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R W Hendler

Publications and source records attributed to R W Hendler.

At least 55 records · Page 3Linked to original sources
PubMed1986

Complete analysis of the cytochrome components of beef heart mitochondria in terms of spectra and redox properties. Cytochromes aa3.

Using newer techniques of data collection that accumulate entire spectra at a series of discrete voltages and newer techniques of analysis that utilize the additional data, we have re-examined the redox behavior and corresponding difference spectra of redox centers responsible for the alpha absorbance features of cytochromes aa3 in beef heart mitochondria. Our analysis reveals three Nernstian components with Em values of 200, 260, and 340 mV with n values of 2, 2, and 1, respectively. The maximum alpha absorbance in the difference spectra for each of these species is located at 602, 605, and 607 nm respectively. Titrations in the presence of carbon monoxide led to the identification of the lowest voltage species as cytochrome a3. The Em of the carbon monoxide-liganded species was not raised. This is contrary to the result expected when a ligand has a much stronger affinity for the reduced form of a redox couple than the oxidized form. It is, however, consistent with a proton-pumping model of cytochrome oxidase in which the binding of ligand results in the dissociation of protons.

Animals↗
PubMed1986

Analysis of the spectra and redox properties of pure cytochromes aa3.

The findings in the current studies with pure cytochrome aa3 confirm the findings in an accompanying paper pertaining to cytochrome aa3 in mitochondria (Reddy et al., 1985). In both cases, three Nernstian titrations are seen with Em values near 200, 260, 340 mV with n values of 2, 2, and 1. Similarly, the alpha absorption features of the difference spectra in both cases were centered near 602, 605, and 607 mn. The component with Em approximately 200 mV was identified as heme a3 on the basis of experiments conducted in an atmosphere of carbon monoxide, and in both cases, the carbon monoxide-liganded species did not display an elevated Em. In the current studies, unique Soret absorbance features are added to the difference spectra for the three Nernstian transitions. Specifically, absorption peaks at 429, 446, and 448 nm go with the alpha peaks seen respectively at 602, 605, and 607 nm. Evidence was presented to support the hypothesis that the redox state of heme alpha may control the redox potential of heme a3.

Animals↗
PubMed1985

The H+/O ratio of proton translocation linked to the oxidation of succinate by mitochondria. Reply to a commentary.

Costa, L.E., Reynafarje, B. and Lehninger, A.L. [(1984) J. Biol. Chem. 259, 4802-4811] have reported 'second-generation' measurements of the H+/O ratio approaching 8.0 for vectorial H+ translocation coupled to succinate oxidation by rat liver mitochondria. In a Commentary in this Journal [Krab, K., Soos, J. and Wikström, M. (1984) FEBS Lett. 178, 187-192] it was concluded that the measurements of Costa et al. significantly overestimated the true H+/O stoichiometry. It is shown here that the mathematical simulation on which Krab et al. based this claim is faulty and that data reported by Costa et al. had already excluded the criticism advanced by Krab et al. Also reported are new data, obtained under conditions in which the arguments of Krab et al. are irrelevant, which confirm that the H+/O ratio for succinate oxidation extrapolated to level flow is close to 8.

Animals↗
PubMed1985

Thermodynamic and kinetic considerations of Q-cycle mechanisms and the oxidant-induced reduction of cytochromes b.

In coenzyme Q-cycles, it is proposed that one electron from the quinol reduces the Rieske iron sulfur center (Em approximately 280 mV) and the remaining electron on the semiquinone reduces cytochrome br (Em approximately -60 mV). The Em for the two-electron oxidation of the quinol is approximately 60 mV and therefore the reduction of cytochrome bT by quinol is not favorable. As the stability constant for the dismutation of the semiquinone decreases, the calculated Em for the Q/QH. couple is lowered to values below the Em of cytochrome bT. Contemporary coenzyme Q-cycles are based on the belief that the lower value for the Em of the Q/QH. couple compared to the Em for cytochrome bT means that the semiquinone is a spontaneous reducing agent for the b-cytochrome. The analysis in the paper shows that this is not necessarily so and that neither binding sites nor ionization of the semiquinone per se alters this situation. For a Q-cycle mechanism to function, ad hoc provisions must be made to drive the otherwise unfavorable reduction of cytochrome bT by the semiquinone or for the simultaneous transfer of both electrons to cytochrome bT and cytochrome c1 (or the iron sulfur protein). Q-cycle mechanisms with these additional provisions can explain the observation thus far accumulated. A linear path which is functionally altered by conformational changes may also explain the data.

Cytochrome b Group↗
PubMed1983

Complete analysis of the cytochrome components of beef heart mitochondria in terms of spectra and redox properties. The b-type cytochromes.

The b-cytochromes of beef heart mitochondria have been analyzed by procedures which use optical spectra rather than two points for each cytochrome species. The analytical procedures included first derivatives for background behavior, second derivatives for peak features, and singular value decomposition (Shrager, R.I., and Hendler, R.W. (1982) Anal. Chem. 54, 1147-1152) for the entire spectra. Titrations were performed by electrical oxidation and reduction and by chemical reduction. Four b-cytochromes have been identified in terms of their unique difference spectra, midpoint potentials (Em), and number of electrons transferred (n). They are cytochrome bK, Em = 83 mV, n = 1; cytochrome bT2, Em = -17 mV, n = 2; cytochrome bT1; Em = -95 mV, n = 1; and cytochrome b562L, Em = -146 mV, n = 4. The Em values of cytochromes bT1, bT2, and b562L respond to changes in pH indicating a loss of one proton per oxidation. Antimycin and/or ATP do not affect the spectrum or Em value of any of the cytochromes. An apparent red shift for the absorption maximum of cytochrome bK caused by antimycin may be due to an increased level of reduction of cytochrome bT2.

Animals↗
PubMed1983

A cellular factor involved in the formation of a DNA-synthesizing complex from DNA polymerase I in Escherichia coli.

A factor ('E') has been identified which stabilizes an endogenous DNA-synthesizing complex involving DNA polymerase I. The complex is separated from free DNA polymerase by polyacrylamide gel electrophoresis. The factor will reform the complex after it has been dissociated and will convert a preparation of DNA polymerase I to complex. The factor and the DNA-synthesizing complex both appear to be localized at the cell membrane.

DNA Polymerase I↗
PubMed1983

Simultaneous measurements of proton motive force, delta pH, membrane potential, and H+/O ratios in intact Escherichia coli.

An instrument is described that enables the simultaneous monitoring of proton motive force (PMF), membrane potential (delta psi), the delta pH across a membrane, oxidase activity, proton movements, and H+/O ratios. We have studied the relationship existing among these parameters of energy transduction as a critical condition is changed during an experiment. The major findings are: (a) In the pH range of 4.5 to 7.5, increasing the external pH causes an increase in delta psi, internal pH, and oxidase activity, a decrease in H+/O ratio, and a peak-plateau in PMF from pH 5.5 to 6.6 where delta pH is converted to delta psi. (b) An increase in [K+] from 1 to 100 mM, in the presence of 0.5 microM valinomycin, causes the conversion of delta psi to delta pH, a gradual decline in PMF and an increase in H+/O ratio, internal pH, and oxidase activity. (c) Increasing valinomycin concentration from 0 to 2.5 microM, in the presence of 50 mM [K+], causes a decline in delta psi from 125 to 0 mV, and an increase in delta pH from 35 to 70 mV. From 2.5 to 10 microM, the delta pH and the PMF (which it solely represents), stay constant, H+/O ratio increases mainly from 0 to 0.5 microM and much more slowly from 2.5 to 10 microM. (d) Oxygen at only 10% of its concentration in air-saturated buffer can support the generation of 90% or more of the delta psi, delta pH, and PMF generated in an air-saturated solution. (e) The return of extruded protons to the cell (referred to here as "suck-back") represents a complicated process driven by delta psi and influenced by a variety of factors. (f) H+/O ratios measured by the kinetic technique used here are much higher than those measured by standard oxygen pulse techniques.

Cell Membrane↗
PubMed1982

Interference of electron donors in the measurement of the proton gradient and membrane potential by flow dialysis.

The three most commonly used electron donors for flow dialysis measurements of membrane potential lead to the development of an apparent but artifactual membrane potential with the interior negative in the presence or absence of membrane vesicles. The same three electron donors used in flow dialysis determinations of delta pH in the presence or absence of membrane vesicles lead to the development of an apparent but artifactual delta pH with the interior acidic. These artifacts have been evaluated using two probes for membrane potential, namely, TPP+ and rubidium in the presence of valinomycin and for two probes of delta pH, namely, acetate and DMO. Measurements were made over a range of ionic strengths.

Dialysis↗
PubMed1979

Potentiometric analysis of Escherichia coli cytochromes in the optical absorbance range of 500 nm to 700 nm.

The oxidation-reduction potentials of Escherichia coli cytochromes have been studied by a recently described technique for automated electrodic potentiometry (Hendler, R.W., Songco, D., and Clem, T.R. (1977) Anal. Chem. 49, 1908-1913; Hendler, R.W. (1977) Anal. Chem. 49, 1914-1918), where entire spectra are recorded at a series of solution potentials. New techniques for resolution of the spectra versus voltage data have been applied. The results indicate that a 1-electron transport chain conducts electrons from substrate to cytochrome d, which is the cytochrome oxidase. Cytochrome d contains several components which appear to increase electron transfer first to a 2-electron stage and then to a 4-electron stage for the final reduction of a molecule of oxygen to 2 molecules of water.

Cell Membrane↗
PubMed1978

Reconstitution of escherichia coli succinoxidase from soluble components.

1. The membrane-bound succinoxidase of Escherichia coli was fractionated with deoxycholate into three soluble components, viz. succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase complex, and a factor identified as a phospholipid-containing component. 2. The dehydrogenase and cytochrome oxidase complexes were partially purified by filtration on Amicon membranes, Sepharose 4B chromatography, and sucrose gradient centrifugation. 3. Reconstitution of membranous succinoxidase, which catalyzes the oxidation of succinate by molecular oxygen by an integrated CN(-)-sensitive pathway, was achieved by mixing the soluble succinate dehydrogenase.cytochrome b1 complex with the soluble cytochrome oxidase complex in the presence of deoxycholate and then removing the detergent by gel filtration on Sephadex G-75. The phospholipid-containing factor stimulated the formation of succinoxidase by about 100% over that observed with the two complexes. 4. Isopycnic sucrose gradient centrifugation of succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase, and the reconstituted succinoxidase gave buoyant densities (p value) as 1.167, 1.229, and 1.194, respectively. 5. Electron microscopic evidence is presented for the vesicular nature of the reconstituted succinoxidase.

Cell Membrane↗
PubMed1975

Redox properties of beta-type cytochromes in Escherichia coli and rat liver mitochondria and techniques for their analysis.

We describe here apparatus and procedures for conducting potentiometric titrations and for analyzing the collected data in terms of the number of components present, their amounts and their midpoint potentials. Using these procedures we have determined the presence of three forms of cytochrome b1 in Escherichia coli with midpoint potentials at pH 7.1 OF about minus 50, plus 110 and plus 220 mV. We were not able to demonstrate a change in any of these potentials by the addition of phosphate, ATP, or 2, 4-dinitrophenol. We have been able to confirm the presence of two forms of cytochrome b in non-energized mitochondria and the apparent conversion of the low-potential component to a new high potential component upon energization of the mitochondria. However we cite further experimental data that question the actual conversion of one form of cytochrome b to another. An alternative interpretation based on our analysis suggests that the high voltage component may be present in a masked form in the non-energized mitochondria.

Animals↗
PubMed1975

DNA synthesis involving a complexes form of DNA polymerase I in extracts of Escherichia coli.

DNA polymerase I (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) has been recovered as a complex of about 390,000 molecular weight. The complex displays an ATP-stimulated DNA-synthesizing activity that prefers native to heat-denatured DNA. Genetic evidence indicates that the recBC enzyme is associated with the polymerase in the complex. Preliminary evidence for complexes involving DNA polymerases II and III is also presented.

Adenosine Triphosphate↗