6 and 80,000 nM for Z-Val-Pro-Val-CF3 and its alcohol analogue, linear free energy correlations between inhibitory potency and catalytic efficiency for structurally related TFMKs and substrates, and the pH dependence of kon for the inhibition of HLE by Z-Val-Pro-Val-CF3, which is sigmoidal and displays a pKa of 6.9. Hemiketal formation is probably not rate limiting, however. Kinetic solvent isotope effects of unity suggest that kon cannot be rate limited by a reaction step, like hemiketal formation, that is subject to protolytic catalysis. A general mechanism that is consistent with these results is one in which formation of the hemiketal is rapid and is followed or preceded by a slow step that rate limits kon.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
O P Shevchenko O V Eremenko O G Ogloblina E I Sorochinskaia 《Biulleten' eksperimental'no? biologii i meditsiny》1988,105(1):80-82
The activity of granulocyte elastase (GE) was discovered in the preparations of leukocyte thermostable alpha-glycoprotein (LTG) isolated from pus by means of ion-exchange chromatography. The activity of GE was determined according to MeoSuc(Ala)2ProValpNa hydrolysis. The antibodies against LTG were isolated from monospecific antisera. Sepharose with immobilized fraction of pus proteins was utilized as immunosorbent. Isolated antibodies to LTG inhibited the GE activity. An inhibitory effect of antibodies increased with the increase in their concentration. The identity or binding of LTG and GE was suggested. The binding of LTG with pus protein component was discovered, the biological meaning of this phenomenon being unknown. 相似文献
14.
R L Stein A M Strimpler B R Viscarello R A Wildonger R C Mauger D A Trainor 《Biochemistry》1987,26(13):4126-4130
Valine-derived benzoxazinones have been synthesized and found to be competitive, slow-binding inhibitors of human leukocyte elastase (HLE). Steady-state inhibition constants Ki are dependent on aryl substitution and reach a maximum of potency of 0.5 nM with the 5-Cl compound 6. UV-spectral data for the interaction of HLE and the unsubstituted inhibitor 3 indicate that the stable complex formed between enzyme and inhibitor is an acyl-enzyme that can either undergo ring closure, to reform intact benzoxazinone, or hydrolysis, to liberate an N-acylanthranilic acid. "Burst" kinetic data, derived from the direct observation of the interaction of HLE and 3, are consistent with results of the inhibition of catalysis experiments. 相似文献
15.
The effects of 30-min intravenous infusions of ethanol (about 50 mm blood concentration), acetaldehyde (about 100 μm blood concentration), and acetate (equimolar dose to acetaldehyde) were studied in normal and adrenalectomized rats. Blood glucose, plasma free fatty acids (FFA), plasma immunoreactive insulin, and glucagon and hepatic glycogen concentrations were measured. Ethanol itself in the presence of 4-methylpyrazole (4-MP) produced no marked changes in the parameters measured. Its infusion without 4-MP reduced plasma insulin by 35% in the normal rats, but not in the adrenalectomized rats, with no simultaneous changes in blood glucose. Acetaldehyde infusion produced hyperglycemia and relatively slight hyperinsulinemia in the normal rats, but not in the adrenalectomized rats. Equimolar acetate was not as potent a stimulator of glycogenolysis as acetaldehyde. Plasma FFA concentrations were markedly reduced by ethanol (without 4-MP), acetaldehyde and acetate both in the normal and adrenalectomized rats, but in the presence of 4-MP ethanol was without effect. The results indicate that metabolites of ethanol (mostly acetaldehyde) produced during ethanol oxidation in vivo are responsible for the stimulation of glycogenolysis through the release of catecholamines from the adrenal glands. The ethanol-induced decrease in plasma FFA is also attributable to the metabolites of ethanol, acetaldehyde having a more potent depressing action than acetate. The mode of inhibition of lipolysis is not related to hormonal factors. 相似文献
16.
A Baici 《Biological chemistry Hoppe-Seyler》1986,367(3):245-258
Polyclonal antibodies raised in rabbits against human leukocyte elastase contained two distinct populations of enzyme-inhibiting immunoglobulins. The enzyme-catalyzed reaction in the presence of antibodies (both IgG or monovalent Fab fragments) showed a transient state lasting up to several minutes depending on the inhibitor and substrate concentrations, which was followed by a linear steady-state. The transient was a concave upward or concave downward lag phase depending on whether the enzyme had been preincubated with the antibodies or not, respectively. The kinetic analysis of reaction progress curves showed that both antibody populations were slow inhibitors, which completely and reversibly excluded the substrate from binding to the enzyme. For both antibody populations, the formation of the enzyme-inhibitor complex was characterized by an initial rapid interaction followed by a slow isomerization to a catalytically inactive complex. The apparent pseudo first-order rate constant of the transient slow phase was a hyperbolic function of the inhibitor concentration for both antibodies, from which relevant kinetic constants and the half times for enzyme inactivation could be calculated. For instance, with a total antibody concentration of 1 mg/ml (as IgG), leukocyte elastase was inactivated with t1/2 = 0.31s and 24.8s by the faster and the slower of the two antibodies, respectively. It is suggested that the hysteretic response of the enzyme to the inhibitory action of its antibodies may be due to a kind of memory of the antibody molecule for a special inactive enzyme conformation resulting from inhibition by proteinase inhibitors during the immunization procedure. In turn, the purified antibodies would be able to reversibly induce a slow transition of the enzyme molecule from an active to a substrate-excluding conformation ("induced misfit"). 相似文献
17.
Catalysis by human leukocyte elastase: mechanistic insights into specificity requirements 总被引:1,自引:0,他引:1
Steady-state kinetic parameters were determined for the human leukocyte elastase catalyzed hydrolysis of a series of peptide-based thiobenzyl esters and p-nitroanilides. The peptide units are MeOSuc-Val, MeOSuc-Alan-Pro-Val (n = 0-2), and MeOSuc-Alan-Pro-Ala (n = 1 or 2). The results of this study suggest five important mechanistic features for HLE. Few important remote subsite contacts are established in the Michaelis complex. Full recognition and tight binding of the substrate occurs in the transition state for acylation. The P3-S3 interaction is critical during acylation. Subsite contacts are unimportant in deacylation. P1 specificity is regulated by peptide length. An important steady-state kinetic consequence of this specificity is that the rate-limiting step of kc for p-nitroanilide hydrolysis changes from acylation to deacylation as the peptide chain is lengthened. 相似文献
18.
Chondroitin sulfate (Structum) interacts with human leukocyte elastase, a potent mediator of articular cartilage degradation, producing a partial inhibition of the enzyme activity (60% at saturation). Kinetically, the inhibition mechanism can be classified as simple intersecting, hyperbolic noncompetitive and is almost identical to that found earlier for similar compounds. The best inhibitory activity of chondroitin sulfate was found in fractions having at the same time a high proportion of chondroitin-6-sulfate relative to the corresponding 4-isomer and a high molecular mass. Thus, a fraction with high Mr and containing 92% of isomer 6 inhibited leukocyte elastase with Ki = 1.8 micrograms/ml, whereas a fraction with low Mr and almost equal composition of the 4- and 6-isomer had Ki = 140 micrograms/ml. Ki for unfractionated chondroitin sulfate was 3.4 micrograms/ml. It is suggested, that the modulation of the extracellular activity of cartilage-degrading enzymes by cartilage-derived factors may explain, at least in part, the beneficial effects of some therapeutically used chondroprotective agents. 相似文献
19.
W B Knight A L Maycock B G Green B M Ashe P Gale H Weston P E Finke W K Hagmann S K Shah J B Doherty 《Biochemistry》1992,31(21):4980-4986
The cephalosporin derivatives L 658758 [1-[[3-(acetoxymethyl)-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-en-2-yl]carbonyl]proline S,S-dioxide] and L 659286 [1-[[7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo- 1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-aza-(6R)-bicyclo[4.2.0]-o ct-2-en-2-yl]carbonyl]pyrrolidine S,S-dioxide] are mechanism based inhibitors of human leukocyte elastase (HLE). The mechanism involves initial formation of a Michaelis complex followed by acylation of the active site serine. The group on the 3'-methylene is liberated during the course of these reactions, followed by partitioning of an intermediate between hydrolysis to regenerate active enzyme and further modification to produce a stable HLE-inhibitor complex. The partition ratio of 2.0 obtained for the reaction with L 658758 approaches that of an optimal inhibitor. These compounds are functionally irreversible inhibitors as the recovery of activity after inactivation is slow. The half-lives at 37 degrees C of the L 658758 and L 659286 derived HLE-I complexes were 9 and 6.5 h, respectively. The complexes produced by both inhibitors are similar chemically since the thermodynamic parameters for activation to regenerate active enzyme are essentially identical. The free energy of activation for this process is dominated primarily by the enthalpy term. The stability of the final complexes likely arises from Michael addition on the active site histidine to the 3'-methylene. 相似文献