Moises Eisenberg

The kinetic mechanism of action of an uncoupler of oxidative phosphorylation.

SummaryThe chemiosmotic hypothesis predicts that the mechanism by which weak acids uncouple oxidative phosphorylation in mitochondria is identical to the mechanism by which they transport hydrogen ions across artificial bilayer membranes. We report here the results of a kinetic study of uncoupler-mediated hydrogen ion transport across bilayer membranes. We made electrical relaxation measurements on black lipid membranes exposed to the substituted benzimidazole 5,6-dichloro-2-trifluoromethylbenzimidazole. The simplest model consistent with our experimental data allowed us to deduce values for adsorption coefficients and rate constants. Our major conclusions are that the back diffusion of the neutral species is the rate limiting step for the steady state transport of hydrogen ions, that both the neutral and charged forms of the uncoupler adsorb strongly to the interfaces, and that the reactions at the membrane-solution interfaces occur sufficiently rapidly for equilibrium to be maintained. Independent measurements of the adsorption coefficients of both the neutral and anionic forms of the weak acid and also of the permeability of the membrane to the neutral form agreed well with the values deduced from the kinetic study.

A fluorescence quenching technique for the measurement of paramagnetic ion concentrations at the membrane/water interface. Intrinsic and X537A-mediated cobalt fluxes across lipid bilayer membranes

We have characterized the quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine by Co2+ in egg phosphatidylcholine (PC) lipid bilayer vesicles. The quenching constant obtained is 59 M-1. We demonstrate one use of this fluorescence quenching technique by measuring intrinsic and X537A-mediated transmembrane Co2+ fluxes in large unilamellar PC vesicles. The intrinsic rate constant for Co2+ flux we measure is 3 X 10(-6) S-1. We confirm that the neutral Co approximately (X537A)2 complex is the main component of the X537A-mediated cobalt flux. Since this method measures the concentration of Co2+ at the site of the fluorophore, it is generally applicable to the measurement of paramagnetic ion concentrations in the region of the membrane/water interface.

Recognition and repair of 8-oxoguanine and formamidopyrimidine lesions in DNA.

Reactive oxygen species, including hydroxyl radicals, arise during the enzymatic and chemical reduction of molecular oxygen in cells. Hydroxyl radicals are also formed when cells are exposed to ionizing radiation, tumor promoters, and certain chemical carcinogens. Reactive oxygen species produce a variety of complex lesions in DNA. 8-Oxoguanine (8-oxodG) and formamidopyrimidine (Fapy) are among the more abundant products of oxidative DNA damage. Both lesions are actively repaired in prokaryotic and eukaryotic cells. The mutagenic potential of 8-oxodG is reflected in its miscoding properties. Primer extension reactions, catalyzed by DNA polymerases, show that dAMP and dCMP can be incorporated on DNA templates during tranlesional synthesis and readily extended from the 3{prime} terminus. In contrast, Fapy lesions block the progression of DNA polymerase. Plasmid vectors containing a single lesion were used to establish the mutational frequency and specificity of 9oxodG in bacteria and mammalian cells. 9-OxodG is weakly mutagenic in both systems; G:C{l_arrow}T:A transversions are targeted to the site of the lesion. Mutagenic properties have not been reported for Fapy.

Analysis and prediction of hydrogen bonding in protein‐DNA complexes using parallel processors

A number of essential biological functions are controlled by proteins that bind to specific sequences in genomic DNA. In this article we present a simplified model for analyzing DNA‐protein interactions mediated exclusively by hydrogen bonds. Based on this model, an optimized algorithm for geometric pattern recognition was developed. The large number of local energy minima are efficiently screened by using a geometric approach to pattern matching based on a square‐well potential. The second part of the algorithm represents a closed form solution for minimization based on a quadratic potential. A Monte Carlo method applied to a modified Lennard‐Jones potential is used as a third step to rank DNA sequences in terms of pattern matching. Using protein structures derived from four DNA‐protein complexes with three‐dimensional coordinates established by X‐ray diffraction analysis, all possible DNA sequences to which these proteins could bind were ranked in terms of binding energies. The algorithm predicts the correct DNA sequence when at least two hydrogen bonds per base pair are involved in binding to the protein, providing a partial solution to the three‐dimensional docking problem. This study lays a framework for future refinements of the algorithm in which the number of assumptions made in the present analysis are reduced.