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Biomedical subjects

M J Waring

Publications and source records attributed to M J Waring.

At least 163 records · Page 9Linked to original sources
PubMed1976

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.

Binding Sites↗
PubMed1976

Specific release of ribosomal proteins by nucleic acid-intercalating agents.

Increasing concentrations of ethidium bromide cause progressive inactivation of ribosomes, apparently by binding to double-stranded regions of the rRNA. At low drug concentrations (10(-4)M) the partial inhibition detected is due to specific release of proteins L7 and L12; activity can be restored by addition of an excess of these two proteins. At higher concentrations the inactivation is not reversed by supplementation with released proteins. The presence of ethanol affects the extent of ethidium binding and also the release of ribosomal proteins. In all tests the proteins most sensitive to the presence of the drug are L7 and L12, followed by L8/9, L11, L27, L28, L29 and L30. Despite the fact that L7 and L12 are the first two proteins released by ethidium they are never totally missing from drug-treated ribosomes, though the other proteins can be displaced completely. About 50% of proteins L7 and L12 remain on the ribosomes at the highest drug concentrations tested, possibly indicating heterogeneity in the binding sites for the several copies present in the ribosome.

Binding Sites↗
PubMed1975

A solvent-partition method for measuring the binding of drugs to DNA. Application to the quinoxaline antibiotics echinomycin and triostin A.

The development of a novel solvent-partition method for measuring the interaction between nucleic acids and drugs of limited water solubility is described. Factors relevant to the choice of a suitable water-immiscible solvent are summarised. i-Amyl acetate was selected for studying the binding of echinomycin and triostin A to DNA. Details of the experimental determination of extinction and partition coefficients are given; in the i-amyl acetate/buffer system employed for most experiments, the partition coefficients for echinomycin and triostin A were 111 +/- 4 and 943 +/- 23, respectively. Equilibration of echinomycin between the organic and aqueous phases was 90% complete within a few minutes, and a period of 2 h shaking was found satisfactory to ensure full attainment of equilibrium. Representative results are presented showing specific binding of the quinoxaline antibiotics to DNA, strong preference for double-helical as opposed to heat-denatured or single-stranded DNA, and restricted uptake by closed circular duplex PM2 DNA. The method is potentially applicable, with appropriate modifications, to the study of interactions between other ligands and DNA.

Animals↗
PubMed1975

Stereochemical aspects of the interaction between steroidal diamines and DNA.

A complete series of stereoisomeric quaternised diaminoandrostanes, differing in their stereochemistry at the 3,5 and 17 positions, has been examined for effects on the thermal denaturation of calf thymus DNA and for the ability to remove and reverse the supercoiling of closed circular duplex PM2 DNA. In both types of test the eight isomers rank in the same order of effectiveness. The preferred stereochemistry for the quaternary substituents at positions 3 and 17 is beta; of these the orientation of the 17- substituent is more critical. Folding of the steroid skeleton between the A and B rings, as in 5beta-androstanes, diminishes effectiveness but does not necessarily abolish the effect on supercoiling. The over-riding importance of the C-D ring end of the steroid nucleus bearing a 17beta-amino substituent is confirmed by a comparison of the effects of five mon-amino androstanes. Relative helix=unwinding angles per bound steroidal diamine molecule have been determined for four of the isomers; for three 17beta compounds the estimated values are similar to that previously reported for irehdiamine A. For a fourth isomer the angle is 0.22 times that of ethidium, the lowest yet determined for any DNA-binding drug. The results lend further support to the argument that intercalation can be reled out, and alternative models for the binding mechanism are discussed.

Amines↗
PubMed1975

Intercalative binding of ellipticine to DNA.

Ellipticine (NSC 71795), a plant alkaloid with antitumor activity, is a weakly basic polycyclic molecule with dimensions similar to those of proflavin. Like proflavin, ellipticine exhibits hypocromic and bathochromic changes in absorption spectrum in the presence of DNA. It binds preferentially to helical DNA by intercalation, but the strength of binding is substantially greater than that of proflavin. The evidence for intercalation is based on effects on the sedimentation and viscosity of sheared DNA fragments, removal and reversal of the supercoiling of closed circular DNA, and electric dichroism measurements. The sedimentation and viscosity changes are quantitatively similar to those produced by proflavin. The unwinding angle on binding to supercoiled DNA is estimated to be 7.9 degrees, similar to that of proflavin. Electric dichroism shows the plane of the bound ellipticine molecule to be oriented parallel (plus or minus 7 degrees) to the plane of the bases in helical DNA. Ellipticine differs from proflavin in that it is uncharged at neutral pH and becomes protonated under mildly acid conditions. This feature may influence the intracellular distribution of the drug. Ellipticine bound to DNA is probably in its protonated form.

Alkaloids↗
PubMed1974

Stablilzation of two-standard ribohomopolymer helices and destabilzation of a three-stranded helix by ethidium bromide.

Thermal-denaturation profiles of helical polynucleotides have been measured in the presence of increasing concentrations of ethidium bromide. The poly(A).poly(U) helix is strongly stabilized by binding of ethidium, to much the same extent as is DNA, but the stabilizing effect on poly(I).poly(C) is much smaller. In the poly(A).2poly(U) system the drug selectively destabilizes and eventually destroys the triple helix, leaving only the double-helix-to-coil transition.

DNA↗