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Biomedical subjects

A Pingoud

Publications and source records attributed to A Pingoud.

At least 127 records · Page 7Linked to original sources
PubMed1983

Does the specific recognition of DNA by the restriction endonuclease EcoRI involve a linear diffusion step? Investigation of the processivity of the EcoRI endonuclease.

The time course of the EcoRI endonuclease catalysed cleavage of three substrates, two plasmid DNAs and one oligonucleotide, each with two EcoRI sites, was measured. The two plasmid DNAs with the EcoRI sites 318 and 96 base pairs apart are cut in a distributive fashion, while the oligonucleotide with the EcoRI sites 8 base pairs apart is cut in a partially processive manner. It is concluded that a linear diffusion of the EcoRI endonuclease on its substrate across long stretches of DNA is not likely to be operative during the recognition process. Microscopic dissociation-reassociation processes, however, increase the probability of the enzyme to attack further sites located in the immediate vicinity of a given site.

Base Composition↗
PubMed1982

The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP.

Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP. Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin. The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP. In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1. While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations. A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected. The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E. coli ribosome.

Escherichia coli↗
PubMed1982

The antibiotics kirromycin and pulvomycin bind to different sites on the elongation factor Tu from Escherichia coli.

Pulvomycin and kirromycin, two antibiotics which inhibit protein biosynthesis in Escherichia coli by complex formation with the elongation factor Tu (EF-Tu), bind to different sites on the protein. While only one molecule of kirromycin can be bound to one molecule of EF-Tu, more than one molecule of pulvomycin interacts with a molecule of EF-Tu. This has been deduced from experiments in which the aminoacyl-tRNA binding and the GTPase activity of EF-Tu were measured in the presence of varying amounts of both antibiotics. These experiments are interpreted to mean that pulvomycin but not kirromycin can replace the other antibiotic in its respective site. Our conclusions are supported by circular dichroism spectroscopy.

Aminoglycosides↗
PubMed1982

Two identical subunits of the EcoRI restriction endonuclease Co-operate in the binding and cleavage of the palindromic substrate.

The cleavage of radioactively labelled double-stranded d(G-G-A-A-T-T-C-C) was studied in single turnover experiments with substrate and enzyme both being in the micromolar range. The reaction rate was found to increase with enzyme concentration until a ratio of one tetrameric enzyme to two double-stranded substrates was reached, further increase of the enzyme concentration then leads to a sharp decline of the reaction rate. These findings are interpreted in the following manner. (a) Two subunits of the EcoRI endonuclease co-operate in binding and possibly also in cleaving the palindromic substrate. (b) The enzymatic action of the EcoRI endonuclease is inhibited by excess enzyme, possibly due to unspecific binding of the enzyme-substrate complex. The self-inhibition of EcoRI endonuclease has also been observed with macromolecular substrates.

Binding, Competitive↗
PubMed1981

The effect of several nucleic acid binding drugs on the cleavage of d(GGAATTCC) and pBR 322 by the Eco RI restriction endonuclease.

The endonucleolytic action of the EcoRI restriction enzyme on the double-stranded oligonucleotide d(GGAATTCC) and the supercoiled plasmid DNA pBR 233 is inhibited by actinomycin D, ethidium bromide, proflavin, distamycin A and netropsin. Half-maximal inhibition is observed at around 100 microM concentrations for the intercalating drugs, and around 0.1 to 1 microM concentrations for netropsin and distamycin A. The inhibitory activity of these drugs can be correlated with their affinity to the oligonucleotide and the plasmid DNA. Since at high concentrations of the drugs a complete inhibition is observed, it is concluded that the effect of the drugs on the stereochemistry of the EcoRI site is such that recognition is excluded.

Circular Dichroism↗
PubMed1981

Transient cleavage kinetics of the Eco RI restriction endonuclease measured in a pulsed quench-flow apparatus: enzyme concentration-dependent activity change.

We report measurements of the cleavage rate of pBR 322 plasmid DNA by the restriction endonuclease Eco RI as a function of enzyme and DNA concentration. The reaction, which at high excess of enzyme over DNA occurs between 0.2 and 5 seconds, was studied by the means of a microprocessor controlled pulsed quench-flow apparatus. Enzyme concentrations were between 1 and 100 nM with DNA concentrations being 3 to 6 nM (specific Eco RI sites). The catalytic constants for cleavage of the first and second phosphodiester bonds as measured at high enzyme concentration both have the same value of 0.35 sec-1 and 21 degrees C. At enzyme concentrations comparable to or less than DNA concentration, the rate of the first cleavage is proportional to enzyme concentration, while the second step is independent of concentration. At approx. 10 nM Eco RI endonuclease concentration, a rate increase shows up in both the first and the second cleavage. We suggest that this increase is due to the tetramerization reported by Modrich & Zabel1, which occurs in this concentration range.

DNA Restriction Enzymes↗
PubMed1981

The elongation factor Tu . guanosine tetraphosphate complex.

The elongation factor Tu (EF-Tu) isolated from Escherichia coli cells that have undergone the stringent response is predominantly complexed with guanosine 5'-diphosphate 3'-diphosphate. This complex can be separated by anion-exchange chromatography from Ef-Tu . GDP. Unlike EF-Tu complexed with guanosine 5'-triphosphate 3'-diphosphate, the complex with guanosine 5'-diphosphate 3'-diphosphate cannot form a ternary complex with aminoacyl-tRNAs as can be shown directly by hydrolysis protection experiments.

Bacterial Proteins↗
PubMed1981

Interaction of myelin basic protein with mononuclear cells: the primary reaction for the MEM and EMT tests.

The primary reaction of the macrophage electrophoretic mobility test (MEM) and its modifications (viz. the interaction of myelin basic protein (MBP) and mononuclear cells) has been investigated. The binding of MBP to mononuclear cells is rather weak, and on incubation with mononuclear cells the MBP is proteolytically degraded. A fast process leads to fragments with mol. wts in the range 9000-14,000, followed by a slower process leading to peptides smaller than 5000. Both binding and proteolytic degradation are the same for mononuclear cells from cancer patients and from control individuals.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

The pharmacokinetics, bioequivalence and bioavailability of different formulations of metoclopramide in man.

The pharmacokinetics of 4-amino-5-chloro-N-(2-diethylaminoethyl)-2-methoxybenzamide (metoclopramide, Paspertin) after a single dose of the drug in five different dosage forms: ampoules, tablets, drops, dragées and suppositories, were studied. The clinical investigations were carried out on 10 healthy subjects under carefully controlled conditions. The analysis of metoclopramide in plasma was carried out using reverse-phase high-pressure liquid chromatography. Bioavailabilities between 76 and 79% were determined for the oral application forms and 53% for the rectal application form. Significant differences between maximum plasma levels were found for different individuals. The initial distribution of the drug is very rapid, and the elimination half-lives are comparable for all formulations and ranged from 3.9 and 5.3 h. The apparent volume of distribution is 3.1 l/kg body weight and the total body clearance is 38.4 l/h.

Adolescent↗
PubMed1980

Inhibition of Eco RI action by polynucleotides. A characterization of the non-specific binding of the enzyme to DNA.

The cleavage of the plasmid pBR322 by the restriction endonuclease Eco RI has been studied in the presence of various polynucleotides and the double stranded octanucleotide d-(GGAATTCC) in order to clarify whether there is a preferential interaction of Eco RI with DNA sequences other than -GAATTC-. The steady state kinetic analysis shows that all polynucleotides investigated with the possible exception of poly-dG.poly-dC inhibit the cleavage competitively with Ki values in the range of 10(-4) to 10(-5) [M nucleotides]. The Ki of d-(GGAATTCC) is 1.5.10(-6) [M nucleotides], indicating that the specific binding is approx. 2 orders of magnitude stronger than non-specific binding.

DNA Restriction Enzymes↗
PubMed1980

Aminoacyl transfer ribonucleic acid binding site of the bacterial elongation factor Tu.

Hydrolysis protection experiments were used for a quantitative determination of the binding of several aminoacyl-tRNAs to the Escherichia coli elongation factor Tu. The observed differences could not be rationalized in terms of structural properties of the tRNAs. The experimental results support, however, a model according to which the differences in the affinity of naturally occurring aminoacyl-tRNAs are determined mainly by the nature of the amino acid esterified to the tRNA. Aminoacyl-tRNAs with polar amino acid side chains are bound less strongly than those with apolar ones. This model is substantiated by results obtained with misacylated and modified aminoacyl-tRNAs. Furthermore, it could be shown that the aminoacyl group of the aminoacyl-tRNA must be in the L configuration; EF-Tu in this way prevents blocking of the ribosomal A site or even incorporation of D-amino acids into protein. The data have been used for a schematic description of the structure of a part of the aminoacyl-tRNA binding site of the bacterial elongation factor Tu.

Carbon Radioisotopes↗
PubMed1979

Numerical analysis of binding studies: a direct procedure avoiding the pitfalls of a Scatchard analysis of equilibrium data for unknown binding models.

It is shown on theoretical grounds that the straightforward analysis of binding data according to Scatchard may lead to erroneous results, especially when more complicated binding schemes are involved. We have demonstrated this point by presenting Scatchard plots with slight variation of experimental parameters. These inherent difficulties of Scatchard analyses can be avoided by applying a direct procedure. We have developed a program, which compares the measured quantity and the theoretical value directly and which considers the following binding models: (i) independent equivalent binding of n ligands; (ii) independent unequivalent binding of 2 ligands; (iii) positive or negative cooperative binding of 2 ligands. Other binding schemes can easily be implemented. We have used this procedure for the evaluation of equilibrium data on the complex formation of tRNA-Tyr and tyrolyl tRNA synthetase from E. coli in terms of different binding models.

Amino Acyl-tRNA Synthetases↗
PubMed1978

The binding of kirromycin to elongation factor Tu. Structural alterations are responsible for the inhibitory action.

The influence of kirromycin on the elongation factor Tu (EF-Tu) in its binary and ternary complexes was investigated. The equilibrium constant for the binding of the antibiotic to EF-Tu . GDP and EF-Tu . GTP was determined by circular dichroism titrations to be 4 x 10(6) M-1, and to EF-Tu . GTP . aa-tRNA by a combination of circular dichroism titrations and hydrolysis protection experiments to be 2 x 10(6) M-1. In the presence of kirromycin the binding of aminoacyl-tRNAs to EF-Tu . GTP is weakened by a factor of two. The antibiotic changes the conformation of the ternary complex in such a way that the aminoacyl moiety of the aminoacyl-tRNA is more accessible to the non-enzymatic hydrolysis. It is concluded that this structural alteration is responsible for the inhibitory action of the antibiotic.

Anti-Bacterial Agents↗
PubMed1977

Fluoresceinylthiocarbamyl-tRNATyr: a useful derivative of tRNATyr (E.coli) for physicochemical studies.

Fluoresceinylthiocarbamyl-tRNATyr (FTC-tRNATyr) is prepared from tRNATyr and fluoresceinisothiogyanate (FITC) under mildly alkaline conditions. Labelling occurs specificly at the base Q of tRNATyr. The modified tRNA is fully active in the aminoacylation assay; when aminoacylated it is recognized by the elongation factor Tu (EF-Tu). Codon-anticodon interaction, however, is severely affected by the modification.

Anticodon↗