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Biomedical subjects

W Stoeckenius

Publications and source records attributed to W Stoeckenius.

At least 19 recordsLinked to original sources
PubMed1991

Alternative translocation of protons and halide ions by bacteriorhodopsin.

Bacteriorhodopsin (bR568) in purple membrane near pH 2 shifts its absorption maximum from 568 to 605 nm forming the blue protein bRacid605, which no longer transports protons and which shows no transient deprotonation of the Schiff base upon illumination. Continued acid titration with HCl or HBr but not H2SO4 restores the purple chromophore to yield bRHCl564 or bRHBr568. These acid purple forms also regain transmembrane charge transport, but no transient Schiff base deprotonation is observed. In contrast to bR568, no rate decrease of the bRacidpurple transport kinetics is detected in 2H2O; however, the transport rate decreases by a factor of approximately 2 in bRHBr568 compared with bRHCl564. The data indicate that in the acid purple form bR transports the halide anions instead of protons. We present a testable model for the transport mechanism, which should also be applicable to halorhodopsin.

Anions↗
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↗
PubMed1977

Photoreactions of bacteriorhodopsin.

Bacteriorhodopsin is a membrane-bound light energy transducer which generates an electrochemical proton gradient. It undergoes a cyclic photoreaction in which five intermediates have been identified. During the cycle it releases a proton from one surface of the membrane and takes up a proton on the opposite surface. The active chromophore consists of retinal bound through a Schiff base to the protein. The Schiff base is deprotonized during the photoreaction cycle and appears to be involved in the transport of protons through the membrane. The retinal may also undergo an isomerization.

Bacteriorhodopsins↗
PubMed1977

Molecular weight of bacteriorhodopsin solubilized in Triton X-100.

Bacteriorhodopsin from Halobacterium halobium has been solubilized in the nonionic detergent Triton X-100. The circular dichroic spectrum and hydrodynamic properties indicate that the structure of this protein in the detergent is not significantly altered from that of the native membrane-bound form. Bacteriorhodopsin is monomeric under the conditions of solubilization with a molecular weight of 24,250+/-2,000 and binds approximately one micelle of Triton X-100.

Bacteriorhodopsins↗
PubMed1976

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane.

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side. In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21degreesC. In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds. Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds. The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.

Bacteriorhodopsins↗