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.

Theory and procedures for finding a correct kinetic model for the bacteriorhodopsin photocycle.

In this paper, we present the implementation and results of new methodology based on linear algebra. The theory behind these methods is covered in detail in the Supporting Information, available electronically (Shragerand Hendler). In brief, the methods presented search through all possible forward sequential submodels in order to find candidates that can be used to construct a complete model for the BR-photocycle. The methodology is limited only to forward sequential models. If no such models are compatible with the experimental data,none will be found. The procedures apply objective tests and filters to eliminate possibilities that cannot be correct, thus cutting the total number of candidate sequences to be considered. In the current application,which uses six exponentials, the total sequences were cut from 1950 to 49. The remaining sequences were further screened using known experimental criteria. The approach led to a solution which consists of a pair of sequences, one with 5 exponentials showing BR* f L(f) M(f) N O BR and the other with three exponentials showing BR* L(s) M(s) BR. The deduced complete kinetic model for the BR photocycle is thus either a single photocycle branched at the L intermediate or a pair of two parallel photocycles. Reasons for preferring the parallel photocycles are presented. Synthetic data constructed on the basis of the parallel photocycles were indistinguishable from the experimental data in a number of analytical tests that were applied.

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.

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.

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.

O2-binding to fully reduced cytochrome aa3 reexamined using SVD analysis of simulated and real data.

Using new time-resolved multichannel experimental and simulated data, analyzed by SVD, it is concluded that the second order binding rate constant for O2-binding to fully reduced mammalian cytochrome aa3 is approximately five times higher than the currently accepted value of approximately 1.5 x 10(8) M-1 s-1.

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.

13C/31P NMR studies on the mechanism of insulin resistance in obesity.

The mechanism of insulin resistance in obesity was examined in ten obese (BMI 33 +/- 1 kg/m2) and nine lean (BMI 22 +/- 1 kg/m2) Caucasian women during a hyperglycemic-hyperinsulinemic clamp using 13C and 31P nuclear magnetic resonance (NMR) spectroscopy to measure rates of muscle glycogen synthesis and intramuscular glucose-6-phosphate (G-6-P) concentrations. Under similar steady-state plasma concentrations of glucose (approximately 11 mmol/l) and insulin (approximately 340 pmol/l), rates of muscle glycogen synthesis were reduced approximately 70% in the obese subjects (52 +/- 8 micromol/[l muscle-min]) as compared with the rates in the lean subjects (176 +/- 22 micromol/[l muscle-min]; P < 0.0001). Basal concentrations of intramuscular G-6-P were similar in the obese and lean subjects; but during the clamp, G-6-P failed to increase in the obese group (deltaG-6-P obese 0.044 +/- 0.011 vs. lean 0.117 +/- 0.011 mmol/l muscle; P < 0.001), reflecting decreased muscle glucose transport and/or phosphorylation activity. We conclude that insulin resistance in obesity can be mostly attributed to impairment of insulin-stimulated muscle glycogen synthesis due to a defect in glucose transport and/or phosphorylation activity.

Laparoscopic treatment of gastroesophageal reflux disease.

Laparoscopic fundoplication is technically feasible in treating gastroesophageal reflux disease (GERD). Although medication is the primary treatment for GERD, not all patients respond completely or are able to adhere to a medical regimen. In the present series, 59 patients were laparoscopically treated for GERD at three centers using a standardized technique. All patients had been medically treated prior to referral, although 84 per cent had heartburn and 2 per cent had laryngitis despite 20 to 40 mg/day of omeprazole. Fifteen per cent of patients were intolerant of or would no longer take omeprazole. Patients were evaluated by esophageal manometry (in 100%) and 24-hour pH studies (in 66%). Seventy-six per cent of patients had lower-esophageal sphincter pressure <15 mm Hg. Five patients had low esophageal body peristaltic pressures (<35 mm Hg). These patients underwent Toupet partial fundoplication, whereas 54 patients underwent Nissen fundoplication. Mean operative time was 158 +/- 7 minutes, and three patients (5%) were converted to an open procedure. Operative complications were minor and occurred in 13 per cent. In 45 patients evaluated 1 year after surgery, heartburn had resolved in 98 per cent. Thirty-nine of 56 patients (70%) had mild early (<1 month postoperatively) dysphagia, and 9 (19%) had severe early dysphagia, which improved in 7 after nonoperative dilatation. Two of these had continued mild dysphagia. Two patients had severe dysphagia and were laparoscopically converted from Nissen to Toupet fundoplications, which resulted in marked improvement. Early gas bloat symptoms occurred in 45 per cent and dropped to 5 per cent at 1 year. Laparoscopic treatment of GERD is safe and effective in preventing reflux symptoms. Although mild dysphagia occurs after the procedure, this is transient in most patients. Patients with severe dysphagia can be treated with nonoperative dilatation or laparoscopic partial fundoplication and maintain the antireflux characteristics of the wrap.

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.

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.

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.

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.

Laparoscopic reoperation on failed antireflux procedures: report of two patients.

Two cases are presented that demonstrate the feasibility of using the laparoscopic transabdominal approach for failed antireflux procedures. The first patient had two previous Belsey hemifundoplications that had both failed and had intractable reflux unresponsive to conservative treatment. The Belsey operation was taken down and a floppy Nissen fundoplication performed; the patient had only transient postoperative dysphagia. The second patient had a standard floppy Nissen fundoplication performed over a large bougie and developed a postoperative motility problem that failed to respond to medication and dilation. The Nissen was taken down laparoscopically and converted to a Toupet procedure; the patient had total relief of the dysphagia. Neither patient had intra-abdominal complications, and both have had total relief of their heartburn and regurgitation.

Lipid-induced conformational changes of an integral membrane protein: an infrared spectroscopic study of the effects of Triton X-100 treatment on the purple membrane of Halobacterium halobium ET1001.

Exposure of purple membrane from Halobacterium halobium to sublytic concentrations of Triton X-100 results in significant changes in the bacteriorhodopsin (BR) photocycle (Mukhopadhyay et al., 1994). Infrared spectra of purple membrane samples exposed briefly to Triton indicate that this change in protein function accompanies the preferential release of purple membrane glycolipids and squalenes, an association of Triton with purple membrane, and a perturbation of specific lipid headgroup interactions within the membrane. Specifically, the bilayer alterations induced by Triton entail a disruption of lipid headgroup hydrogen bonding in addition to protein conformational changes involving a loss in beta-turn and alphaII-helical structures in BR. We propose that the purple membrane glycolipids and squalenes are critical for the normal functioning of the BR photocycle and that perturbations of these lipids cause the profound photocycle changes induced by exposure to Triton. Lipid reconstitution studies demonstrated that although several of the infrared spectral parameters characteristic of the structural changes induced by Triton were reversed, the photocycle characteristics of BR in native purple membrane were not regained. The observed changes in the vibrational spectra induced by lipid-mediated bilayer perturbations suggest a useful approach for clarifying structure-function relationships of intrinsic membrane proteins exhibiting transmembrane helices.

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.

The effects of weight reduction to ideal body weight on body fat distribution.

Obesity is a well-known health risk factor. Several studies have demonstrated that upper-body fat distribution plays a major role in the association between increased adiposity and metabolic disorders. The present study was undertaken to evaluate changes in intraabdominal and subcutaneous fat areas in obese subjects undergoing a weight reduction to their ideal body weight (IBW), as defined by a body mass index (BMI) no greater than 21 or body fat less than 30%, and compare the fat distribution at IBW with that of never-obese control subjects. We studied 33 obese women (151% +/- 1% of IBW; BMI, 31.6 +/- 2.5 [mean +/- SE]) before and after weight loss and a control group of 16 never-obese women (101.0% +/- 1.0% of IBW; BMI, 21.2 +/- 1.1). Eighteen obese women successfully achieved and stabilized at IBW for at least 2 months. Nonsuccessful obese subjects were significantly younger than reduced-weight subjects, but other physical characteristics were similar. In obese, reduced-obese, and never-obese groups, weight was 85 +/- 2.0, 62 +/- 1, and 58 +/- 1 kg; percent body fat was 41% +/- 1%, 24% +/- 2%, and 23% +/- 1%; intraabdominal fat area was 82 +/- 5, 28 +/- 3, and 25 +/- 4 cm2; waist subcutaneous fat area was 275 +/- 15, 120 +/- 9, and 81 +/- 7 cm2; hip subcutaneous fat area was 416 +/- 17, 204 +/- 10, and 195 +/- 7 cm2; and waist to hip ratio (WHR) was 0.84 +/- 0.02, 0.77 +/- 0.01, and 0.73 +/- 0.01, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.