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T G Dewey

Publications and source records attributed to T G Dewey.

29 records · Page 2Linked to original sources
PubMed1987

Chemical relaxation in a chemiosmotic-coupled system: driving the calcium adenosinetriphosphatase with bacteriorhodopsin.

Phase-lifetime spectroscopy has been used to measure chemical relaxation processes in a chemiosmotic-coupled system. In this experiment the calcium ATPase and bacteriorhodopsin were coreconstituted into phospholipid vesicles. Upon illumination the bacteriorhodopsin pumps protons into the vesicles and forms a membrane potential. This membrane potential alters the activity of the internal calcium and thus perturbs the equilibrium of ATP hydrolysis/synthesis coupled to calcium transport. Mechanically chopping the actinic light provides a periodic perturbation to the system, and small response signals can be observed by using phase-sensitive detection. It is shown that this periodic perturbation occurs about a steady-state membrane potential that is independent of chopping frequency. The amplitude dispersion curve for the fluorescence of a calcium indicator was observed and analyzed in terms of the relaxation time for the ATPase-catalyzed calcium transport. Thus, this technique provides a method of measuring ion transport kinetics against a constant chemiosmotic potential. The calcium ATPase showed a single relaxation time on this time scale. The dependence of this relaxation time on ADP and phosphate concentration was measured and analyzed with a random sequential mechanism. This analysis gave dissociation constants for ADP and phosphate of 3.2 mM and 1.4 mM, respectively. These binding steps are followed by slow isomerization steps with forward and reverse rate constants (in the direction of ATP synthesis) of 67 s-1 and 227 s-1, respectively. These results demonstrate that highly accurate kinetic data can be obtained with this modulation relaxation technique.

Adenosine Diphosphate↗
PubMed1987

A fluorescence investigation of the nucleotide binding sites of the Ca ATPase.

Competitive binding and fluorescence energy transfer experiments were used to examine the binding of 2'[3']-O-(2,4,6-trinitrophenyl) adenosine-5'-diphosphate (TNP-ADP) to the catalytic site of Ca ATPase. Fluorescein isothiocyanate (FITC), which is known to covalently bind near the catalytic site (13), was shown to exclude all TNP-ADP binding. TNP-ADP, in turn, will protect against FITC labeling of the Ca ATPase in its native state and in both phosphoenzyme forms. The competitive nature of these probes indicates that TNP-ADP binds to the catalytic site exclusively. Fluorescence energy transfer studies using TNP-ADP as an energy acceptor and 1,N6-ethenoadenosine-5'-diphosphate (epsilon-ADP) as an energy donor were used to estimate the distance between nucleotide binding sites in the enzyme complex. A lower limit for the distance measured was 44 A. This is interpreted to be the distance between catalytic sites on adjacent monomers of a dimer unit. The results of this work are consistent with a single nucleotide site per ATPase monomer.

Adenosine Diphosphate↗
PubMed1986

Resonance energy transfer study of membrane-bound aggregates of the sarcoplasmic reticulum calcium ATPase.

The aggregation of the membrane-bound calcium ATPase from sarcoplasmic reticulum has been studied by resonance energy transfer. The temperature dependence of resonance energy transfer from a fluorescent membrane lipid donor to an acceptor covalently linked to the Ca2+ ATPase was observed for the native sarcoplasmic reticulum vesicles and for purified protein reconstituted into phospholipid vesicles. The efficiency of energy transfer in these systems increases as the size of protein aggregates decrease. This is due to the increased exposure of the protein in the lipid domain that results in the shortening of distances between donors and acceptors. The degree of aggregation was observed to decrease with increasing temperature. Aggregates rea h a limiting size at low temperature (5 degrees C) but not a high temperatures (45 degrees C). For the reconstituted system, the aggregate size showed a continuous, smooth decrease with increasing temperature. Sarcoplasmic reticulum vesicles showed a decrease in aggregation except for a region from 20 to 30 degrees C in which no change occurred. Arrhenius plots of the calcium transport activities for both systems do not reflect these differences, but instead show similar discontinuities and activation energies. A theoretical model is used to analyze the resonance energy transfer results for the reconstituted vesicles. The average radius of the ATPase aggregate is obtained from this analysis. The limiting, low temperature value of the aggregate radius is consistent with the formation of a tetramer. This structure breaks down to smaller, functional units at higher temperatures.

Calcium↗
PubMed1985

Steady state kinetics of ATP synthesis and hydrolysis coupled calcium transport catalyzed by the reconstituted sarcoplasmic reticulum ATPase.

The steady state kinetics of calcium transport driven by ATP hydrolysis and ATP synthesis catalyzed by purified, reconstituted calcium ATPase has been investigated as a function of the transmembrane calcium gradient. Purified calcium ATPase was reconstituted into phospholipid vesicles enabling control of the transmembrane calcium gradient. Calcium transport was monitored spectrophotometrically by the calcium indicator, Arsenazo III. Thus, only the enzymatic activity of coupled transport was measured. It was shown under conditions of low external calcium that ATP hydrolysis and synthesis follow simple Michaelis-Menten kinetics and that Michaelis constants obtained for both processes appear independent of the calcium gradient. The maximum velocities for both hydrolysis and synthesis strongly depend on the transmembrane calcium gradient. Based on these results, a mechanism is proposed in which a random addition of substrates for ATP synthesis is followed by random release of ATP and calcium. By measuring the ATP hydrolysis and synthesis under identical conditions, determination of the equilibrium constant for ATP hydrolysis as a function of the transmembrane calcium gradient was possible. Our results indicate that the thermodynamics of the catalytic cycle can be totally accounted for by the energetics of transport of 2.2 +/- 0.3 calciums and the hydrolysis of 1 ATP. An equilibrium constant for ATP hydrolysis in the absence of a calcium gradient was determined to be 4.0 X 10(4).

Adenosine Triphosphate↗
PubMed1984

Statistical mechanical model of the energetics of coated vesicle formation.

Adsorptive endocytosis is mediated by a structural transformation of a two-dimensional hexagonal lattice to a polyhedron or coated vesicle. Clathrin is the structural protein involved in this process. A theoretical model that focuses on the energetics of clathrin in coated vesicle formation is presented. Fisher's cluster model of phase transitions is applied to this problem. The equilibrium constant for the process of converting a large two-dimensional patch to a coated vesicle and a smaller patch is calculated. There are three energetic contributions to be considered. They are the surface energy, the interior or lattice energy, and a loop entropy. Features of the Ising model are introduced into this model by scaling the critical exponents. In determining the configurational partition functions required for equilibrium constant calculations, the polyhedron is represented as a planar graph with no surfaces. The equilibrium constants are extremely sensitive to changes in the lattice and surface energies. The loop entropy contributions favor bimodal distributions in vesicle size under certain conditions. Conditions can also be established where the energy required to form the vesicle is small.

Clathrin↗
PubMed1981

Steady state kinetics of ATP synthesis and hydrolysis catalyzed by reconstituted chloroplast coupling factor.

The steady state kinetics of ATP synthesis and hydrolysis catalyzed by the chloroplast dicyclohexylcarbodiimide-sensitive ATPase reconstituted into phospholipid vesicles were studied as a function of the transmembrane proton gradient. Bacteriorhodopsin also was incorporated into the vesicles so that a constant pH gradient could be maintained by continuous illumination of the liposomes. The dependence of the initial rates of ATP synthesis and hydrolysis on substrate concentrations is consistent with Michaelis-Menten kinetics, with enzyme, ADP, and Pi forming a ternary complex. The Michaelis constants for both synthesis and hydrolysis are essentially independent of the pH gradient, while the maximum velocities depend strongly on it. The equilibrium constant for hydrolysis was calculated from the steady state kinetic parameters, and the dependence of the equilibrium constant on the pH gradient indicates that 3 protons are transported per ATP synthesized or hydrolyzed. The dependence of the steady state kinetic parameters on the pH gradient can be described by a mechanism in which the binding of substrates occurs before the transport of protons and the transport of the 3 protons is sequential rather than concerted.

Adenosine Triphosphate↗
PubMed1981

Solvent effects on the kinetics and thermodynamics of stacking in poly(cytidylic acid).

The Raman laser temperature-jump technique has been used to measure the kinetics of the coil to helix reaction of poly(cytidylic acid) [poly(C)] in aqueous cosolvent mixtures. The rate of helix formation has a low activation energy and is proportional to reciprocal solvent viscosity. The observations suggest helix formation is rotationally diffusion controlled. The rate of coil formation in poly(C) has an activation energy of approximately 11 kcal/mol, presumably reflecting the electronic stacking interactions which stabilize the helix. Viscous cosolvents, glycerol or sucrose, slow down the rate of coil formation; acetonitrile and formamide at 5 mol % increase the rate relative to that in water. The polar cosolvents may specifically attack a cytosine stack. The absorbance vs. temperature profiles for poly(C) are analyzed with the one-dimensional Ising model. When only optical data are used, the cooperativity parameter, sigma, and the enthalpy, delta H, cannot be uniquely determined. A method is proposed that allows determination of sigma by combining spectroscopic and calorimetric data. The values of sigma derived for poly(C) are between 0.8 and 1.0, and delta H is about -9 kcal/mol of stack. An alternative method using integration of the excess heat capacity curve and extrapolation to fully stacked and random coil species yields a delta H of -7 kcal/mol of stack.

Acetonitriles↗
PubMed1981

Method for studying kinetics of light-induced transport across membranes.

A technique based on phase spectrophotometry is described for studying the rates of elementary processes associated with the light-driven transport of ions and molecules across membranes. The light-induced pumping of protons by bacteriorhodopsin reconstituted into phospholipid vesicles and by chloroplast thylakoids has been studied to illustrate the potential of this technique. The exciting light is modulated by a mechanical chopper over the frequency range 5 Hz to 2 kHz. The internal pH of the membrane vesicles is modulated at the same frequency as the exciting light but differs in phase because of the finite rate of proton pumping. Measurement of this phase difference or of the frequency dispersion of the amplitude of the internal pH modulation is accomplished by use of a lock-in amplifier. The results can be interpreted in terms of relaxation times characterizing the chemical steps in proton pumping. The shortest relaxation time that can be measured is about 50 microseconds, although the time resolution could be easily extended by use of faster light chopping techniques. At pH 8.0, two relaxation processes are associated with proton pumping by bacteriorhodopsin reconstituted into phospholipid vesicles; the relaxation times are 2 and 28 msec. Two relaxation processes also were found with chloroplast thylakoids at pH 7.8, with relaxation times of 2 and 16 msec. The former can be associated with photosystem II and the latter, with photosystem I.

Bacteriorhodopsins↗
PubMed1980

Laser temperature jump study of solvent effects of poly(adenylic acid) stacking.

The dynamics of the single-stranded, helix--coil transition of poly(adenylic acid) have been investigated by using the Raman laser temperature jump technique. The driving forces of this conformational transition have been probed by varying the cosolvent in mixed aqueous solutions. The rate of helix formation correlates well with the inverse of the solution viscosity. This correlation and the low activation barrier of approximately 4 kcal/mol for this process indicate a rotational diffusion controlled reaction. The rate of coil formation has a much higher activation barrier, presumably due to the strength of base stacking in the helix. This unstacking rate is virtually unaffected by alcohol cosolvents. A significant increase in this rate occurs when polar cosolvents such as urea, formamide, or acetonitrile are added. Absorbance changes with temperature suggest that urea specifically solvates the adenine base. The polar cosolvents appear to break up the stacked, helical conformation by interacting with the bases.

Cacodylic Acid↗
PubMed1980

Calculation on fluorescence resonance energy transfer on surfaces.

A general method for estimating fluorescence resonance energy transfer between distributions of donors and acceptors on surfaces is presented. Continued fraction approximants are obtained from equivalent power series expansions of the change in quantum yield in terms of the fluorescent lifetimes or the steady-state fluorescence. These approximants provide analytic equations for the analysis of energy transfer and error bounds for the approximants. Specific approximants are derived for five models of interest for membrane biochemistry: (a) an infinite plane, (b) parallel infinite planes, (c) the surface of a sphere, (d) the surfaces of concentric spheres, and (e) the surfaces of two separated spheres. Recent experimental results in the literature are analyzed with the equations obtained.

Energy Transfer↗