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M J Waring

Publications and source records attributed to M J Waring.

At least 127 records · Page 7Linked to original sources
PubMed1984

Evidence of different binding sites for nogalamycin in DNA revealed by association kinetics.

The kinetics of association between nogalamycin and DNA have been measured by stopped-flow spectrometry. With a naturally occurring DNA (calf thymus) the reaction profile requires not less than three exponentials for its complete description. By contrast, binding to poly(dA-dT) is fully described by two exponentials which correspond to the two faster components seen with the natural DNA, whereas binding to poly(dG-dC) is a single exponential process whose time constant is about the same as the slowest component measured with calf thymus DNA. In all cases the amplitude of each component in the decay varies considerably with polynucleotide concentration. The results are consistent with a model in which the antibiotic is only able to bind directly to regions of the DNA which are transiently perturbed, probably non-basepaired. As a result, the antibiotic interacts much faster with AT-rich rather than GC-rich DNA sequences, which may provide a basis for its apparent sequence selectivity.

Animals↗
PubMed1984

Sequence preferences in the binding to DNA of triostin A and TANDEM as reported by DNase I footprinting.

Six or seven triostin-binding sites have been identified in a 160-base-pair DNA restriction fragment containing the tyr T promoter sequence. Each is centred round a CpG step, and the minimum binding site-size appears to be six base pairs. The sites are practically the same as those reported for echinomycin by DNase I digestion. Only two sites are protected by binding of TANDEM, the des-N-tetramethyl analogue of triostin A; they are centred around the sequences ATA or TAT.

Binding Sites↗
PubMed1984

Sequence-specific binding of echinomycin to DNA: evidence for conformational changes affecting flanking sequences.

The technique of DNAase I footprinting has been used to investigate preferred binding sites for echinomycin on a 160-base-pair DNA fragment from E. coli containing the tyr T promoter sequence. Six binding sites have been precisely located in the sequence; a seventh has been partially identified. The minimum site-size is six base pairs. All the binding sites contain the dinucleotide sequence CpG but no other regularities can be discerned. When the protected regions on each complementary strand are compared it is evident that they are staggered by 2-3 base-pairs towards the 3' end at each site. Footprinting with DNAase II reports a similar, though less precise, pattern of protection. Cutting by both enzymes is markedly enhanced at AT-rich regions flanking the antibiotic-binding sites. This increased susceptibility to nuclease attack can be attributed to an altered helix conformation in the vicinity of the bis-intercalated echinomycin molecule. It seems that certain sequences, mainly runs of A or runs of T, switch from a nuclease-resistant to a nuclease-sensitive form when echinomycin binds nearby.

Base Composition↗
PubMed1984

Stopped-flow kinetic studies on the interaction between echinomycin and DNA.

The kinetics of association between the quinoxaline antitumor antibiotic echinomycin and DNA have been studied by stopped-flow methods. With natural DNAs, the reaction profile is completely described by a single exponential, the time constant for which varies linearly with the DNA concentration. This bimolecular rate constant is similar for both calf thymus and Micrococcus lysodeikticus DNA (k = 6 X 10(4) M-1 s-1 at 25 degrees C, I = 0.01) and is probably dominated by interaction with relatively weak but abundant binding sites from which the antibiotic dissociates fairly quickly. The observed single exponential suggests a molecular mechanism of binding in which both chromophores of the antibiotic become intercalated simultaneously rather than sequentially; no transition from a mono-intercalated state to a bis-intercalated state could be detected. The reaction is slowed by a factor of about 3 on raising the salt concentration from I = 0.01 to I = 0.5. Binding to poly(dA-dT) is also described by a single exponential, the time constant for which is about 3 times faster than that seen with natural DNAs. By contrast, the interaction with poly-(dG-dC) requires two exponentials for a proper description, the faster of which is similar to that seen with natural DNAs. This may reflect the initial interaction of the antibiotic with two types of sequences, tentatively identified as GpC and CpG, from which it dissociates at very different rates. The differences in kinetic behavior may be explicable on the basis of an alternating B structure for poly(dA-dT) and a more classical B form for poly(dG-dC).

Animals↗
PubMed1984

Directed biosynthesis of novel derivatives of echinomycin by Streptomyces echinatus. I. Effect of exogenous analogues of quinoxaline-2-carboxylic acid on the fermentation.

Streptomyces echinatus A8331 cultured on a maltose minimal salts medium normally produces a single antibiotic, echinomycin (quinomycin A), containing two quinoxaline-2-carbonyl chromophores. Echinomycin is powerfully active against experimental tumours and can be assayed by its activity against Gram-positive bacteria. Grown in the presence of aromatic carboxylic acids related to quinoxaline, S. echinatus responds in favourable circumstances by incorporating the added material into analogues of the natural antibiotic having replacement chromophores. Both mono- and bis-substituted derivatives are formed. With quinoline-2-carboxylic acid as precursor, large quantities of analogues are produced, and the time course of synthesis, extraction, purification, assay, and characterization of the derivatives are described. Twenty-two other aromatic acids have been tested as potential substrates for antibiotic analogue biosynthesis. Half of them did not significantly affect growth and echinomycin production. Five appeared to stimulate antibiotic synthesis, while the remainder proved inhibitory. New biologically active antibiotics were detected in cultures supplemented with 7-chloroquinoxaline-2-carboxylic acid; 1,2,4-benzo-as-triazine-3-carboxylic acid; thieno[3,2-b]pyridine-5-carboxylic acid; and 6-methylquinoline-2-carboxylic acid.

Echinomycin↗
PubMed1984

Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part I. The synthesis of echinomycin.

Washed suspensions of Streptomyces echinatus, and protoplasts derived from them, have been shown to synthesise echinomycin in the absence of growth. Protoplast suspensions free from significant contamination with unlysed mycelia are obtained by incubation with lysozyme followed by filtration through layers of tightly packed glass wool. Although physiologically young cells produce a better yield of protoplasts, optimal antibiotic biosynthesis is achieved with protoplasts prepared from mycelia about to enter the stationary phase of growth i.e., approximately 24 h after inoculation into a nutrient broth--salts seed medium. As judged by the incorporation of label from L-[methyl-14C]methionine, echinomycin synthesis proceeds for about 1 h after preparation of washed suspensions, but the kinetics of incorporation by intact cells and protoplasts are different. Uptake of labelled methionine by protoplasts is critically dependent upon the presence of sucrose as osmotic stabiliser and is drastically reduced if galactose, calcium, or magnesium is omitted from the suspending buffer. Uptake by intact, washed cells is essentially independent of nutrients in the medium. Small quantities of 11 materials other than echinomycin are detectable in chloroform extracts after labelling with L-[methyl-14C]methionine; some of these may represent precursors in the biosynthesis of the antibiotic. All amino acid constituents of echinomycin as well as tryptophan, a putative precursor of the quinoxaline chromophores, are actively incorporated into echinomycin by protoplasts and resting cells, but not with equal efficiency.

Amino Acids↗
PubMed1984

Directed biosynthesis of novel derivatives of echinomycin. II. Purification and structure elucidation.

New antibiotics produced by Streptomyces echinatus A8331 cultured in the presence of heterocyclic aromatic acids can be separated and purified by high-performance liquid chromatography using reversed phase columns. Natural quinoxaline antibiotics and certain quinoline derivatives can also be efficiently separated in normal phase systems. Details of purification procedures are described together with experiments to characterise the new antibiotics by field desorption mass spectrometry and proton magnetic resonance. Mono- and bis-substituted derivatives of echinomycin containing the following replacement chromophores have been isolated: 7-chloroquinoxaline-2-carbonyl, thieno[3,2-b]pyridine-5-carbonyl, and 6-methylquinoline-2-carbonyl. With a 6-methylquinoline-2-carboxylic acid precursor the analogues containing one or two replacement chromophores are each separable into two distinct components. One of the bis-substituted 6-methylquinoline products appears inactive in an antibacterial assay and behaves as a triostin analogue, presumably an immediate precursor of the corresponding echinomycin derivative.

Chemical Phenomena↗
PubMed1983

Interaction between a 3-nitrobenzothiazolo (3,2-a) quinolinium antitumour drug and deoxyribonucleic acid.

The interaction of 3-nitrobenzothiazolo (3,2-a) quinolinium (NBQ) perchlorate with DNA was studied by u.v.-visible and fluorescence spectrophotometry as well as by hydrodynamic methods. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts, and the fluorescence was quenched. Binding parameters, determined from spectrophotometric measurements by Scatchard analysis according to an excluded-site model, indicated a binding constant of 2.4 X 10(5)M-1 for calf thymus DNA at ionic strength 0.01. The interaction was markedly suppressed by increasing the salt concentration. Binding to the GC-rich DNA of Micrococcus lysodeikticus was weaker than the binding to calf thymus DNA at ionic strength 0.01 NBQ increased the viscosity of sonicated rod-like DNA fragments, producing a calculated increment in length of 2.4 A/bound drug molecule. It removed and reversed the supercoiling of closed circular duplex plasmid pBR322 DNA by virtue of a helix-unwinding angle estimated as approximately 13 degrees/bound ligand molecule. We conclude that the binding of NBQ to DNA occurs by a mechanism of intercalation, which probably accounts for its reported antitumor activity.

Animals↗
PubMed1983

The use of radiolabelled triostin antibiotics to measure low levels of binding to deoxyribonucleic acid.

Triostin antibiotics, which contain a cyclic peptide with a disulphide bridge, have been prepared by growing Streptomyces triostinicus in the presence of inorganic [35S]-sulphate. The labelled triostin A has been shown to behave in all respects similarly to the authentic natural product and to enable a much more sensitive radiochemical adaptation of the solvent-partition method for determining antibiotic binding to DNA. By this means, binding isotherms at low, biologically relevant levels (down to one antibiotic molecule per gene) have been measured. The results indicate the existence of some tight binding sites in natural DNA species that are preferentially occupied at low concentrations. No evidence has been found for any allosteric transitions provoked by interaction between these antibiotics and natural DNA species, though there is evidence for co-operativity in the binding of triostin A to poly(dA-dT). For the first time accurate isotherms have been determined for the binding of triostin C to DNA; its binding constants for a variety of polydeoxynucleotides are uniformly tighter than those of triostin A but fall into the same ranking order when different species of natural DNA are compared.

Animals↗
PubMed1983

Preparation and DNA-binding properties of substituted triostin antibiotics.

Novel derivatives of the triostin group of antibiotics were prepared by supplementing cultures of the producing organism Streptomyces triostinicus with a variety of aromatic carboxylic acids. Five new antibiotics, each having both the natural quinoxaline chromophores replaced by a substituted ring system, were purified to homogeneity and characterized by high-pressure liquid chromatography and nuclear magnetic resonance. Their antibacterial activities and DNA-binding properties were investigated. Addition of a halogen atom at position 6 of the quinoxaline ring or an amino group at position 3 had little effect on either the biological activity or the DNA-binding characteristics. The bis-3-amino derivative is fluorescent, and its fluorescence is strongly quenched by calf thymus DNA and polydeoxyguanylate-polydeoxycytidylate but not by polydeoxyadenylate-polydeoxythymidylate, suggesting that it binds preferentially to guanosine-cytosine-rich sequences in natural DNA. Binding constants for the bis-6-chloro and bis-3-amino derivatives do not differ greatly from those of unsubstituted triostin A. The analogs having two quinoline chromophores or a chlorine atom in position 7 of the quinoxaline ring display little or no detectable antibacterial activity, in marked contrast to the other congeners. Bis-7-chloro-triostin A binds conspicuously more tightly to polydeoxyadenylate-polydeoxythymidylate than to any other polynucleotide tested.

Anti-Bacterial Agents↗
PubMed1983

Effect of phosphate and amino acids on echinomycin biosynthesis by Streptomyces echinatus.

Streptomyces echinatus produces only echinomycin (quinomycin A), in contrast to other streptomycetes, which produce families of quinoxaline antibiotics differing in the amino acid composition of the oligopeptide (quinomycins A, B, B0, C, D, and E) or the structure of the sulfur-containing cross bridge (triostins A, B, and C). Attempts were made to establish conditions for directed biosynthesis with S. echinatus. The lability of the peptide lactone to alkaline pH was obviated by using high levels of phosphate or HEPES [4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid] buffer in the production medium. Maintaining the pH below 7.5 resulted in an apparent stimulation of production. Amino acids which serve as structural components or as precursors of echinomycin were employed singly or in combination with nitrate in a chemically defined medium. Based on specific yield (micrograms of echinomycin per milligram of mycelia [dry weight]), D- and L-serine, D-alanine, L-valine, and L-phenylalanine produced equivalent yields of antibiotic which were approximately twofold greater than yields obtained with nitrate alone. In contrast, L-alanine, beta-alanine, and L-threonine produced a three- to fourfold stimulation of production. Although the other amino acids diminished antibiotic production, L-isoleucine, which ostensibly was inhibitory to production, supported the accumulation of a quinoxaline antibiotic in which the cross-bridge sulfur lacked a methyl group.

Amino Acids↗
PubMed1983

Conversion of triostins to quinomycins by protoplasts of Streptomyces echinatus.

Protoplasts of Streptomyces echinatus have been used to investigate the biosynthesis of echinomycin (quinomycin A). It has been shown that this organism has the capacity to convert a series of triostins to the corresponding quinomycins by a mechanism involving methylation. Evidence is presented which suggests that triostin A is the natural precursor of echinomycin. Conversion of tetra-N-demethyl analogues of triostin A to corresponding analogues of echinomycin was not detected.

Anti-Bacterial Agents↗
PubMed1982

Differential inhibition of a restriction enzyme by quinoxaline antibiotics.

The inhibition of cleavage by HpaI at two well-defined restriction sites in linearised phi X174-RF DNA by quinoxaline antibiotics has been investigated. Echinomycin, which displays a certain preference for binding to GC basepairs, inhibits cleavage at one site much more than the other, whereas triostin A, which displays less pronounced sequence-selectivity, inhibits both sites about equally. Other congeners inhibit reaction at the two sites with varying effectiveness. The results demonstrate the usefulness of studying inhibition of cleavage at specific sites by restriction enzymes as a means of exploring the specificity of DNA-ligand interactions.

Anti-Bacterial Agents↗
PubMed1982

Equilibrium and kinetic studies on the binding of des-N-tetramethyltriostin A to DNA.

The interaction between TANDEM (a des-methyl analogue of triostin A) and poly(dA-dT) results in extension of the helix by 6.8 A for each ligand molecule bound, exactly as predicted for a bis-intercalation reaction. Cooperativity is evident in Scatchard plots for the interaction at ionic strengths of 0.2 and 1.0, where the binding constant is diminished compared to that which pertains at low salt concentrations. Binding to a natural DNA (calf thymus), already considerably weaker than binding to poly(dA-dT), is also sensitive to increased ionic strength. With a self-complementary octanucleotide d(G-G-T-A-T-A-C-C) the binding curve indicates the presence of a single des-N-tetramethyltriostin A binding site per helical fragment with a non-cooperative association constant about 6 . 10(6) M-1. Detergent-induced dissociation of des-N-tetramethyltriostin A-poly(dA-dT) complexes results in a simple exponential decay at all levels of binding, but the time constant of decay is dependent upon the initial binding ratio. This behavior cannot directly explain the cooperativity of equilibrium binding isotherms but suggests the occurrence of relatively long-lived perturbations of the helical structure by binding of the ligand. [Ala3, Ala7]des-N-tetramethyltriostin A, which has a more flexible octapeptide ring lacking the disulphide cross-bridge, dissociates from poly(dA-dT) much faster than des-N-tetramethyltriostin A. Dissociation of des-N-tetramethyltriostin A from calf thymus DNA is more rapid than dissociation of triostin A or other quinoxaline antibiotics, which may account for its low antimicrobial activity.

Animals↗
PubMed1982

The interaction between prothidium dibromide and DNA at the molecular level.

The interactions in solution and in the fibre state of the trypanocidal drug, prothidium dibromide, with DNA have been investigated using a number of biophysical techniques. The binding parameters at 0.11 ionic strength were determined by spectroscopic means. Sedimentation studies show that the drug is able to unwind closed circular DNA in solution, but X-ray diffraction and linear dichroism experiments indicate that it is uncertain whether this unwinding can be attributed to intercalation in the classical sense. On the basis of our results, we propose that the primary mode of binding is "sideways" intercalation, supplemented by electrostatic binding along the sugar-phosphate chains and interstrand binding involving hydrogen bonding.

Animals↗
PubMed1982

Structure and conformation of fourteen antibiotics of the quinoxaline group determined by 1H NMR.

Nuclear magnetic resonance has been employed to characterize fourteen new antibiotics belonging to the quinoxaline group, produced by feeding aromatic acids to Streptomyces echinatus. Twelve of the antibiotics are the expected substituted quinomycins and adopt conformations very similar to that of echinomycin. This is discussed in relation to their different DNA-binding characteristics. The other two antibiotics are triostins, supporting the proposal that triostins serve as biosynthetic precursors of the quinomycins.

Anti-Bacterial Agents↗