Search PubMed⌕ Search

Biomedical subjects

W Stoeckenius

Publications and source records attributed to W Stoeckenius.

At least 55 records · Page 3Linked to original sources
PubMed1981

Walsby's square bacterium: fine structure of an orthogonal procaryote.

The "square" bacterium, first described by Walsby from brine collected at the Red Sea shore [A. E. Walsby, Nature (London) 283:69-71, 1980] was examined by electron microscopy. The cells appeared as flat rectangular boxes in scanning electron micrographs. In sections and freeze-fracture preparation, the edges looked more rounded. The thickness apparently remains constant as the cells grow and divide. Their sides were a few micrometers long, but the cells were only 0.25 micrometers thick. They showed typical procaryote structure, with a regular cell wall and a gas vacuole fine structure similar to that of other halophilic procaryotes. The inner fracture faces of the cell membrane showed a much denser population of intramembrane particles than the outer fracture faces, but no patches of purple membrane, despite the presence of bacteriorhodospin-like pigment in the cell suspension. Morphologically identical cells have been found in brine from Baja California, Mexico.

Bacteria↗
PubMed1980

Effect of protein-protein interaction on light adaptation of bacteriorhodopsin.

Triton X-100 solubilized monomers of bacteiorhodopsin (bR) show a decrease in the extent of light adaptation; the red shift and the absorbance increase of the visible absorption band are reduced no less than half the values observed in purple membrane (p.m.) with a corresponding reduction in the isomerization of 13-cis- to all-trans-retinal. Cross-linking of bR with glutaraldehyde before exposure to Triton prevents dissociation of the lattice and reduction in light adaptation. Experiments with cross-linked and lipid-extracted p.m. show that Triton effectively substitutes for the native membrane lipids and that the lattice structure apparently stabilizes the light-adapted state of bR under illumination. In lipid vesicles at molar lipid protein ratios greater than or equal to 80, bR exists as monomers above the lipid-phase transition and aggregates below the phase transition. Above the lipid-phase transition and aggregates below the phase transition. Above the lipid-phase transition light adaptation in the monomers, measured as either the red shift of the visible absorbance maximum or the isomerizaiton o 13-cis- to all-trans-retinal, is also reduced to less than half of the extent observed in intact purple membrane or in the bR aggregates formed in lipid vesicles below the plhase transition. At very high lipid to protein ratios, bR molecules cannot aggregate when the temperature is decreased below the phase transition, and these monomers in a solid lipid phase show the same reduced extent of light adaptation as monomers above the phase transition, thus confirming that this effect is mainly due to the absence of protein-protein interaction and not to the state of the lipid. The extent of the red shift upon light adaptation may be used as a convenient indicator to distinguish the aggregated and monomeric states of bR.

Bacteriorhodopsins↗
PubMed1980

Light-regulated retinal-dependent reversible phosphorylation of Halobacterium proteins.

By (32P)orthophosphate pulse-labeling in vivo, we have identified a class of phosphoproteins in Halobacterium halobium. Exposing labeled whole cells to light results in a rapid dephosphorylation of two of the proteins, which are rapidly rephosphorylated upon darkening of the cells. The light sensitivity of these two proteins is abolished by the retinal synthesis inhibitor nicotine and is restored upon addition of exogenous retinal to the cells. This indicates that the dephosphorylation depends on the rhodopsin-like (retinal-containing) photoreceptors of this organism. Retinal pigments, one of which is the retinyldene protein bacteriorhodopsin, are responsible for both photoenergy and photosensory transduction by Halobacterium. The light-regulated phosphoproteins are of molecular weights of approximately 80,000 and 100,000. Acid and hydroxylamine resistance of the phosphate bonds in both proteins suggests phosphoserine or phosphothreonine linkages.

Bacterial Proteins↗
PubMed1980

Action spectrum and quantum efficiency for proton pumping in Halobacterium halobium.

The action spectrum and quantum efficiency (phi H+) for proton ejection from Halobacterium halobium have been determined under conditions chosen to minimize light-triggered proton influx which is usually observed in intact cells. The action spectrum for the carotenoid-containing strain, R1, shows that light energy absorbed by the carotenoids does not contribute to the proton ejection. After correction for shielding by the carotenoids and other cell pigments, the action spectrum closely follows the absorption spectrum of bacteriorhosopsin. Values determined for phi H+ in H. halobium cells and cell envelopes range from 0.4 to 0.7. These values are significantly higher than the currently accepted value for the quantum efficiency for the photoreaction cycle of bacteriorhodopsin in isolated purple membrane, suggesting that at least in intact cells and envelopes more than one proton is pumped during the bacteriorhodopsin photocycle. A new nondestructive assay for bacteriorhopopsin in intact cells and envelopes which also contain other pigments was used in this work.

Bacteriorhodopsins↗
PubMed1980

Light-dark adaptation of bacteriorhodopsin in triton-treated purple membrane.

Solubilization of purple membrane with Triton X-100 yields Triton micelles containing bacteriorhodopsin monomers. The absorption maximum of dark-adapted solubilized bacteriorhodopsin is blue-shifted to 549 nm. Light adaption increases the absorbance by 4% and shifts the absorption maximum to 553 nm, i.e., the extent of light adaptation is considerably less than in intact purple membrane. Extraction of dark-adapted bacteriorhodopsin in Triton yields a 13-cis- to all-trans-retinal ratio of 58 : 42 which changes after light adaptation to 38 : 62. It has been shown by Sperling et al. (Sperling, W., Carl, P., Rafferty, Ch.N. and Dencher, N.A. (1977) Biophys. Struct. Mech. 3, 79-94) that light adaptation in intact purple membrane occurs through a branching of the 13-cis photoreaction cycle, so that part of the pigment during each cycle crosses over into the all-trans photoreaction cycle. We explain the decreased extent of light adaptation in solubilized bacteriorhodopsin by assuming a significant back reaction from the all-trans to the 13-cis cycle. This assumption predicts a wavelength dependence of the extent of light adaptation, which is born out by experiment.

Bacteriorhodopsins↗
PubMed1980

Exchange kinetics of the Schiff base proton in bacteriorhodopsin.

Using rapid mixing techniques and resonance Raman spectroscopy, we have found that the 1H/2H exchange time for the Schiff base proton of bacteriorhodopsin in purple membrane is 4.7 msec, when experiments are carried out at pH 2 or pH 7 at room temperature in the dark. We argue that diffusion of neutral water into the membrane is fast on this time scale. Also, model Schiff bases in solution have a pKa between 6 and 7, and we show that such model Schiff bases have much faster exchange rates. Therefore, we conclude that the Schiff base proton in bacteriorhodopsin is protected from interaction with the medium, probably by interaction with a protein group, and this would account for a pKa considerably higher than 6-7.

Bacteriorhodopsins↗
PubMed1980

Location of the chromophore in bacteriorhodopsin.

We present a location for the retinylidene chromophore in dark-adapted bacteriorhodopsin based on the differences in neutron scattering between purple membrane preparations reconstituted with retinal and with deuterated retinal. The Fourier difference density map contains more peaks than expected, and additional arguments are introduced to exclude artificial peaks, caused by the reconstitution techniques or the limited resolution of the diffraction data. The membrane preparation used is necessarily dark-adapted and therefore contains 13-cis- and all-trans-retinal isomers in roughly equal amounts. However, we find only a single position for both isomers. Presumably, the difference in conformation caused by isomerization around the C13-C14 double bond is minimized by rotation around other bonds. The retinal is located between alpha-helical segments of the protein and its nearest neighbor (intratrimer) distance is 26 A; the next-nearest neighbor (intertrimer) distance is 38 A.

Apoproteins↗
PubMed1979

Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin.

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.

Bacteriorhodopsins↗
PubMed1978

Transient photovoltages in purple membrane multilayers. Charge displacement in bacteriorhodopsin and its photointermediates.

The photovoltaic properties of bacteriorhodopsin molecules and their photochemical intermediates have been investigated in an experimental cell consisting of multilayered films of highly oriented, dry fragments of purple membrane and lipid sandwiched between two metal (Pd) electrodes. The electrical time constant of these sandwich cells containing between 5 and 30 layers is less than 10(-5) S. Bright illumination of these cells with actinic flashes of approximately 1 ms duration generates transient photovoltages. These photovoltages, which make the extracellular surface of purple membrane positive with respect to the intracellular surface, follow the time course of the flash with no detectable latency. The amplitude of the photovoltages increases linearly with light intensity and their action spectrum matches the absorption spectrum of the light-adapted state of bacteriorhodopsin, BR570. In these dry multilayer cells, the slow photointermediates of bacteriorhodopsin, M412, N520 and O640 are long lived. Illumination of the sandwich cells with long duration (200 ms) pulses of light results, therefore, in the formation of photomixtures containing all these slow photointermediates. Flash illumination of the sandwich cells immediately following the conditioning pulse produces photovoltages whose action spectra match the absorption spectra of the M412 and N520 photointermediates. The M412 photovoltages, like the BR570 photovoltages, follow the time course of the actinic flash with no detectable latency and increase in amplitude linearly with light intensity. But, unlike the BR570 photovoltage, the M412, N520 and O640 photovoltages make the extracellular surface of purple membrane negative with respect to the intracellular surface. Through the of their specific photovoltaic signals, M412 and N520 are shown to be kinetically distinct photointermediates of bacteriorhodopsin. Detection of fast photovoltages with these characteristics in the absence of any ionic solution, and in parallel with spectrophotometric changes, suggest that they arise from charge displacements in the bacteriorhodopsin molecules and their photointermediates as they undergo photochemical conversion in response to the absorption of photons.

Bacteriorhodopsins↗
PubMed1978

Picosecond and steady state, variable intensity and variable temperature emission spectroscopy of bacteriorhodopsin.

The bacteriorhodopsin emission lifetime at 77 degrees K has been obtained for different regions of the emission spectrum with single-pulse excitation. The data under all conditions yield a lifetime of 60 +/- 15 ps. Intensity effects on this lifetime have been ruled out by studying the relative emission amplitude as a function of the excitation pulse energy. We relate our lifetime to previously reported values at other temperatures by studying the relative emission quantum efficiency as a function of temperature. These variable temperature studies have indicated that an excited state with an emission maximum at 670 nm begins to contribute to the spectrum as the temperature is lowered. Within our experimental error the picosecond data seem to suggest that this new emission may arise from a minimum of the same electronic state responsible for the 77 degrees K emission at 720 nm. A correlation is noted between a 1.0-ps formation time observed in absorption by Ippen et al. (Ippen, E.P., C.V. Shank, A. Lewis, and M.A. Marcus. 1978. Subpicosecond spectroscopy of bacteriorhodopsin. Science [wash. D.C.]. 200:1279-1281 and a time extrapolated from relative quantum efficiency measurements and the 77 degrees K fluorescence lifetime that we report.

Bacteriorhodopsins↗
PubMed1978

Oriented adsorption of purple membrane to cationic surfaces.

We have investigated the orientation of isolated fragments of Halobacterium halobium purple membrane (PM) adsorbed to poly-L-lysine-treated glass (PL-glass), by quanitative electron microscopy. Three lines of evidence support the conclusion that the cytoplasmic side of the membrane is preferentially absorbed. First, monolayer freeze-fracture reveals nonrandom orientation; more fracture faces (89%) are particulate than smooth. Second, the amount of each membrane surface present can be assayed using polycationic ferritin; 90% of all adsorbed membrane fragments are labeled. Third, it is possible to distinguish two surfaces, "cracked" (the extracellular surface) and "pitted" (the cytoplasmic surface) , in slowly air-dried, platinum-carbon-shadowed membranes. When applied under standard conditions, more than 80% appear cracked. Selection for the cytoplasmic by the cationic substrate suggests that the isolated PM, buffered at pH 7.4 and in the light, has a higher negative charge on its cytoplasmic surface than on its extracellular surface. Nevertheless, cationic ferritin (CF) preferentially adsorbs to the extracellular surface. Orientation provides a striking example of biomembrane surface asymmetry as well as the means to examine the chemical reactivity and physical properties of surfaces of a purified, nonvesicular membrane fragment.

Adsorption↗
PubMed1977

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films.

A suspension of purple membrane fragments in a solution of soya phosphatidyl-choline in hexane is spread at an air-water interface. Surface pressure and surface potential measurements indicate that the membrane fragments and lipids organize at the interface as an insoluble film. Electron microscopy of shadow-cast replicas of the film reveal that in the bacteriorhodopsin to soya PC weight ratio range of 2:1 to 10:1, these films consist of nonoverlapping membrane fragments which occupy approximately 35% of the surface area and are separated by a lipid monolayer. Furthermore, the membrane fragments are oriented with their intracellular surface towards the aqueous subphase. Nearly all the bacteriorhodopsin molecules at the interface are spectroscopically intact and exhibit visible spectral characteristics identical to those in aqueous suspensions of purple membrane and in intact bacteria. In addition, bacteriorhodopsin in air-dried interface films show spectral changes upon dark-adaptation and upon flash illumination similar to those observed in aqueous suspensions of purple membrane, but with slower kinetics. The kinetics of the spectral changes in interface films can be made nearly the same as in aqueous suspension by immersing the films in water.

Air↗
PubMed1977

Proton transport by bacteriorhodopsin through an interface film.

Interface films of purple membrane and lipid containing spectroscopically intact and oriented bacteriorhodopsin have been used as a model system to study the function of this protein. Small positive charges in surface potential (less than 1 mV) are detected upon illumination of these films at the air-water interface. These photopotentials are not affected by overlaying the interface film with a thin layer (0.3 mm) of decane. However, they are dramatically increased when lipid soluble proton carriers FCCP or DNP are added to the decane. The polarity of the photopotential indicates that, in the light, positive charges are transported through the interface from the aqueous to the organic phase. The action spectrum of the photopotential is identical to the absorption spectrum of bacteriorhodopsin. Since bacteriorhodopsin molecules are oriented with their intracellular surface towards the aqueous subphase, the characteristics of the photopotential indicate that in the light bacteriorhodopsin translocates protons from its intracellular to its extracellular surface. The kinetics of the photopotential reveal that the rate and extent of proton transport are proportional both to the fraction of bacteriorhodopsin molecules excited and to the concentration of proton acceptor. The photopotentials result from changes in the ionic distribution across the decane-water interface and can be cancelled by lipid soluble anions.

Air↗
PubMed1977

Freeze-fractured purple membrane particles: protein content.

Optical diffraction and image reconstruction can be used to correlate the electron microscope image of the biological membrane with its electron density projection. Such correlation shows that a single purple membrane particle contains 9 to 12 protein molecules--63 to 84 transmembrane alpha helices--a complexity two to ten times greater than that previously suggested for membrane particles.

Bacteriorhodopsins↗
PubMed1977

Purple membrane vesicles: morphology and proton translocation.

Purple membrane vesicles prepared by different techniques differ widely in their morphology and ability to establish a proton gradient in the light. The procedures used to prepare active vesicles do not completely dissociate the purple membrane and thus preserve a preferential orientation of the protein, while most of the lipid is exchanged for added lipid. Responses to illumination are largely determined by the size of the vesicles and the degree to which bacteriorhodopsin is preferentially oriented. Any attempt to compare the interaction of different lipids with bacteriorhodopsin by measuring the pH response must take these factors into account. With an improved technique we have obtained vesicles of rather uniform size and bacteriorhodopsin orientation, which accumulate protons with an initial rate of 160 ng H+ sec-1 mg-1 protein at light intensities of 10(6) erg cm-2 sec-1. The kinetics of the process are complex and at present insufficiently understood.

Bacteriorhodopsins↗
PubMed1977

Identification of retinal isomers isolated from bacteriorhodopsin.

The purple membrane of Halobacterium halobium contains the protein bacteriorhodopsin which resembles the visual pigment, rhodopsin, in many aspects. The isomeric configurations of its chromophore, retinal, were studied by a combination of methylene chloride extraction and analysis by high-pressure liquid chromatography. The light-adapted form bR570LA yields solely all-trans-retinal, while the dark-adapted form of bacteriorhodopsin, bR560DA, yields a mixture of 13-cis and all-trans with a ratio of similar to 1;1. The photointermediate M412 in a membrane modified by ether at high NaCl concentration also yields an approximately 1:1 mixture of 13-cis-and all-trans-retinals, while a similar M405 species produced by illumination in 2 M guanidine hydrochloride at high pH yields mainly 13-cis-retinal. These results indicate that the photochemical cycle of bR570LA may involve an isomerization of the retinal chromophore from the all-trans to the 13-cis form.

Bacteriorhodopsins↗