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Biomedical subjects

D J Patel

Publications and source records attributed to D J Patel.

At least 253 records · Page 14Linked to original sources
PubMed1977

Sequence specificity of mutagen-nucleic acid complexes in solution: intercalation and mutagen-base pair overlap geometries for proflavine binding to dC-dC-dG-dG and dG-dG-dC-dC self-complementary duplexes.

The complex formed between the mutagen proflavine and the dC-dC-dG-dG and dG-dG-dC-dC self-complementary tetranucleotide duplexes has been monitored by proton high resolution nuclear magnetic resonance spectroscopy in 0.1 M phosphate solution at high nucleotide/drug ratios. The large upfield shifts (0.5 to 0.85 ppm) observed at all the proflavine ring nonexchangeable protons on complex formation are consistent with intercalation of the mutagen between base pairs of the tetranucleotide duplex. We have proposed an approximate overlap geometry between the proflavine ring and nearest neighbor base pairs at the intercalation site from a comparison between experimental shifts and those calculated for various stacking orientations. We have compared the binding of actinomycin D, propidium diiodide, and proflavine to self-complementary tetranucleotide sequences dC-dC-dG-dG and dG-dG-dC-dC by UV absorbance changes in the drug bands between 400 and 500 nm. Actinomycin D exhibits a pronounced specificity for sequences with dG-dC sites (dG-dG-dC-dC), while propidium diiodide and proflavine exhibit a specificity for sequences with dC-dG sites (dC-dC-dG-dG). Actinomycin D binds more strongly than propidium diiodide and proflavine to dC-dG-dC-dG (contains dC-dG and dG-dC binding sites), indicative of the additional stabilization from hydrogen bonding and hydrophobic interactions between the pentapeptide lactone rings of actinomycin D and the base pair edges and sugar-phosphate backbone of the tetranucleotide duplex.

Acridines↗
PubMed1976

NMR studies of the structure and stability of the 1 : 2 actinomycin D-d-pG-C complex in aqueous solution.

We describe NMR studies at superconducting fields which characterize aspects of the structure and stability of the 1 : 2 actinomycin-d-pG-C complex in solution as monitored at the Watson-Crick base pairs and backbone phosphate groups. Two guanine N1H resonances (12.17 and 11.66 ppm) are observed in the 360 MHz proton NMR spectra of the complex in water at -4 degrees C. These slowly exchangeable resonances, which demonstrate the presence of two Watson-Crick G + C base pairs in the complex, broaden in a sequential manner with increasing temperature. The terminal and internucleotide phosphates of both d-pG-C molecules are observable in the 145.7 MHz 31P spectra of the 1 : 2 actinomycin-d-pG-C complex at 0 degrees C. The internucleotide phosphate resonance at 1.905 ppm broadens prior to that at 2.385 ppm with increasing temperature, consistent with a sequential breakage of the G + C base pairs in the complex. The lifetime of the complex (4.5 +/- 0.5 X 10(-4) s, 33 degrees C) was deduced from the variation of the d-pG-D internucleotide 31P resonance line width on gradual addition of the antibiotic in solution.

Binding Sites↗
PubMed1976

Ethidium bromide-(dC-dG-dC-dG)2 complex in solution: intercalation and sequence specificity of drug binding at the tetranucleotide duplex level.

The binding of ethidium bromide (EtdBr) to the dC-dG-dC-dG self-complementary duplex has been monitored at the resolvable drug and nucleic acid protons and backbone phosphates at high nucleotide/drug (N/D) ratios by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. We observe averaged resonances (25 degrees-95 degrees) for the nucleic acid and drug nonexchangeable protons in the presence of excess tetranucleotide (N/D = 24), indicative of rapid exchange relative to the chemical shifts in the free and complexed states. Complex formation results in upfield shifts for the base protons at the terminal and internal base pairs and an increase in the transition midpoint for the duplex-to-strand conversion. We observe upfield chemical shift changes of 1.2 ppm at the Watson-Crick guanine N-1 proton(s) on complex formation (N/D = 24), with slow exchange between (dC-dG-dC-dG)2 and EtdBr-(dC-dG-dC-dG)2 relative to this chemical shift difference at-5 degrees. The EtdBr phenanthridine ring protons shift upfield by about 0.9 ppm (H-2, H-4, H-7, H-9) and greater than 0.5 ppm (H-1, H-10) on complex formation, with the chemical shifts versus temperature plots (25 degrees-95 degrees) monitoring the dissociation of the EtdBr-(dC-dG-dC-dG)2 structure. These upfield shifts at the exchangeable and nonexchangeable base protons and phenanthridine ring (but not side chain) protons demonstrate intercalation of the phenanthridine ring of EtdBr into the dC-dG-dC-dG duplex in solution. The intercalation model may be supported by the observation of downfield shifts (up to 1ppm) at the internucleotide phosphate(s) of the tetranucleotide duplex on addition of EtdBr at low temperatures. We observe stronger binding of EtdBr to the self-complementary dC-dG-dC-dG (2 dC-dG intercalation sites) and dC-dC-dG-dG (1 dC-dG site) duplexes compared to the dG-dG-dC-dC (no dC-dG sites) as monitored by UV absorbance changes at 480 nm. These studies extend to the tetranucleotide duplex level earlier observations that EtdBr exhibits a selectivity for formation of complexes to dinucleoside monophosphates with a pyrimidine (3'-5') purine sequence in the crystal and in solution. The experimental proton NMR upfield shifts at the phenanthridine protons on formation of the EtdBr-(dC-dG-dC-dG)2 complex compare favorably with calculated values (atomic diamagnetic anisotropy and ring current contributions) based on the overlap geometry for EtdBr intercalated into the pyrimidine (3'-5') purine dinucleoside monophosphate duplex in the crystal.

Ethidium↗
PubMed1976

Free acid, anion, alkali, and alkaline earth complexes of lasalocid a (X537A) in methanol: structural and kinetic studies at the monomer level.

We report below on nuclear magnetic resonance investigations of the structure and exchange kinetics for the free acid, anion, sodium complex, and barium complex of the ionophore lasalocid A (X537A) in methanol solution. A comparison between the proton and carbon longitudinal relaxation times of lasalocid in nonpolar and polar solvents demonstrates that the free acid (HX) is a monomer in methanol solution. Parallel proton and carbon relaxation measurements demonstrate that the anion (X-), sodium complex (NaX), and barium complex (BaX+) are also monomeric in methanol solution. These results are in contrast to the Na2X2 dimer and the BaX2-H20 dimer observed in crystals and in nonpolar (cyclohexane and methylene chloride) solutions. Large downfield shifts on complex formation (X- to NaX and BaX+) are detected for protons located on the polar face of the ionophore with their C-H bonds directed towards and proximal to the metal ion. The exchange of lasalocid anion between free (X-) and complexed (BaX+) states in methanol can be monitored from the temperature-dependent line shapes of the proton resonances at superconducting fields. The exchange rates are independent of the reactant concentrations and are characteristic of a rate-determining dissociation of BaX+ in methanol solution with activation parameters delta H++ = 6.5 kcal mol-1 (25 degrees) and delta S++ = -20.0 cal mol-1 degree -1 (1 cal = 4.184 J). The rate constants for dissociation and formation of BaX+ complex in methanol, 25 degrees, are 5.2 X 10(3) sec-1 and 1.5 X 10(10) M-1 sec-1, respectively. These studies were extended to derive the activation parameters for the exchange of lasalocid anion between BaX+ and NaX and between BaX+ and HX in methanol, while the exchange among HX, X-, and NaX is too rapid to be monitored on the time scale of nuclear magnetic resonance.

Anti-Bacterial Agents↗
PubMed1976

Nuclear magnetic resonance studies of the helix-coil transition of poly (dA-dT) in aqueous solution.

The well-resolved base and sugar proton resonances in the high resolution proton nuclear magnetic resonance (NMR) spectra of poly(dA-dT) can be monitored during the helix-coil transition. The observable resonances shift upfield on helix formation and the temperature-dependent chemical shifts exhibit a melting temperature t 1/2 = 69.9 +/- 0.3 degrees for 18.8 mM (with respect to phosphorus) poly(dA-dT) in 0.5 M Tris, 0.1 M cacodylate, D2O, pH 7.05. The observable protons are in fast exchange throughout the poly (dA-dT) helix-coil transition. The adenine H2 resonance that shifts upfield by about 1 ppm on helix formation exhibits uncertainty broadening in the fast exchange region...

Magnetic Resonance Spectroscopy↗
PubMed1976

Structural and kinetic studies of lasalocid A (X537A) and its silver, sodium, and barium salts in nonpolar solvents.

The ionophore lasalocid A (X537A) and its metal salts have been investigated by high resolution (270 MHz and 360 MHz) proton nuclear magnetic resonance spectroscopy to obtain structural and kinetic information in nonpolar solution. The proton resonances were assigned from double resonance studies on lasalocid A and on its salts, homologs, isomers, and chemically modified derivatives. Studies of proton and carbon longitudinal relaxation time suggest that lasalocid A exists as a monomer, whereas the sodium and barium salts exist as dimers in nonpolar solvents. A study of the magnitude of the vicinal proton coupling constants and the chemical shifts and linewidths of the hydroxyl resonances suggest that the backbone conformation and intramolecular hydrogen bonds are similar for lasalocid A and its sodium and barium salts in nonpolar solvents. Nuclear magnetic resonance studies on the role of bound solvent molecules suggest a tightly bound water molecule in the barium complex dimer (crystallized from water-ethanol) and a weakly bound ethanol molecule in the lasalocid A monomer (crystallized from ethanol) in cyclohexane. The selective changes in proton chemical shift on complexation [where the polar faces of two lasalocid anions coordinate the metal cation(s) in nonpolar solvents have been analyzed in terms of the proximity of the resonances to the cation, their linkage to the coordinating oxygen atoms, and the magnetic anisotropy effects of the polar groups of one ligand on the resonances of its partner in the dimer. The nuclear magnetic resonance studies in solution are compared with earlier observations on lasalocid A and its salts in the crystalline state. Thus, the short Ag-C5 distance in the crystal structure of silver complex dimer is also observed in the solution structure. The kinetic parameters associated with the exchange between lasalocid A and its barium complex in chloroform have been measured from an analysis of the resonance line shapes as a function of temperature.

Anti-Bacterial Agents↗
PubMed1975

Assignment of the imidazole ring nitrogen protons of histidine 48 in the proton NMR spectrum of ribonuclease A in water solution.

Several exchangeable resonances, designated a, b, c and d are observed in the 11-14 ppm (from 2,2-dimethyl-2-silapentane-5-sulfonate) region of the proton spectrum of ribonuclease A in water solution. We describe a number of lines of evidence suggesting the assignment of peaks b and c to the N1 and N3 protons of His 48, which occupies an interior position in the protein remote from the active site. This evidence includes the observation that the binding of Cu(II) and 3'-CMP (cytidine 3'-monophosphate) has no effect on these resonances. Further evidence includes pH titration data showing a pKa of approx. 2 for these protons, solvent exchange rates in the native state and with disulfide bridges IV-V and III-VIII cleaved, the observation of the carboxymethylated enzymes CM-His12-RNAase A and CM-His119-RNAase A, and of the modified enzymes Des(1-21)-RNAase A (S-protein) and Des(119-124)-RNAase A.

Amino Acid Sequence↗
PubMed1975

Proton nuclear magnetic resonance investigations of fraying in double-stranded d-ApTpGpCpApT in H2O solution.

The chemical shifts and line widths of the Watson-Crick ring NH resonances of the self-complementary duplex of d-ApTpGpApT have been monitored at low ionic strength and in the presence of Mg ions at neutral pH in aqueous solution to determine the thermodynamic parameters associated with fraying (D. J. Patel (1974), Biochemistry 13, 2396) at the terminal and internal base pairs as a function of temperature and pH. From studies in H2O-MeOH (3:2), the fraying process persists down to approximately -20 degrees for the internal TA base pair and down to and probably beyond -30 degrees for the terminal AT base pair. The observed average chemical shift at each of these base pairs as a function of temperature suggests rapid exchange on the nuclear magnetic resonance (NMR) time scale between helix and coil (chemical shift separation of 3.2 ppm) and have been utilized to determine the dissociation constant at the terminal and internal base pairs. Comparison of the reaction enthalpies elucidated from the chemical shift parameters with those reported from optical studies suggests that the symmetry related internal TA base pairs break in a coupled manner at low ionic strength, with the coupling removed in the presence of Mg ions and high salt. By contrast, the symmetry related terminal AT base pairs break independently of each other in the absence and presence of Mg ions and high salt. The terminal base pair exhibits a Tm of 10-15 degrees lower than that of the internal base pair in the hexanucleotide, with divalent Mg ions and high salt stabilizing the double helix as reflected in the Tm values of these base pairs. The observed line width changes as a function of temperature provide an estimate of the exchange rate of the proton from the coil form with water. The exchange reaction from the coil state is base catalyzed with rate constants in the diffusion limit.

Binding Sites↗
PubMed1975

Proton nuclear magnetic resonance investigations of the nucleation and propagation reactions associated with the helix-coil transition of d-ApTpGpCpApT in H2O solution.

The chemical shifts and line widths of the Watson-Crick ring NH resonances of the self-complementary duplex of d-ApTpGpCpApT have been monitored in the presence of 0.1 M phosphate at neutral pH in aqueous solution. While the resonance positions of the terminal and internal AT base pairs shift upfield and broaden as average resonances with increasing temperature (helix and coil exchange several times prior to exchange with water from the coil form), those of the central GC base pairs broaden in the absence of upfield shifts (exchange with water occurs each time helix converts to coil). The line-width changes at the AT base pairs monitor the lifetime of the coil state at these positions prior to exchange with water while the line-width changes at the GC base pairs monitor the lifetime of the helix prior to dissociation to strands. This permits the separation of the propagation reaction at the AT base pairs from the nucleation reaction at the GC base pairs during helix formation. The experimental data have been quantitatively analyzed to yield (at 20 degrees) a nucleation formation rate of approximately 10(3) sec-1 for the GC base pairs (bimolecular rate constant of approximately 6 times 10(6) l. mol-1 sec-1) and a dissociation rate of 6 times 10(2) sec-1 at these same base pairs (unimolecular dissociation to strands). The unimolecular propagation reactions at the terminal and terminal base pairs are associated with reaction rates greater than 10(4) sec-1. These values are consistent with a slow formation of a stable nucleus at the GC base pairs followed by a rapid propagation reaction at the AT base pairs. The line width of the (GC) central base pairs in the presence of phosphate is a direct measure of the lifetime of the total helix and yields an activation energy of 45 kcal for helix to coil conversion measured over a narrow temperature range. The exchange from the coil form with water is catalyzed by 0.1 M phosphate with a rate constant kHPO2-/4 = 0.2 times 10(6) 1. mol-1 sec-1.

Hydrogen-Ion Concentration↗
PubMed1975

The predicted secondary structure of the N-terminal sequence of the lac repressor and proposed models for its complexation to the lac operator.

Rules for the prediction of protein conformation (Chou, P. Y., and Fasman, G. D. (1974), Biochemistry 13, 211, 222) have been applied to the N-terminal sequence 1-60 of the lac repressor. This analysis predicts beta structure at sequences 4-9 and 15-20, helices at 26-32, 38-45, and 52-57, and beta turns at 48-51 and 14-17. Repressor mutants lacking operator binding capacity in which Pro replaces Ser-16 and Ala replaces Thr-19 (Weber, K., Platt, T., Ganem, D., and Miller, J. H. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 3624) have no effect on the prediction of beta structure at residues 15 to 20, which suggests that the polar side chains of Ser-16, Tyr-17, Gln-18, and Thr-19 participate in intermolecular hydrogen bonding with conplementary polar groups on the lac operator. The loss of operator binding capacity on replacement of Ala by Val at position 53 in the repressor results from a predicted secondary structural change from helix to beta structure for residues 52-57 which can be transmitted to the N-terminal sequence via a beta turn at residues 48-51. The basic residues at positions 33, 35, and 37 between the helical regions 26-32 and 38-45 probably bind to the phosphate groups on the operator on complexation. It is proposed that complex formation involves the interaction of either a beta structure (residues 15-20) or a right-hand twisted antiparallel beta-pleated sheet (residues 4-9 and 12-20) with operator DNA.

Amino Acid Sequence↗
PubMed1975

Static linear and nonlinear elastic properties of normal and arterialized venous tissue in dog and man.

Ten normal and four transplanted canine jugular vein segments and four human saphenous vein segments were studied to determine the in vitro static elastic properties of venous tissue and their modification by transplantation into the arterial system. Both the intraluminal pressure and the longitudinal force were varied, and the resulting dimensions were recorded photographically. Venous segments manifested a hysteresis response but showed minimum tendency to creep. The pressure-strain relationships were curvilinear with an initial, highly compliant phase over the physiological venous pressure range followed by a relatively noncompliant phase. This transition occurred at lower pressures for jugular segments than it did for saphenous segments. In contrast, comparable-sized canine carotide artery segments did not show this essentially noncompliant phase over the pressure range studied (0 to 200 cm H2O). At comparable pressures and strains, the jugular vein segments were stiffer than the saphenous vein segments in both the circumferential and the longitudinal directions. At comparable strains, the saphenous vein moduli were similar to those in the carotid artery segments. Jugular segments transplanted into arterial circuits became virtually noncompliant and markedly inhomogeneous, with wall thickening and a histologic picture of intimal proliferation. They showed no tendency to "arterialize," that is, they failed to assume either the elastic or the histologic characteristics of arterial tissue.

Biomechanical Phenomena↗