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R H Shafer

Publications and source records attributed to R H Shafer.

At least 73 records · Page 4Linked to original sources
PubMed1982

Biosynthesis and characterization of [15N]actinomycin D and conformational analysis by nitrogen-15 nuclear magnetic resonance.

We describe the production and characterization of actinomycin D labeled with 15N at all twelve nitrogen positions. Cultures of Streptomyces parvulus were incubated in the presence of racemic [15N]glutamic acid and, following an initial delay, labeled antibiotic was produced. Evidence is presented that the D enantiomorph of glutamic acid was ultimately used for actinomycin biosynthesis. The 15N NMR spectrum at 10.14 and 20.47 MHz of the labeled drug in CDCl3 is presented. All nitrogens except the phenoxazone chromophore nitrogen are inverted when spectra are obtained under broad-band proton irradiation conditions. All 15N resonances have been assigned, and the proton-nitrogen one-bond coupling constants were determined in CDCl3 to be 92.5 +/- 0.3 Hz for the valine and threonine amide protons by both 1H and 15N NMR. 15N NMR spectra were also obtained in dimethyl sulfoxide, methanol, and water in order to probe solvent interactions with the peptide nitrogens and carbonyl groups. Large downfield shifts (greater than 5 ppm) were seen for the Pro, sarcosine, and methylvaline resonances when the solvent was changed from dimethyl sulfoxide to water. Smaller downfield shifts were observed for the Val and Thr peaks. These results are discussed in terms of a model for the solution conformation of the actinomycin pentapeptide rings based on different hydrogen-bonding interactions in the monomer in organic solvents and the dimer which is formed in water.

Dactinomycin↗
PubMed1982

High-resolution proton nuclear magnetic resonance analysis of conformational properties of biosynthetic actinomycin analogues.

High-resolution proton nuclear magnetic resonance (1H NMR) has been used to study the conformation and dynamics of biosynthetic analogues of actinomycin D that have been substituted at one or both of the 3'-amino acids with azetidine-2-carboxylic acid, pipecolic acid, or 4-ketoproline for one or both of the prolines. The results of measurements made in organic solvents indicate that the amino acid residues on the alpha and beta pentapeptide lactone rings experience different magnetic environments in all the analogues studied. Analysis of the J alpha NH coupling constants and temperature coefficients for the valine and threonine amide protons indicates that substitution at the 3' position has little effect on the conformational and hydrogen-bonding properties of the amino acids at the 1' or 2' position. Examination of the low-field position of the H alpha proton of the 3'-amino acid reveals that the biosynthetic substitutions were made at a unique site on either the alpha or the beta pentapeptide ring and that only one conformation of proline, azetidine-2-carboxylic acid, or pipecolic acid is observed. In D2O, the dynamics of the prolines appear to differ; one remains in a preferred conformation while the other fluctuates at a rate that is intermediate on the NMR time scale. A possible relationship between the conformational and dynamic properties and the DNA binding and kinetic properties is discussed.

Dactinomycin↗
PubMed1982

Role of actinomycin pentapeptides in actinomycin-deoxyribonucleic acid binding and kinetics.

Results are reported on equilibrium and kinetic experiments probing the DNA binding properties of a series of actinomycin analogues differing at the 3'-amino acid position. While the parent compound, actinomycin D, contains proline at this position on both pentapeptide lactone rings, the analogues under consideration here contain either azetidine-2-carboxylic acid, pipecolic acid, or 4-ketoproline on one or both pentapeptide rings. This study extends our earlier results on doubly substituted analogues [Shafer, R.H., Burnett, R. R., & Mirau, P.A. (1980) Nucleic Acids Res. 8, 1121]. DNA binding constants were determined from Scatchard plots constructed from visible absorption data and covered the range of (0.3-9) X 10(6) M-1 for the whole series of analogues. The thermal denaturation temperature of calf-thymus DNA was increased by 3-17 degrees C. DNA dissociation kinetics, along with enthalpies and entropies of activation, were also determined. The time constant for the slowest dissociation process ranged from 278 to 10 900 s. The strongest DNA binding analogue, in terms of the largest binding constant, the largest increase in DNA thermal denaturation temperature, and the slowest DNA dissociation rate, was actinomycin V, which has 4-ketoproline in the beta peptide ring, while the weakest DNA binding analogue has pipecolic acid on both peptide rings. Evidence is presented for one peptide ring exerting a greater influence than the other in the interaction with DNA. Also, the possible role of cis-trans isomerization about one or two peptide bonds in determining the slow DNA binding kinetics is discussed.

Animals↗
PubMed1982

Binding of 5-fluorotryptamine to polynucleotides as a model for protein-nucleic acid interactions: fluorine-19 nuclear magnetic resonance, absorption, and fluorescence studies.

Fluorine-19 nuclear magnetic resonance (19F NMR), optical absorption, and fluorescence spectroscopy have been used to study the interaction of 5-fluorotryptamine (5FTA) with polynucleotides as a model for protein--nucleic acid interactions. In the presence of DNA, denatured DNA, poly(A), and poly(A).poly(U), the 19F resonance of 5FTA shifted 0.3-0.6 ppm upfield while the presence of poly(I).poly(C) had little effect on the chemical shift. Differences in the 19F chemical shift induced upon changing from H2O to 2H2O indicate differences in the solvent accessibility of 5FTA bound to the various polynucleotides. 19F NMR relaxation experiments were carried out for free 5FTA and in its nucleic acid complexes, and the results were interpreted by using a two correlation time model that included contributions to relaxation from dipolar coupling and chemical shift anisotropy. Values for the internal motion correlation time and the overall motion correlation time are reported. The effect of 5FTA on the melting transition of the double-stranded polynucleotides and on the quenching of 5FTA fluorescence was also studied. The 19F NMR results support the model of partial intercalation of the 5FTA chromophore into the polynucleotides, and the implications for protein-nucleic acid interactions are discussed.

Fluorine↗
PubMed1980

Spectroscopic analysis of the equilibrium and kinetic DNA binding properties of several actinomycin analogs.

Experiments are described that measure DNA dissociation kinetics and thermal denaturation temperatures for a series of actinomycin analogs containing, in the 3' amino acid position, pipecolic acid, proline or azetidine-2-carboxylic acid. Also included are studies on actinomycin C3. Analysis of the temperature dependence of the slowest rate constant for DNA dissociation shows that both the enthalpy and entropy of ativation increase as the ring size of the 3' amino acid decreases from six to five to four. All compounds increase the DNA melting temperature to the same extent except for the analog containing pipecolic acid, which shows a smaller effect. These results are discussed in terms of a possible role for conformational changes in the actinomycin pentapeptide lactone rings in determining the slow DNA dissociations rates for this class of intercalators. It is suggested that cis-trans isomerization of proline may be important in this regard.

Binding Sites↗
PubMed1980

DNA damage in rat 9L cells treated with nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea assayed by viscoelastometry and S1 nuclease.

The techniques of viscoelastometry and S1 nuclease digestion were applied to the analysis of DNA damage in rat 9L cells treated with nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Results of the S1 nuclease assay permitted quantitation of the amount of single-strand (or alkali-labile) break formation as well as DNA interstrand cross-link formation. In the presence of 2% detergent, only cells treated with nitrogen mustard showed evidence of DNA cross-link formation as determined by this assay. Viscoelastic analysis of cell lysates under denaturing conditions (pH 12.15) showed that cells treated with nitrogen mustard led to substantial increases in both the viscoelastic retardation time and recoil, consistent with the presence of DNA cross-links, while treatment with BCNU led to decreases in these two properties, consistent with the induction of single-strand breaks. Viscoelastic analysis of cell lysates under nondenaturing conditions (pH 11.15) showed that nitrogen mustard produced an increase in retardation time, consistent with single-strand break induction, along with a fast recoiling component that eventually led to gel-like behavior, suggesting the possibility of drug-induced intermolecular DNA-DNA cross-links. BCNU treatment resulted in a decrease in retardation time. This decrease in consistent with induction of DNA interstrand cross-links by BCNU and shows that the single-strand breaks observed at denaturing conditions were due to the presence of alkali-labile sites rather than true strand breaks. While other methods using denaturing conditions have resulted in evidence for DNA cross-links following BCNU treatment, both viscoelastic and S1 nuclease experiments showed negative results in this regard. Further work is needed to clarify this point.

Animals↗
PubMed1979

Viscoelastic behavior of mammalian DNA.

The viscoelastic behavior of rat 9L cellular DNA was studied as a function of the detergent used for lysis, the pH and duration of lysis, and gamma ray dose. For nondenaturing lysis conditions, a model of the DNA was proposed to account for the effects of these agents on the viscoelastic retardation time. It was concluded that these agents affect the hydrodynamic radius of the DNA rather than its molecular weight. For denaturing lysis conditions, molecular weights calculated from the relaxation time were consistent with those calculated from alkaline sucrose sedimentation profiles.

Animals↗
PubMed1976

Radial migration of DNA molecules in cylindrical flow. III. Circles and the effect of non-gaussian polymer statistics.

We have previously shown that DNA will migrate radially inward in a concentric-cylinder shear flow apparatus. We assumed gaussian chain statistics, and we considered only linear molecules. In this paper, we extend the analysis to closed circular molecules, and we consider non-gaussian statistics for both linears and circles. We find that, in good solvents, the inward radial migration velocity is more sensitive to the molecular weight than M5/2, which we previously reported for gaussian chains. Furthermore, linears migrate radially inward 8 times faster than do circles of the same molecular weight. This suggests the possibility of separating linear from circular DNA in solution.

DNA↗