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M D Barkley

Publications and source records attributed to M D Barkley.

25 records · Page 2Linked to original sources
PubMed1986

Comparison of approaches to the instrumental response function in fluorescence decay measurements.

Deconvolution of pulse fluorometry data requires knowledge of the instrumental response, which is not directly observable in some circumstances. Various procedures for approaching the instrumental response function were evaluated for nanosecond fluorescence decay data analyzed by nonlinear least squares, including the commonly used time shift correction and several reference fluorophore methods. A new reference fluorophore technique using a Monte Carlo convolution is introduced and tested. The correction for scattered light in several reference techniques is also presented. The random convolution and one other reference fluorophore method consistently gave superior results over a wide range of experimental conditions.

Computers↗
PubMed1985

Tryptophan fluorescence of terminal deoxynucleotidyl transferase: effects of quenchers on time-resolved emission spectra.

Terminal deoxynucleotidyl transferase (EC 2.7.7.31) is a eucaryotic DNA polymerase that does not require a template. The tryptophan environments in calf thymus terminal transferase were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was separated by time-resolved emission spectroscopy. Nanosecond fluorescence decays at 296-nm excitation and various emission wavelengths were deconvolved by global analysis, assuming that the lifetimes but not the relative weighting factors were independent of emission wavelength. The data were fit to three exponentials of lifetimes tau 1 = 1.4 ns, tau 2 = 4.5 ns, and tau 3 = 7.7 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 328, 335, and 345 nm. The accessibility of individual tryptophan environments to polar and nonpolar fluorescence quenchers was examined in steady-state and time-resolved experiments. In the presence of iodide and acrylamide, the steady-state emission spectra shift to the blue. However, at low quencher concentrations, the emission from the 7.7-ns component (maximum 345 nm) is hardly affected, suggesting that this hydrophilic tryptophan environment is buried within the protein. On the other hand, the red shift in the steady-state emission spectrum in the presence of trichloroethanol indicates that the 1.4-ns component (maximum 328 nm) is an exposed hydrophobic tryptophan environment. The results are consistent with an inside-out model for terminal transferase protein, with the more hydrophobic tryptophan(s) near the surface and the most hydrophilic tryptophan(s) in the core.

Animals↗
PubMed1985

Fluorimetric assay for terminal deoxynucleotidyl transferase activity.

A fluorimetric assay for measuring terminal deoxynucleotidyl transferase activity in purified and crude enzyme preparations has been developed. Etheno-substituted deoxynucleotides are shown to be substrates of the enzyme. The assay involves polymerization of the fluorescent nucleotide 1,N6-ethenodeoxyadenosine triphosphate (epsilon dATP) on an oligodeoxynucleotide initiator, [poly(deoxyadenylic acid) with an average chain length of 50 residues] under the reaction conditions used in the standard radiometric assay. The incorporation of epsilon dATP into polymer is quantitated by fluorescence after isolation and nuclease digestion of the product. The enzymological properties of etheno substrates were also determined. Epsilon dATP binds about twofold tighter than dATP to terminal transferase, but has a twofold-lower catalytic rate. However etheno substitution does not affect initiator binding. The fluorimetric assay is suitable for clinical analysis of terminal transferase in human leukemias, and may be a useful adjunct to recently developed immunochemical methods which detect protein, not activity.

Animals↗
PubMed1981

Salt dependence of the kinetics of the lac repressor-operator interaction: role of nonoperator deoxyribonucleic acid in the association reaction.

The kinetics of binding of lac repressor protein and operator deoxyribonucleic acid (DNA) have been studied as a function of monovalent and divalent cation concentration. The salt dependence of the association and dissociation rate constants has been interpreted in light of recent theoretical analyses based on Manning's counterion condensation model. The bell-shaped dependence of the association rate constant on salt concentration evidences a role for nonoperator DNA binding in the repressor's search for the operator site on a large DNA molecule. At intermediate mono- or divalent cation concentrations, the association rate goes through a maximum. At lower cation concentrations, it decreases and becomes dependent on DNA concentration; the high affinity of repressor for nonoperator DNA confines the protein to the DNA. At higher cation concentrations, the association rate decreases and becomes dependent on the weak affinity of repressor for nonoperator DNA. The kinetic data are fit to the theory of Berg and Blomberg [Berg, O. G., & Blomberg, C. (1978) Biophys. Chem. 8, 271] for the salt dependence of association kinetics with coupled diffusion, using published values of the affinity for nonoperator DNA. From this fit, one-dimensional diffusion of repressor along the DNA chain is estimated to be about 4 times faster on MgDNA than on NaDNA. At higher cation concentrations, the salt dependence of the association and dissociation rate constants is consistent with a preequilibrium mechanism for the association reaction [Lohman, T. M., deHaseth, P. L., & Record, M. T., Jr. (1978) Biophys. Chem. 8, 281]. Agreement between literature values (corrected for the presence of Mg2+) and experimental values of the rate constants in the presence of monovalent salt is quite good.

DNA, Viral↗
PubMed1981

Ion effects on the lac repressor--operator equilibrium.

The effects of ions on the interaction of lac repressor protein and operator DNA have been studied by the membrane filter technique. The equilibrium association constant was determined as a function of monovalent and divalent cation concentrations, anions, and pH. The binding of repressor and operator is extremely sensitive to the ionic environment. The dependence of the observed equilibrium constant on salt concentration is analyzed according to the binding theory of Record et al. [Record, M. T., Jr., Lohman, T. M., & deHaseth, P. L. (1976) J. Mol. Biol. 107, 145]. The number of ionic interactions in repressor--operator complex is deduced from the slopes of the linear log-log plots. About 11 ionic interactions are formed between repressor and DNA phosphates at pH 7.4 and about 9 ionic interactions at pH 8.0, in reasonable agreement with previous estimates. A favorable nonelectrostatic binding free energy of about 9-12 kcal/mol is estimated from the extrapolated equilibrium constants at the 1 M standard state. The values are in good accord with recent results for the salt-independent binding of repressor core and operator DNA. The effects of pH on the repressor--operator interaction are small, and probably result from titration of functional groups in the DNA-binding site of the protein. For monovalent salts, the equilibrium constant is slightly dependent on cation type and highly dependent on anion type. At constant salt concentration, the equilibrium constant decreases about 10000-fold in the order CH3CO2- greater than or equal to F- greater than Cl- greater than Br- greater than NO3- greater than SCN- greater than I-. The wide range of accessible equilibrium constants provides a useful tool for in vitro studies of the repressor--operator interaction.

Anions↗
PubMed1975

Interaction of effecting ligands with lac repressor and repressor-operator complex.

The equilibrium association constants for the binding of a wide variety of effecting ligands of the lac repressor were measured by equilibrium dialysis. Also, detailed investigations of the apparent rate of dissociation of repressor-operator comples as a function of ligand concentration were carried out for several inducers and anti-inducers. The affinity of repressor-ligand comples for operator DNA was evaluated from the specific rate constants at saturating concentrations of effecting ligand. By fitting the experimental data depicting the functional dependence of the rate of dissociation upon ligand concentrations to calculated curves, assuming simple models of the induction mechanism, the equilibrium association constant for the binding of effecting ligand to repressor-operator comples was determined. Inducers reduce the affinity of lac repressor for operator DNA by a factor of approximately 1000 under standard conditions; the extent of destabilization depends on Mg2+ ion concentration. Anti-inducers increase the affinity of repressor for operator at most a factor of five. Only one neutral ligand, which binds to repressor without altering the stability of repressor-operator comples, was found. No homotropic or heterotropic interactions in the binding of effecting ligands either to repressor or to repressor-operator complex are evident.

Binding Sites↗