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R W Hendler

Publications and source records attributed to R W Hendler.

At least 19 recordsLinked to original sources
PubMed2001

Importance of lipids for bacteriorhodopsin structure, photocycle, and function.

This review begins with a brief history of early studies on the involvement of lipids in certain bacteriorhodopsin (BR) properties. Such properties include the regulation of the pK for the purple to blue transition caused by deionization, and the reformation of trimers from monomers after exposure of the purple membrane to Triton X-100. Most of the review is devoted to newer studies which indicate an important role for the neutral lipid squalene in the functional stability of the fast-decaying M-intermediate, for its decay through a pathway involving the O-intermediate, and for the regulation of the relative amounts of slow-decaying and fast-decaying forms of M. Participation of a peripheral acidic amino acid in the overall expression of fast-decaying M is also discussed. Initial studies suggest that the acidic amino acid may be Asp36 and/or Asp38.

Bacteriorhodopsins↗
PubMed2000

On the kinetics of voltage formation in purple membranes of Halobacterium salinarium.

The kinetics of the bacteriorhodopsin photocycle, measured by voltage changes in a closed membrane system using the direct electrometrical method (DEM) of Drachev, L.A., Jasaitus, A.A., Kaulen, A.D., Kondrashin, A.A., Liberman, E.A., Nemecek, I.B., Ostroumov, S.A., Semenov, Yu, A. & Skulachev, V.P. (1974) Nature 249, 321-324 are sixfold slower than the kinetics obtained in optical studies with suspensions of purple membrane patches. In this study, we have investigated the reasons for this discrepancy. In the presence of the uncouplers carbonyl cyanide m-chlorophenylhydrazone or valinomycin, the rates in the DEM system are similar to the rates in suspensions of purple membrane. Two alternative explanations for the effects of uncouplers were evaluated: (a) the 'back-pressure' of the Deltamicro;H+ slows the kinetic steps leading to its formation, and (b) the apparent difference between the two systems is due to slow major electrogenic events that produce little or no change in optical absorbance. In the latter case, the uncouplers would decrease the RC time constant for membrane capacitance leading to a quicker discharge of voltage and concomitant decrease in photocycle turnover time. The experimental results show that the primary cause for the slower kinetics of voltage changes in the DEM system is thermodynamic back-pressure as described by Westerhoff, H.V. & Dancshazy, Z. (1984) Trends Biochem. Sci. 9, 112-117.

Bacteriorhodopsins↗
PubMed1999

Regulation of the bacteriorhodopsin photocycle and proton pumping in whole cells of Halobacterium salinarium.

Single-turnover kinetics of the bacteriorhodopsin photocycle and proton-pumping capabilities of whole cells were studied. It was found that the Delta mu (tilde)H+ of the cell had a profound influence on the kinetics and components of the cycle. For example, comparing the photocycle in whole cells to that seen in PM preparations, we found that (1) the single-turnover time of the cycle was increased approximately 10-fold, (2) the mole fraction of M-fast (at high actinic light) decreased from 50 to 20%, and (3) the time constant for M-slow increased significantly. The level of Delta mu(tilde)H+ was dependent on respiration, ATP formation and breakdown, and the magnitude of a pre-existing K+ diffusion gradient. The size of the Delta mu(tilde)H+ could be manipulated by additions of HCN, nigericin, and DCCD (N,N'-dicyclohexylcarbodamide). At higher levels of Delta mu(tilde)H+, further changes in the photocycle were seen. (4) Two slower components of M-decay appeared as major components. (5) The apparent conversion of the M-fast to the O intermediate disappeared. (6) A partial reversal of an early photocycle step occurred. The photocycle of intact cells could be changed to that seen in purple membrane suspensions by the energy-uncoupler CCCP or by lysis of the cells. In fresh whole cells, light-induced proton pumping was not seen until the K+ diffusion potential was dissipated and proton accumulation facilitated by use of a K+-H+ exchanger (nigericin), respiration was inhibited by HCN, and ATP synthesis and breakdown were inhibited by DCCD. In stored cells, the pre-existing K+ diffusion gradient was diminished through slow diffusion, and only DCCD and HCN were required to elicit proton extrusion.

Bacteriorhodopsins↗
PubMed1998

Importance of specific native lipids in controlling the photocycle of bacteriorhodopsin.

Brief treatment of purple membrane (PM) with dilute detergent can cause major disruption of the BR photocycle without disrupting the trimer structure of BR [Mukhopadhyay et al. (1996) Biochemistry 35, 9245-9252]. Normal photocyle behavior can be recovered by incubating the damaged membranes with a total extract of the five types of native lipids present in PM. It is shown here that full restoration can also be obtained with combinations of squalene (SQ) and phosphatidyl glycerophosphate (PGP) which act synergistically. The addition of SQ to suboptimal levels of PGP induces complete reconstitution, principally by restoring the characteristics of the fast M intermediate, Mf (as defined in Mukhopadhyay et al. (1996) Biochemistry 35, 9245-9252). The addition of small amounts of PGP to SQ, which alone is ineffective, also induces full reconstituion. At very high levels, full reconstitution can be obtained with PGP alone. These results, in combination with earlier studies which implicate an acidic amino acid residue [Bose et al. (1997) J. Phys. Chem. B 101, 10584-10587], suggest that a crucial interaction between a particular amino acid residue and a SQ-PGP lipid complex may be essential for normal BR photocycle activity.

Bacteriorhodopsins↗
PubMed1998

Some pitfalls in curve-fitting and how to avoid them: a case in point.

When curve-fitting is used to support a complex nonlinear model containing several exponential terms, some of which have closely-spaced time constants, a particular burden of proof must be assumed. Most important, the uniqueness of the solution must be explored and discussed. Statistical tests for the degree of error and independence of the parameters should be provided, as well as information relating to the steps actually used in the fitting procedures. As an example of the need for the procedures we recommend in this communication, we have chosen an important case in point that has been published recently, and which deals with the kinetics of electron transfer from fully-reduced cytochrome oxidase to O2, analyzed by the method of SVD-based least squares. The problems we deal with in this case are applicable to a wide variety of other cases that involve curve-fitting to mathematical models.

Electron Transport↗
PubMed1997

A high speed optical multichannel analyzer.

An optical multichannel analyzer capable of recording spectra at sampling rates up to 100 kHz is described. The instrument, designed to gather data on the kinetic reaction mechanisms of biological preparations such as cytochrome oxidase and bacteriorhodopsin, features a massively parallel approach in which each photosensing element of the detector array has a dedicated amplifier, integrator, analog to digital converter, and sample buffer. The design has 92 such elements divided in two separate arrays, each of which sits at the focal plane of a 1/4 m Ebert spectrometer. The spectrometers may be tuned to cover independent, 130 nm wide, regions of the spectrum from 350 nm to 900 nm with a dispersion of 2.8 nm per element. Each detection channel has 12-bit resolution with an electronic dark count of 1 count and may be sampled 1024 times during a single experiment with dynamically variable sampling intervals from 10 microseconds to several seconds. Time averaging of up to thousands of consecutive laser-initiated kinetic cycles allows analyses of spectral changes < 0.001 optical density units. A personal computer with custom software provides a number of features: entry of experiment parameters; transfer of data from temporary buffers to permanent files; real time display; multiple spectrum averaging; and control and synchronization of associated system hardware. Optical fibers or lenses provide coupling from a parabolic reflector Xenon arc monitoring light source, through the sample chamber, to the entry slit of the monochromator. The instrument has been used for extensive studies on the rapid kinetics and definition of reaction sequences of the energy-transducing enzymes cytochrome oxidase and bacteriorhodopsin. Some results from these studies are discussed.

Bacteriorhodopsins↗
PubMed1997

Multichannel analysis of single-turnover kinetics of cytochrome aa3 reduction of O2.

The single-turnover kinetics of the oxidation of cytochrome aa3 by O2 have been studied using a new approach. Up to 1000 whole spectra covering both the Soret and alpha regions were sequentially collected at room temperature from single samples with a time resolution of 10 microns. All of the spectral and time information were used in analyses based on singular value decomposition. Four spectral transitions (i.e., intermediates) were distinguished with time constants near 0.01, 0.1, 1.1, and 30 ms. Two different kinds of sequential models were evaluated, one linear and the other branched. Although past kinetic analyses have emphasized the linear sequential model, the complexity of the intramolecular electron transfer in this enzyme suggests that a branched model be considered. This is especially true in a single-turnover experiment where earlier optical and EPR studies have pointed unequivocally to a branched model [Clore et al. (1980) Biochem. J. 185, 139-154; Blair et al. (1985) J. Am. Chem. Soc. 107, 7389-7399]. In the present study, analysis of spectral data in terms of the linear model did not reveal the formation and decay of the expected oxyferryl intermediate, whereas analysis of the branched model did. The results obtained using the branched model are consistent with all of the available evidence from a broad range of physical techniques that have been applied to examine the single-turnover kinetics of the oxidation of reduced cytochrome aa3 by O2.

Animals↗
PubMed1996

A monitoring system for energy transduction by bacteriorhodopsin liposomes.

A computer-controlled system and custom software are described that collect information and perform computations to quantify important parameters of energy transduction during the conversion of photons into a proton electrochemical gradient (delta mu H+) by bacteriorhodopsin (BR)-liposomes. The strong actinic light used to energize the BR-liposomes causes several serious problems for the approaches commonly used to measure these parameters. This paper identifies these problems and presents solutions that permit the acquisition of the desired information, namely, the initial (1st sec) rate and total extent of H+ translocation, rate of H+ leakage (driven by an existing delta mu H+), external, internal and delta pH values, and delta psi values. The system is presented with representative experimental data.

Bacteriorhodopsins↗
PubMed1996

Control of the integral membrane proton pump, bacteriorhodopsin, by purple membrane lipids of Halobacterium halobium.

Brief exposure of purple membrane (PM) to dilute Triton X-100 eliminates the actinic light effect on the relative amounts of fast M (Mf) and slow M (Ms) intermediates and alters the character and kinetics of the photocycle, without destroying the native BR trimers (Mukhopadhyay et al., 1994). Particular membrane lipids are removed during the Triton treatment, and adding back an extract of membrane lipids can repair most of the affected photocycle behavior (Dracheva et al., 1996). This paper defines conditions which are important in the reconstitution procedure, using a group of quantitative parameters which measure the extents of damage and repair. Circular dichroism in both the UV and visible ranges shows that Triton can disturb both the secondary structure of BR and its ability to polymerize into trimers. Whereas the damage to protein conformation could be reversed by lipids alone, the formation of trimers and recovery of normal photocycle behavior required both lipids and a high salt concentration.

Bacteriorhodopsins↗
PubMed1996

Chemical and functional studies on the importance of purple membrane lipids in bacteriorhodopsin photocycle behavior.

In native purple membrane (PM), there are approximately 1 squalene, 2 glycolipid sulfate (GLS), and 6 phospholipid (PL) molecules per bacteriorhodopsin (BR) monomer. Brief (approximately 2 min) exposure to 0.1% Triton X-100 removes about 25%, 20%, and 6% of squalenes, GLS, and PL, respectively (this paper) while causing profound changes in the BR photocycle, including the loss of 'photocooperativity'. The BR photocycle in Triton-treated PM can be restored to near normal behavior by reconstitution with native PM lipids. Isolated squalenes are not effective whereas PL alone partially restores normal photocycle characteristics.

Bacteriorhodopsins↗
PubMed1995

The ability of actinic light to modify the bacteriorhodopsin photocycle. Heterogeneity and/or photocooperativity?

The focus of this paper is on the established observation that the bacteriorhodopsin (BR) photocycle responds to the level of actinic light by altering the proportions of two forms of the M intermediate. The first form of M, called M-fast or MF, decays to the O intermediate. In contrast, the second form of M, called M-slow or MS, decays directly to the ground state, and its decay rate is slower than that of MF. Any proposed scheme for the BR photocycle must account for this light-dependent phenomenon. Several papers have attempted to explain the observation on the basis of photocooperativity, or on the basis of heterogeneous populations. In this paper, we test previously proposed cooperative models with experimental data, and find those models to be inadequate. We show that two new models, one purely cooperative, the other purely heterogeneous, can both fit the data, hence such modelling will not resolve the mechanism. Taking into account the demonstration of heterogeneity, the trimer structure of BR, and certain experimental evidence in favor of cooperativity, it appears likely that both heterogeneity and cooperativity are involved in the adaptation of the BR photocycle to different levels of actinic light.

Bacteriorhodopsins↗
PubMed1995

Functional synergism of the magainins PGLa and magainin-2 in Escherichia coli, tumor cells and liposomes.

Xenopus laevis skin secretion contains a mixture of magainins, which are small positively charged oligopeptides with antimicrobial activity. In this study, we show that two of these peptides, i.e. magainin-2 and PGLa, are much more active in biological functions when added together than when added alone. This synergy applies for the antimicrobial activity of these peptides, and for the toxic effects on tumor cells. We show that this peptide combination is also synergistic when permeabilizing protein-free liposomes for glucose, when dissipating the membrane potential in cytochrome oxidase liposomes and Escherichia coli, and, reversibly, when stimulating respiration in the liposomes. The occurrence of synergy in these diverse systems (complex and simple) suggests that the biological synergy results from synergy in the primary activity of the magainin peptides, namely the permeabilization of free-energy transducing membranes, possibly by forming a multimeric transmembrane pore of mixed peptide composition. The antimicrobial activity of X. laevis skin secretions may be greatly enhanced by the application of this binary weapon.

Amino Acid Sequence↗
PubMed1995

Combination of potentiometry and resonance Raman spectroscopy for the analysis of a redox protein.

This paper describes apparatus and procedures for combining resonance Raman and optical absorption spectroscopies with potentiometry for the study of redox-active heme proteins. A specially designed anaerobic titration cell is described which allows for the laser excitation of the sample and the monitoring of both Raman scattered light and directly transmitted light from an optical source. New procedures for utilization of A/D and D/A converters on a standard I/O computer card are described, which allow for computer-controlled potentiometry and coulometry. The system was tested with cytochrome c, a well-characterized respiratory protein. The correct values for the midpoint potential and electron number of the Nernst equation were obtained both by the optical absorption and resonance Raman measurements.

Cytochrome c Group↗
PubMed1994

Membrane-mediated control of the bacteriorhodopsin photocycle.

The ability of actinic light to modify the proportion of fast and slow forms of the M intermediate (i.e., Mf and M(s)) in the bacteriorhodopsin (BR) photocycle is lost by exposure of the purple membrane (PM) to 0.05% Triton for 1-2 min. The decay path of Mf through the O intermediate is also lost, and new, much slower kinetic forms of M appear. In this brief exposure, the trimer structure for BR, as measured by circular dichroism (CD) exciton coupling and sedimentability, is unaffected. The optical properties of the treated PM are affected within seconds of exposure to the detergent as indicated by an increase in transmittance and a blue shift in the wavelength of maximum absorbance for the ground state. Different concentrations of Triton cause reproducibly different changes in the kinetics of the system. These observations support the view that the BR trimer-membrane interaction is important in controlling the BR photocycle.

Bacteriorhodopsins↗
PubMed1994

Magainin oligomers reversibly dissipate delta microH+ in cytochrome oxidase liposomes.

Magainin peptides present in the skin of Xenopus laevis and identified as antimicrobial agents are shown to decrease the membrane potential in cytochrome oxidase liposomes. They also released respiratory control with a third or higher order concentration dependence. Respiratory control was restored by proteolytic digestion of the added magainin. The amount of magainin required for half-maximal stimulation of respiration was proportional to lipid concentration. At appreciably higher concentrations magainins inhibited uncoupled respiration. The results are discussed in terms of a model in which most of the added magainin adsorbs as a monomer to the membranes but equilibrates with a multimeric pore that causes rather general permeability of membranes. The ensuing ion permeation dissipates membrane potential and stimulates respiration.

Animals↗
PubMed1994

Influence of excitation energy on the bacteriorhodopsin photocycle.

Kinetic curves for the bacteriorhodopsin (BR) photocycle were obtained both at 570 and at 412 nm at a series of increasing levels of intensity of the exciting laser. Singular value decomposition (SVD) of these curves showed two transitions in the kinetic profiles that occurred at specific levels of actinic light. This means that the photocycle was influenced by photon density in two ways. In a separate application of SVD, time-resolved optical spectra were analyzed at each of many levels of exciting laser intensities. The studies showed that the transition at the low level of laser intensity was due principally to an increase in the amount of BR that was turning over. The transition at the higher level of laser intensity showed a fundamental change in kinetics of the photocycle. At low intensity levels, the fast form of M (Mf) predominated, whereas at high levels the slow form of M (Ms) predominated. A distinction was found between Mf and Ms, in that the former decayed directly to the O intermediate whereas the latter decayed directly to BR.

Bacteriorhodopsins↗
PubMed1994

Resonance Raman and optical spectroscopic monitoring of heme a redox states in cytochrome c oxidase during potentiometric titrations.

Resonance Raman spectroscopy is used to monitor the redox state of heme a and heme a3 centers in cyanide-inhibited and native cytochrome oxidase during potentiometric titrations. Specific vibrational modes are resolved for each reduced heme with 441.6-nm excitation while oxidized species show vanishingly small Raman intensities. The voltage dependencies of the Raman intensities of reduced heme a and reduced heme a3 modes are quantitatively measured and used to extract heme a and a3 midpoint potentials. In the cyanide-bound enzyme, in which heme a3 remains in the oxidized state, the Raman data indicate that heme a centers exhibit complex Nernstian behavior with two Em values near 350 and 260 mV. In the native enzyme, this resonance Raman-potentiometric method reveals significantly different redox behavior for the two hemes. Heme a centers are described by two effective Em values near 350 and 220 mV, while heme a3 centers have lower Em values near 260 and 200 mV. Singular value decomposition analysis of optical spectral changes supports the Raman data. These results are in contrast to models of cytochrome oxidase redox behavior in which heme a and heme a3 are thought to have essentially identical midpoint potentials.

Animals↗