scholarly journals Active-site characterization of S1 nuclease I. Affinity purification and influence of amino-group modification

1992 ◽  
Vol 285 (2) ◽  
pp. 489-494 ◽  
Author(s):  
S Gite ◽  
G Reddy ◽  
V Shankar
Keyword(s):  
Active Site ◽  
Affinity Purification ◽  
Simple Procedure ◽  
Binding Studies ◽  
S1 Nuclease ◽  
Lysine Modification ◽  
Group Modification ◽  
Substrate Protection ◽  
Kinetics Of

A simple procedure, involving heat-treatment, DEAE-Sephadex, AMP-Sepharose and Bio-Gel P-60 chromatography, was developed for the purification of S1 nuclease to homogeneity from commercially available Takadiastase powder. Chemical modification of the amino groups of purified S1 nuclease revealed that lysine is essential for single-stranded DNAase, RNAase and phosphomonoesterase activities associated with the enzyme. The kinetics of inactivation suggested the involvement of a single lysine residue in the active site of the enzyme. Additionally, lysine modification was accompanied by a concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of single-stranded DNA, RNA and 3′-AMP. Substrate-protection and inhibitor-binding studies on enzyme modified with 2,4,6-trinitrobenzenesulphonic acid showed that lysine may be involved in the substrate binding.

scholarly journals Active-site characterization of S1 nuclease. II. Involvement of histidine in catalysis

1992 ◽  
Vol 288 (2) ◽  
pp. 571-575 ◽  
Author(s):  
S Gite ◽  
G Reddy ◽  
V Shankar
Keyword(s):  
Methylene Blue ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Histidine Residue ◽  
S1 Nuclease ◽  
Modified Enzyme ◽  
Substrate Protection ◽  
Kinetics Of ◽  
Hydrolysis Of

Modification of the histidine residues of purified S1 nuclease resulted in loss of its single-stranded (ss)DNAase, RNAase and phosphomonoesterase activities. Kinetics of inactivation indicated the involvement of a single histidine residue in the catalytic activity of the enzyme. Furthermore, histidine modification was accompanied by the concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of ssDNA, RNA and 3′-AMP. Substrate protection was not observed against Methylene Blue- and diethyl pyrocarbonate (DEP)-mediated inactivation. The histidine (DEP)-modified enzyme could effectively bind 5′-AMP, a competitive inhibitor of S1 nuclease, whereas the lysine (2,4,6-trinitrobenzenesulphonic acid)-modified enzyme showed a significant decrease in its ability to bind 5′-AMP. The inability of the substrates to protect the enzyme against DEP-mediated inactivation, coupled with the ability of the modified enzyme to bind 5′-AMP effectively, suggests the involvement of histidine in catalysis.

scholarly journals Electrochemical Characterization of a Complex FeFe Hydrogenase, the Electron-Bifurcating Hnd From Desulfovibrio fructosovorans

2021 ◽  
Vol 8 ◽  
Author(s):  
Aurore Jacq-Bailly ◽  
Martino Benvenuti ◽  
Natalie Payne ◽  
Arlette Kpebe ◽  
Christina Felbek ◽  
...  
Keyword(s):  
Active Site ◽  
Enzyme Purification ◽  
Gas Diffusion ◽  
Electrochemical Characterization ◽  
High Potential ◽  
Free Atmosphere ◽  
Protein Film ◽  
Reducing Conditions ◽  
Kinetics Of

Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H2 to both the exergonic reduction of NAD+ and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases. In this study, we describe the purification of the enzyme under O2-free atmosphere and its biochemical and electrochemical characterization. Despite its complexity due to its multimeric composition, Hnd can catalytically and directly exchange electrons with an electrode. We characterized the catalytic and inhibition properties of this electron-bifurcating hydrogenase using protein film electrochemistry of Hnd by purifying Hnd aerobically or anaerobically, then comparing the electrochemical properties of the enzyme purified under the two conditions via protein film electrochemistry. Hydrogenases are usually inactivated under oxidizing conditions in the absence of dioxygen and can then be reactivated, to some extent, under reducing conditions. We demonstrate that the kinetics of this high potential inactivation/reactivation for Hnd show original properties: it depends on the enzyme purification conditions and varies with time, suggesting the coexistence and the interconversion of two forms of the enzyme. We also show that Hnd catalytic properties (Km for H2, diffusion and reaction at the active site of CO and O2) are comparable to those of standard hydrogenases (those which cannot catalyze electron bifurcation). These results suggest that the presence of the additional subunits, needed for electron bifurcation, changes neither the catalytic behavior at the active site, nor the gas diffusion kinetics but induces unusual rates of high potential inactivation/reactivation.

scholarly journals Characterization of ruthenium red-binding sites of the Ca2+-ATPase from sarcoplasmic reticulum and their interaction with Ca2+-binding sites

1992 ◽  
Vol 287 (3) ◽  
pp. 767-774 ◽  
Author(s):  
S Corbalan-Garcia ◽  
J A Teruel ◽  
J C Gomez-Fernandez
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Ruthenium Red ◽  
Cation Binding ◽  
Binding Studies ◽  
High Affinity ◽  
Cation Binding Site ◽  
Kinetics Of ◽  
Fast Release

Sarcoplasmic reticulum Ca(2+)-ATPase has previously been shown to bind and dissociate two Ca2+ ions in a sequential mode. This behaviour is confirmed here by inducing sequential Ca2+ dissociation with Ruthenium Red. Ruthenium Red binds to sarcoplasmic reticulum vesicles (6 nmol/mg) with a Kd = 2 microM, producing biphasic kinetics of Ca2+ dissociation from the Ca(2+)-ATPase, decreasing the affinity for Ca2+ binding. Studies on the effect of Ca2+ on Ruthenium Red binding indicate that Ruthenium Red does not bind to the high-affinity Ca(2+)-binding sites, as suggested by the following observations: (i) micromolar concentrations of Ca2+ do not significantly alter Ruthenium Red binding to the sarcoplasmic reticulum; (ii) quenching of the fluorescence of fluorescein 5′-isothiocyanate (FITC) bound to Ca(2+)-ATPase by Ruthenium Red (resembling Ruthenium Red binding) is not prevented by micromolar concentrations of Ca2+; (iii) quenching of FITC fluorescence by Ca2+ binding to the high-affinity sites is achieved even though Ruthenium Red is bound to the Ca(2+)-ATPase; and (iv) micromolar Ca2+ concentrations prevent inhibition of the ATP-hydrolytic capability by dicyclohexylcarbodi-imide modification, but Ruthenium Red does not. However, micromolar concentrations of lanthanides (La3+ and Tb3+) and millimolar concentrations of bivalent cations (Ca2+ and Mg2+) inhibit Ruthenium Red binding as well as quenching of FITC-labelled Ca(2+)-ATPase fluorescence by Ruthenium Red. Studies of Ruthenium Red binding to tryptic fragments of Ca(2+)-ATPase, as demonstrated by ligand blotting, indicate that Ruthenium Red does not bind to the A1 subfragment. Our observations suggest that Ruthenium Red might bind to a cation-binding site in Ca(2+)-ATPase inducing fast release of the last bound Ca2+ by interactions between the sites.

scholarly journals Parallel sample processing using dispersive INtip micro-purification on programmable multichannel pipettes

BioTechniques ◽  
2020 ◽  
Vol 68 (3) ◽  
pp. 148-154
Author(s):  
Patrick A Kates ◽  
John J Tomashek ◽  
David A Miles ◽  
L Andrew Lee
Keyword(s):  
High Throughput ◽  
Solid Phase ◽  
Affinity Purification ◽  
Manual Intervention ◽  
Parallel Sample ◽  
Spin Column ◽  
Pipette Tip ◽  
Automated Platform ◽  
Kinetics Of

Automation gives researchers the ability to process and screen orders of magnitude higher numbers of samples than manual experimentation. Current biomacromolecule separation methodologies suffer from necessary manual intervention, making their translation to high-throughput automation difficult. Herein, we present the first characterization of biomacromolecule affinity purification via dispersive solid-phase extraction in a pipette tip (INtip). We use commercially available resin and compare efficiency with batch and spin column methodologies. Moreover, we measure the kinetics of binding and evaluate resin binding capacities. INtip technology is effective on, and scalable for, an automated platform (INTEGRA ASSIST). The results suggest that high-throughput biomolecular workflows will benefit from the integration of INtip separations.

scholarly journals Generation and Characterization of a Mutant of Influenza A Virus Selected with the Neuraminidase Inhibitor BCX-140

1998 ◽  
Vol 42 (4) ◽  
pp. 801-807 ◽  
Author(s):  
Shanta Bantia ◽  
Anita A. Ghate ◽  
Sandya L. Ananth ◽  
Y. Sudhakar Babu ◽  
Gillian M. Air ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Binding Site ◽  
Active Site ◽  
Influenza A ◽  
Neuraminidase Inhibitor ◽  
Active Site Residues ◽  
Binding Studies ◽  
Drug Induced ◽  
Guanidino Group

ABSTRACT Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.

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.

Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

1986 ◽  
Vol 56 (03) ◽  
pp. 349-352 ◽  
Author(s):  
A Tripodi ◽  
A Krachmalnicoff ◽  
P M Mannucci
Keyword(s):  
Venous Thromboembolism ◽  
Active Site ◽  
Inhibitory Activity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Molecular Defect ◽  
Affinity Adsorption ◽  
Italian Family ◽  
Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

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