scholarly journals The EcoRI restriction endonuclease with bacteriophage λ DNA. Kinetic studies

1980 ◽  
Vol 191 (2) ◽  
pp. 581-592 ◽  
Author(s):  
S E Halford ◽  
N P Johnson ◽  
J Grinsted
Keyword(s):  
Restriction Endonuclease ◽  
Protein Interactions ◽  
Dna Sequences ◽  
Individual Recognition ◽  
Kinetic Studies ◽  
Restriction Enzymes ◽  
Reaction Rates ◽  
Recognition Site ◽  
Experimental Conditions ◽  
Ecori Restriction

The kinetics of the reactions of the EcoRI restriction endonuclease at individual recognition sites on the DNA from bacteriophage lambda were found to differ markedly from site to site. Under certain conditions of pH and ionic strength, the rates for the cleavage of the DNA were the same at each recognition site. But under altered experimental conditions, different reaction rates were observed at each recognition site. These results are consistent with a mechanism in which the kinetic stability of the complex between the enzyme and the recognition site on the DNA differs among the sites, due to the effect of interactions between the enzyme and DNA sequences surrounding each recognition site upon the transition state of the reaction. Reactions at individual sites on a DNA molecule containing more than one recognition site were found to be independent of each other, thus excluding the possibility of a processive mechanism for the EcoRI enzyme. The consequences of these observations are discussed with regard to both DNA-protein interactions and to the application of restriction enzymes in the study of the structure of DNA molecules.

scholarly journals The EcoRI restriction endonuclease with bacteriophage λ DNA. Equilibrium binding studies

1980 ◽  
Vol 191 (2) ◽  
pp. 593-604 ◽  
Author(s):  
S E Halford ◽  
N P Johnson
Keyword(s):  
Restriction Endonuclease ◽  
Dna Sequences ◽  
Reaction Rates ◽  
Recognition Site ◽  
Binding Studies ◽  
Dna Molecule ◽  
Recognition Sites ◽  
Equilibrium Binding ◽  
Ecori Restriction ◽  
Binding Curve

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.

scholarly journals The reactions of the EcoRi and other restriction endonucleases

1979 ◽  
Vol 179 (2) ◽  
pp. 353-365 ◽  
Author(s):  
S E Halford ◽  
N P Johnson ◽  
J Grinsted
Keyword(s):  
Restriction Endonuclease ◽  
Dna Sequences ◽  
Recognition Site ◽  
Single Strand ◽  
Restriction Endonucleases ◽  
Dna Duplex ◽  
Phosphodiester Bond ◽  
Linear Dna ◽  
Ecori Restriction ◽  
Dna Substrates

The reaction of the EcoRI restriction endonuclease was studied with both the plasmid pMB9 and DNA from bacteriophage lambda as the substrates. With both circular and linear DNA molecules, the only reaction catalysed by the EcoRI restriction endonuclease was the hydrolysis of the phosphodiester bond within one strand of the recognition site on the DNA duplex. The cleavage of both strands of the duplex was achieved only after two independent reactions, each involving a single-strand scission. The reactivity of the enzyme for single-strand scissions was the same for both the first and the second cleavage within its recognition site. No differences were observed between the mechanism of action on supercoiled and linear DNA substrates. Other restriction endonucleases were tested against plasmid pMB9. The HindIII restriction endonuclease cleaved DNA in the same manner as the EcoRI enzyme. However, in contrast with EcoRI, the Sa/I and the BamHI restriction endonucleases appeared to cleave both strands of the DNA duplex almost simultaneously. The function of symmetrical DNA sequences and the conformation of the DNA involved in these DNA–protein interactions are discussed in the light of these observations. The fact that the same reactions were observed on both supercoiled and linear DNA substrates implies that these interactions do not involve the unwinding of the duplex before catalysis.

scholarly journals The SalGI restriction endonuclease. Enzyme specificity

1982 ◽  
Vol 203 (1) ◽  
pp. 93-98 ◽  
Author(s):  
A Maxwell ◽  
S E Halford
Keyword(s):  
Restriction Endonuclease ◽  
Dna Sequences ◽  
Dna Cleavage ◽  
Competitive Inhibition ◽  
Recognition Site ◽  
Enzyme Specificity ◽  
Recognition Sequence ◽  
Kinetics Of ◽  
Endonuclease Enzyme

We have analysed the kinetics of DNA cleavage in the reaction between the SalGI restriction endonuclease and plasmid pMB9. This reaction is subject to competitive inhibition by DNA sequences outside the SalGI recognition site; we have determined the Km and Vmax. for the reaction of this enzyme at its recognition site and the KI for its interaction at other DNA sequences. We conclude that the specificity of DNA cleavage by the enzyme is only partly determined by the discrimination it shows for binding at its recognition sequence compared with binding to other DNA sequences.

Hopping, jumping and looping by restriction enzymes

2001 ◽  
Vol 29 (4) ◽  
pp. 363-373 ◽  
Author(s):  
S. E. Halford
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Cleavage ◽  
Restriction Enzymes ◽  
Test System ◽  
Recognition Site ◽  
Dna Looping ◽  
Type Ii ◽  
Common View ◽  
Recognition Sites

Type II restriction endonucleases recognize specific DNA sequences and cleave both strands of the DNA at fixed locations at or near their recognition sites. Many of these enzymes are dimeric proteins that recognize, in symmetrical fashion, palindromic DNA sequences. They generally catalyse independent reactions at each recognition site on the DNA, although in some cases they act processively; cutting the DNA first at one site, then translocating along the DNA to another site and cutting that before leaving the DNA. The way in which the degree of processivity varies with the length of DNA between the sites can reveal the mechanism of translocation. In contrast with the common view that proteins move along DNA by ‘sliding’, the principal mode of transfer of the EcoRV endonuclease is by ‘hopping’ and ‘jumping’, i.e. the dissociation of the protein from one site followed by its re-association with another site in the same DNA molecule, either close to or distant from the original site. Other type II restriction enzymes require two copies of their recognition sites for their DNA cleavage reactions. Many of these enzymes, such as SfiI, are tetramers with two DNA-binding surfaces. SfiI has no activity when bound to just one recognition site, and instead both DNA-binding surfaces have to be filled before it becomes active. Although the two sites can be on separate DNA molecules, SfiI acts optimally with two sites on the same DNA, where it traps the DNA between the sites in a loop. SfiI thus constitutes a test system for the analysis of DNA looping.

SEM analysis of the change in the reaction rates with deposit structures in the copper-iron cementation system

1978 ◽  
Vol 36 (1) ◽  
pp. 456-457
Author(s):  
V. Annamalai ◽  
L.E. Murr
Keyword(s):  
Structural Characteristics ◽  
Secondary Electron Emission ◽  
Copper Deposit ◽  
Reaction Rates ◽  
Sem Analysis ◽  
Experimental Conditions ◽  
Major Drawback ◽  
Emission Mode ◽  
Scrap Iron ◽  
Image Position

Economical recovery of copper metal from leach liquors has been carried out by the simple process of cementing copper onto a suitable substrate metal, such as scrap-iron, since the 16th century. The process has, however, a major drawback of consuming more iron than stoichiometrically needed by the reaction.Therefore, many research groups started looking into the process more closely. Though it is accepted that the structural characteristics of the resultant copper deposit cause changes in reaction rates for various experimental conditions, not many systems have been systematically investigated. This paper examines the deposit structures and the kinetic data, and explains the correlations between them.A simple cementation cell along with rotating discs of pure iron (99.9%) were employed in this study to obtain the kinetic results The resultant copper deposits were studied in a Hitachi Perkin-Elmer HHS-2R scanning electron microscope operated at 25kV in the secondary electron emission mode.

A Reinvestigation of the Deceptively Simple Reaction of Toluene with ●OH, and the Fate of the Benzyl Radical. I. a Combined Thermodynamic and Kinetic Study on the Competition Between ●OH Addition and Hydrogen Abstraction Reactions.

2019 ◽  
Author(s):  
Zoi Salta ◽  
Agnie M. Kosmas ◽  
Marc E. Segovia ◽  
Martina Kieninger ◽  
Oscar Ventura ◽  
...  
Keyword(s):  
Density Functional ◽  
Room Temperature ◽  
Hydrogen Abstraction ◽  
Reaction Rates ◽  
Composite Model ◽  
Alternative Mechanism ◽  
Radical Intermediate ◽  
Experimental Conditions ◽  
Methyl Group ◽  
Benzyl Radical

This work reports density functional and composite model chemistry calculations performed on the reactions of toluene with the hydroxyl radical. Both experimentally observed H-abstraction from the methyl group and possible additions to the phenyl ring were investigated. Reaction enthalpies and heights of the barriers suggest that H-abstraction is more favorable than ●OH addition to the ring. The calculated reaction rates at room temperature and the radical-intermediate product fractions support this view. This is somehow contradictory with the fact that, under most experimental conditions, cresols are observed in a larger concentration than benzaldehyde. Since the accepted mechanism for benzaldehyde formation involves H-abstraction, a contradiction arises that begs for an explanation. In this first part of our work we give the evidences that support the preference of hydrogen abstraction over ●OH addition and suggest an alternative mechanism which shows that cresols can actually arise also from the former reaction and not only from the latter.

A Reinvestigation of the Deceptively Simple Reaction of Toluene with ●OH, and the Fate of the Benzyl Radical. I. a Combined Thermodynamic and Kinetic Study on the Competition Between ●OH Addition and Hydrogen Abstraction Reactions.

2019 ◽  
Author(s):  
Zoi Salta ◽  
Agnie M. Kosmas ◽  
Marc E. Segovia ◽  
Martina Kieninger ◽  
Oscar Ventura ◽  
...  
Keyword(s):  
Density Functional ◽  
Room Temperature ◽  
Hydrogen Abstraction ◽  
Reaction Rates ◽  
Composite Model ◽  
Alternative Mechanism ◽  
Radical Intermediate ◽  
Experimental Conditions ◽  
Methyl Group ◽  
Benzyl Radical

This work reports density functional and composite model chemistry calculations performed on the reactions of toluene with the hydroxyl radical. Both experimentally observed H-abstraction from the methyl group and possible additions to the phenyl ring were investigated. Reaction enthalpies and heights of the barriers suggest that H-abstraction is more favorable than ●OH addition to the ring. The calculated reaction rates at room temperature and the radical-intermediate product fractions support this view. This is somehow contradictory with the fact that, under most experimental conditions, cresols are observed in a larger concentration than benzaldehyde. Since the accepted mechanism for benzaldehyde formation involves H-abstraction, a contradiction arises that begs for an explanation. In this first part of our work we give the evidences that support the preference of hydrogen abstraction over ●OH addition and suggest an alternative mechanism which shows that cresols can actually arise also from the former reaction and not only from the latter.

scholarly journals Glucuronides Hydrolysis by Intestinal Microbial β-Glucuronidases (GUS) Is Affected by Sampling, Enzyme Preparation, Buffer pH, and Species

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1043
Author(s):  
Christabel Ebuzoeme ◽  
Imoh Etim ◽  
Autumn Ikimi ◽  
Jamie Song ◽  
Ting Du ◽  
...  
Keyword(s):  
Enzyme Preparation ◽  
Reaction System ◽  
Reaction Rates ◽  
Experimental Conditions ◽  
Model Animal ◽  
Fresh Feces ◽  
Time Buffer ◽  
Exogenous Compounds ◽  
Activity Loss ◽  
The Impact

Glucuronides hydrolysis by intestinal microbial β-Glucuronidases (GUS) is an important procedure for many endogenous and exogenous compounds. The purpose of this study is to determine the impact of experimental conditions on glucuronide hydrolysis by intestinal microbial GUS. Standard probe 4-Nitrophenyl β-D-glucopyranoside (pNPG) and a natural glucuronide wogonoside were used as the model compounds. Feces collection time, buffer conditions, interindividual, and species variations were evaluated by incubating the substrates with enzymes. The relative reaction activity of pNPG, reaction rates, and reaction kinetics for wogonoside were calculated. Fresh feces showed the highest hydrolysis activities. Sonication increased total protein yield during enzyme preparation. The pH of the reaction system increased the activity in 0.69–1.32-fold, 2.9–12.9-fold, and 0.28–1.56-fold for mouse, rat, and human at three different concentrations of wogonoside, respectively. The Vmax for wogonoside hydrolysis was 2.37 ± 0.06, 4.48 ± 0.11, and 5.17 ± 0.16 μmol/min/mg and Km was 6.51 ± 0.71, 3.04 ± 0.34, and 0.34 ± 0.047 μM for mouse, rat, and human, respectively. The inter-individual difference was significant (4–6-fold) using inbred rats as the model animal. Fresh feces should be used to avoid activity loss and sonication should be utilized in enzyme preparation to increase hydrolysis activity. The buffer pH should be appropriate according to the species. Inter-individual and species variations were significant.

scholarly journals Development of a target identification approach using native mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miaomiao Liu ◽  
Wesley C. Van Voorhis ◽  
Ronald J. Quinn
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Target Identification ◽  
Native Mass Spectrometry ◽  
Pharmaceutical Drugs ◽  
Ligand Complex ◽  
Experimental Conditions ◽  
Protein Mixture ◽  
Native Ms ◽  
Drug Target Identification

AbstractA key step in the development of new pharmaceutical drugs is the identification of the molecular target and distinguishing this from all other gene products that respond indirectly to the drug. Target identification remains a crucial process and a current bottleneck for advancing hits through the discovery pipeline. Here we report a method, that takes advantage of the specific detection of protein–ligand complexes by native mass spectrometry (MS) to probe the protein partner of a ligand in an untargeted method. The key advantage is that it uses unmodified small molecules for binding and, thereby, it does not require labelled ligands and is not limited by the chemistry required to tag the molecule. We demonstrate the use of native MS to identify known ligand–protein interactions in a protein mixture under various experimental conditions. A protein–ligand complex was successfully detected between parthenolide and thioredoxin (PfTrx) in a five-protein mixture, as well as when parthenolide was mixed in a bacterial cell lysate spiked with PfTrx. We provide preliminary data that native MS could be used to identify binding targets for any small molecule.

scholarly journals TBP and SNAP50 transcription factors bind specifically to the Pr77 promoter sequence from trypanosomatid non-LTR retrotransposons

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Francisco Macías ◽  
Raquel Afonso-Lehmann ◽  
Patricia E. Carreira ◽  
M. Carmen Thomas
Keyword(s):  
Transcription Factors ◽  
Protein Interactions ◽  
Dna Sequences ◽  
Direct Interaction ◽  
Promoter Sequence ◽  
Nuclear Proteins ◽  
Hill Coefficient ◽  
Small Nuclear Rna ◽  
Mobile Dna ◽  
Mobility Shift

Abstract Background Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5′ ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. However, transcription factors that mediates this process have still not been reported. Methods TATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) recombinant proteins and specific antibodies raised against them were generated. Protein capture assay, electrophoretic mobility-shift assays (EMSA) and EMSA competition assays carried out using these proteins and nuclear proteins of the parasite together to specific DNA sequences used as probes allowed detecting direct interaction of these transcription factors to Pr77 sequence. Results This study identified TBP and SNAP50 as part of the DNA-protein complex formed by the Pr77 promoter sequence and nuclear proteins of Trypanosoma cruzi. TBP establishes direct and specific contact with the Pr77 sequence, where the DPE and DPE downstream regions are docking sites with preferential binding. TBP binds cooperatively (Hill coefficient = 1.67) to Pr77 and to both strands of the Pr77 sequence, while the conformation of this highly structured sequence is not involved in TBP binding. Direct binding of SNAP50 to the Pr77 sequence is weak and may be mediated by protein–protein interactions through other trypanosomatid nuclear proteins. Conclusions Identification of the transcription factors that mediate Pr77 transcription may help to elucidate how these retrotransposons are mobilized within the trypanosomatid genomes and their roles in gene regulation processes in this human parasite. Graphic abstract

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