scholarly journals Characterization of the active site of human multicatalytic proteinase

1990 ◽  
Vol 265 (2) ◽  
pp. 479-484 ◽  
Author(s):  
R W Mason
Keyword(s):  
Cysteine Proteinase ◽  
Tight Binding ◽  
Serine Proteinase ◽  
Activity Profile ◽  
Reversible Inhibitors ◽  
Multicatalytic Proteinase ◽  
Proteinase Inhibition ◽  
Kinetics Of ◽  
Specific Inhibitors

The activity of multicatalytic proteinase against synthetic substrates and the kinetics of its inhibition by a range of class-specific inhibitors have been investigated. The enzyme was found to have a broader pH activity profile than previously noted, being active against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin optimally at pH 4.5 and against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin optimally at pH 10.5. Neither activity was inhibited by the class-specific inhibitors 1,10-phenanthroline, EDTA, pepstatin, di-isopropyl fluorophosphate, peptidyl chloromethanes, peptidyl diazomethanes or L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64), indicating that the enzyme is not a typical metallo-, aspartic, serine or cysteine proteinase. Inhibition by HgCl2, iodoacetamide and N-ethylmaleimide suggests that free thiols are necessary for the enzyme to maintain activity, but that these thiols are not particularly reactive as is the case for cysteine proteinases of the papain superfamily. The peptidyl aldehydes chymostatin and leupeptin were found to be reversible inhibitors of multicatalytic proteinase. Chymostatin inhibited activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin at pH 4.5 (Ki 160 +/- 22 microM) whereas leupeptin (200 microM) was not inhibitory. Inhibition of activity against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin by these compounds was more complex, in that they behaved as slow tight-binding inhibitors. kon values were determined to be 12 +/- 2 M-1.s-1 and 1290 +/- 125 M-1.s-1 for chymostatin and leupeptin, respectively. The upper limit for Ki values for these two inhibitors was estimated as 5 +/- 1.5 microM and 25 +/- 5 nM, respectively. The different inhibition characteristics for each substrate were also apparent at an intermediate pH of 8.5, showing that the two activities are distinct. Dichloroisocoumarin, a mechanism-based inhibitor of serine proteinases, did inhibit activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin with a rate constant of 250 M-1.s-1, suggesting that multicatalytic proteinase is an atypical serine proteinase.

Characterization of Cystatin C from Bovine Parotid Glands: Cysteine Proteinase Inhibition and Antiviral Properties

1996 ◽  
Vol 377 (1) ◽  
pp. 19-24 ◽  
Author(s):  
Nina Cimerman ◽  
Marinka Drobnič Košorok ◽  
Bruce D. Korant ◽  
Boris Turk ◽  
Vito Turk
Keyword(s):  
Cystatin C ◽  
Cysteine Proteinase ◽  
Parotid Glands ◽  
Proteinase Inhibition

scholarly journals Purification and characterization of a tetrameric α-macroglobulin proteinase inhibitor from the gastropod mollusc Biomphalaria glabrata

1996 ◽  
Vol 316 (3) ◽  
pp. 893-900 ◽  
Author(s):  
Randall C. BENDER ◽  
Christopher J. BAYNE
Keyword(s):  
Quaternary Structure ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Serine Proteinase ◽  
Biomphalaria Glabrata ◽  
Sedimentation Equilibrium ◽  
Flight Mass Spectrometry ◽  
Isolation And Characterization ◽  
Α2 Macroglobulin

The α-macroglobulin proteinase inhibitors (αMs) are a family of proteins with the unique ability to inhibit a broad spectrum of proteinases. Whereas monomeric, dimeric and tetrameric αMs have been identified in vertebrates, all invertebrate αMs characterized so far have been dimeric. This paper reports the isolation and characterization of a tetrameric αM from the tropical planorbid snail Biomphalaria glabrata. The sequence of 18 amino acids at the N-terminus indicates homology with other αMs. The subunit mass of approx. 200 kDa was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and SDS/PAGE. The quaternary structure was determined by sedimentation equilibrium centrifugation and native pore-limit electrophoresis. Evidence for a thioester is provided by the fact that methylamine treatment prevents the autolytic cleavage of the snail αM subunit and results in the release of 4 mol of thiols per mol of snail αM. The snail αM inhibited the serine proteinase trypsin, the cysteine proteinase bromelain and the metalloproteinase thermolysin. The spectrum of proteinases inhibited, together with the demonstration of steric protection of the proteinase active site and a ‘slow to fast’ conformational change after reacting with trypsin, all suggest that the inhibitory mechanism of the snail αM is similar to the ‘trap mechanism’ of human α2-macroglobulin.

Characterization of three types of calcium channel in the luminal membrane of the distal nephron

2004 ◽  
Vol 82 (1) ◽  
pp. 30-37 ◽  
Author(s):  
M G Brunette ◽  
M Leclerc ◽  
D Couchourel ◽  
J Mailloux ◽  
Y Bourgeois
Keyword(s):  
Calcium Transport ◽  
Distal Tubule ◽  
Distal Nephron ◽  
High Affinity ◽  
Luminal Membrane ◽  
Kinetics Of ◽  
Substrate Concentrations ◽  
Renal Calcium Transport ◽  
Specific Inhibitors

We previously reported a dual kinetics of Ca2+ transport by the distal tubule luminal membrane of the kidney, suggesting the presence of several types of channels. To better characterize these channels, we examined the effects of specific inhibitors (i.e., diltiazem, an L-type channel; ω-conotoxin MVIIC, a P/Q-type channel; and mibefradil, a T-type channel antagonist) on 0.1 and 0.5 mM Ca2+ uptake by rabbit nephron luminal membranes. None of these inhibitors influenced Ca2+ uptake by the proximal tubule membranes. In contrast, in the absence of sodium (Na+), the three channel antagonists decreased Ca2+ transport by the distal membranes, and their action depended on the substrate concentrations: 50 µM diltiazem decreased 0.1 mM Ca2+ uptake from 0.65 ± 0.07 to 0.48 ± 0.06 pmol·µg–1·10 s–1 (P < 0.05) without influencing 0.5 mM Ca2+ transport, whereas 100 nM ω-conotoxin MVIIC decreased 0.5 mM Ca2+ uptake from 1.02 ± 0.05 to 0.90 ± 0.05 pmol·µg–1·10 s–1 (P < 0.02) and 1 µM mibefradil decreased it from 1.13 ± 0.09 to 0.94 ± 0.09 pmol·µg–1·10 s–1 (P < 0.05); the latter two inhibitors left 0.1 mM Ca2+ transport unchanged. Diltiazem decreased the Vmax of the high-affinity channels, whereas ω-conotoxin MVIIC and mibefradil influenced exclusively the Vmax of the low-affinity channels. These results not only confirm that the distal luminal membrane is the site of Ca2+ channels, but they suggest that these channels belong to the L, P/Q, and T types.Key words: renal calcium transport, calcium channels, diltiazem, mibefradil, ω-conotoxin.

Identification and characterization of the cysteine and serine proteinases of the trematode,Haplometra cylindraceaand determination of their haemoglobinase activity

Parasitology ◽  
1994 ◽  
Vol 108 (5) ◽  
pp. 595-601 ◽  
Author(s):  
S. J. Hawthorne ◽  
D. W. Halton ◽  
B. Walker
Keyword(s):  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Serine Proteinase ◽  
Hydrolytic Activity ◽  
Molecular Weights ◽  
Affinity Reagent ◽  
Turn Over

SummaryThe excreted/secreted proteinases ofHaplometra cylindraceamaintainedin vitro, were found to hydrolyse the fluorogenic substrates, Z-ArgArg-NHMec and Z-PheArg-NHMec. This activity was shown to be typically that of cysteine proteinases, as turn-over of both substrates could be blocked by pre-incubation with peptidyl diazomethyl ketones. The biotinylated affinity reagent, biotin-Phe Ala-DMK, used in combination with Z-PheTyr(OBut)-DMK, was employed for the labelling and characterization of these cysteine proteinase activities. Three cathepsin B-like species were detected, with molecular weights of 48, 22–23 and 14 kDa, together with a cathepsin L-like enzyme, with a molecular weight of 55 kDa. The proteinases were also found to have hydrolytic activity towards the substrate, Z-GlyGlyArg-NHMec, which could be blocked by pre-incubation with either of the serine proteinase-selective reagents, Z-ArgP(OPh)2, or biotin-LysP(OPh)2, showing the activity to be trypsin-like. Using the biotinylated affinity label to characterize the trypsin-like enzymes revealed two molecular species with molecular weights of 20 and 24 kDa.

scholarly journals Inactivation of papain by antithrombin due to autolytic digestion: a model of serpin inactivation of cysteine proteinases

1998 ◽  
Vol 335 (3) ◽  
pp. 701-709 ◽  
Author(s):  
Ingemar BJÖRK ◽  
Kerstin NORDLING ◽  
Elke RAUB-SEGALL ◽  
Ulf HELLMAN ◽  
Steven T. OLSON
Keyword(s):  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Serine Proteinase ◽  
Peptide Bond ◽  
Enzyme Inactivation ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
Sds Page ◽  
Reaction Proceeds ◽  
Proteinase Inhibition

Cross-class inhibition of cysteine proteinases by serpins differs from serpin inhibition of serine proteinases primarily in that no stable serpin–cysteine proteinase complex can be demonstrated. This difference in reaction mechanism was elucidated by studies of the inactivation of the cysteine proteinases, papain and cathepsin L, by the serpin antithrombin. The two proteinases were inactivated with second-order rate constants of (1.6±0.1)×103 and (8.6±0.4)×102 M-1·s-1 respectively. An antithrombin to papain inactivation stoichiometry of ∼ 3 indicated extensive cleavage of the inhibitor concurrent with enzyme inactivation, a behaviour verified by SDS/PAGE. N-terminal sequence analyses showed cleavage predominantly at the P2–P1 bond, but also at the P2´–P3´ bond of antithrombin. The papain band in SDS/PAGE progressively disappeared on reaction of the enzyme with increasing amounts of antithrombin, but no band representing a stable antithrombin–papain complex appeared. SDS/PAGE with 125I-labelled papain showed that the disappearance of papain was caused by cleavage of the enzyme into small fragments. These results suggest a mechanism in which papain attacks a peptide bond in the reactive-bond loop of antithrombin adjacent to that involved in serine proteinase inhibition. The reaction proceeds, similarly to that between serpins and serine proteinases, to form an inactive acyl-intermediate complex, although with the substrate pathway dominating in the papain reaction. In this complex, papain is highly susceptible to proteolysis and is degraded by still active papain, which greatly decreases the lifetime of the complex and results in liberation of fragmented, inactive enzyme. This model may have relevance also for the inactivation of physiologically or pathologically important cysteine proteinases by serpins.

Irradiation behavior of pyrolytic silicon carbide

1983 ◽  
Vol 41 ◽  
pp. 72-73
Author(s):  
R. J. Lauf
Keyword(s):  
Silicon Carbide ◽  
Fission Products ◽  
Thermodynamics And Kinetics ◽  
Neutron Fluences ◽  
Fuel Particles ◽  
Sic Coatings ◽  
Irradiation Behavior ◽  
Kinetics Of ◽  
Htgr Fuel

Fuel particles for the High-Temperature Gas-Cooled Reactor (HTGR) contain a layer of pyrolytic silicon carbide to act as a miniature pressure vessel and primary fission product barrier. Optimization of the SiC with respect to fuel performance involves four areas of study: (a) characterization of as-deposited SiC coatings; (b) thermodynamics and kinetics of chemical reactions between SiC and fission products; (c) irradiation behavior of SiC in the absence of fission products; and (d) combined effects of irradiation and fission products. This paper reports the behavior of SiC deposited on inert microspheres and irradiated to fast neutron fluences typical of HTGR fuel at end-of-life.

Elaboration and Characterization of the Properties of Refractory Cr Base Alloys

2010 ◽  
Vol 72 ◽  
pp. 46-52 ◽  
Author(s):  
Laurent Royer ◽  
Stéphane Mathieu ◽  
Christophe Liebaut ◽  
Pierre Steinmetz
Keyword(s):  
Mechanical Properties ◽  
Melting Point ◽  
Molten Glass ◽  
Energy Production ◽  
Creep Properties ◽  
Refractory Alloys ◽  
Industrial Use ◽  
Kinetics Of ◽  
Selection Of

For energy production and also for the glass industry, finding new refractory alloys which could permit to increase the process temperatures to 1200°C or more is a permanent challenge. Chromium base alloys can be good candidates, considering the melting point of Cr itself, and also its low corrosion rate in molten glass. Two families of alloys have been studied for this purpose, Cr-Mo-W and Cr-Ta-X alloys (X= Mo, Si..). A finer selection of compositions has been done, to optimize their chemical and mechanical properties. Kinetics of HT oxidation by air, of corrosion by molten glass and also creep properties of several alloys have been measured up to 1250°C. The results obtained with the best alloys (Cr-Ta base) give positive indications as regards the possibility of their industrial use.

scholarly journals New Trimethoprim-Like Molecules: Bacteriological Evaluation and Insights into Their Action

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 709
Author(s):  
Marta Jorba ◽  
Marina Pedrola ◽  
Ouldouz Ghashghaei ◽  
Rocío Herráez ◽  
Lluis Campos-Vicens ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Synergistic Effects ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Detailed Characterization ◽  
Low Concentrations ◽  
Pump Activity ◽  
Bacteriological Evaluation ◽  
Kinetics Of

This work reports a detailed characterization of the antimicrobial profile of two trimethoprim-like molecules (compounds 1a and 1b) identified in previous studies. Both molecules displayed remarkable antimicrobial activity, particularly when combined with sulfamethoxazole. In disk diffusion assays on Petri dishes, compounds 1a and 1b showed synergistic effects with colistin. Specifically, in combinations with low concentrations of colistin, very large increases in the activities of compounds 1a and 1b were determined, as demonstrated by alterations in the kinetics of bacterial growth despite only slight changes in the fractional inhibitory concentration index. The effect of colistin may be to increase the rate of antibiotic entry while reducing efflux pump activity. Compounds 1a and 1b were susceptible to extrusion by efflux pumps, whereas the inhibitor phenylalanine arginyl β-naphthylamide (PAβN) exerted effects similar to those of colistin. The interactions between the target enzyme (dihydrofolate reductase), the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH), and the studied molecules were explored using enzymology tools and computational chemistry. A model based on docking results is reported.

scholarly journals Simultaneous Kinetics of Selenite Oxidation and Sorption on δ-MnO2 in Stirred-Flow Reactors

2021 ◽  
Vol 18 (6) ◽  
pp. 2902
Author(s):  
Zheyong Li ◽  
Yajun Yuan ◽  
Lin Ma ◽  
Yihui Zhang ◽  
Hongwei Jiang ◽  
...  
Keyword(s):  
Rate Constant ◽  
Photoelectron Spectroscopy ◽  
Oxide Surfaces ◽  
Kinetic Rate Constant ◽  
Flow Reactors ◽  
Mn Oxide ◽  
Remediation Strategies ◽  
Process Of Se ◽  
Kinetics Of

Selenium (Se) is an essential and crucial micronutrient for humans and animals, but excessive Se brings negativity and toxicity. The adsorption and oxidation of Se(IV) on Mn-oxide surfaces are important processes for understanding the geochemical fate of Se and developing engineered remediation strategies. In this study, the characterization of simultaneous adsorption, oxidation, and desorption of Se(IV) on δ-MnO2 mineral was carried out using stirred-flow reactors. About 9.5% to 25.3% of Se(IV) was oxidized to Se(VI) in the stirred-flow system in a continuous and slow process, with the kinetic rate constant k of 0.032 h−1, which was significantly higher than the apparent rate constant of 0.0014 h−1 obtained by the quasi-level kinetic fit of the batch method. The oxidation reaction was driven by proton concentration, and its rate also depended on the Se(IV) influent concentration, flow rate, and δ-MnO2 dosage. During the reaction of Se(IV) and δ-MnO2, Mn(II) was produced and adsorbed strongly on Mn oxide surfaces, which was evidenced by the total reflectance Fourier transform infrared (ATR-FTIR) results. The X-ray photoelectron spectroscopy (XPS) data indicated that the reaction of Se(VI) on δ-MnO2 produced Mn(III) as the main product. These results contribute to a deeper understanding of the interface chemical process of Se(IV) with δ-MnO2 in the environment.

scholarly journals Synthesis and Polymerization Kinetics of Rigid Tricyanate Ester

Polymers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1686
Author(s):  
Andrey Galukhin ◽  
Roman Nosov ◽  
Ilya Nikolaev ◽  
Elena Melnikova ◽  
Daut Islamov ◽  
...  
Keyword(s):  
Kinetic Analysis ◽  
Single Step ◽  
Polymerization Kinetics ◽  
Scanning Calorimetry ◽  
Modulated Differential Scanning Calorimetry ◽  
Diffusion Controlled ◽  
Polymerization Product ◽  
The One ◽  
Kinetics Of

A new rigid tricyanate ester consisting of seven conjugated aromatic units is synthesized, and its structure is confirmed by X-ray analysis. This ester undergoes thermally stimulated polymerization in a liquid state. Conventional and temperature-modulated differential scanning calorimetry techniques are employed to study the polymerization kinetics. A transition of polymerization from a kinetic- to a diffusion-controlled regime is detected. Kinetic analysis is performed by combining isoconversional and model-based computations. It demonstrates that polymerization in the kinetically controlled regime of the present monomer can be described as a quasi-single-step, auto-catalytic, process. The diffusion contribution is parameterized by the Fournier model. Kinetic analysis is complemented by characterization of thermal properties of the corresponding polymerization product by means of thermogravimetric and thermomechanical analyses. Overall, the obtained experimental results are consistent with our hypothesis about the relation between the rigidity and functionality of the cyanate ester monomer, on the one hand, and its reactivity and glass transition temperature of the corresponding polymer, on the other hand.

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