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W Stoeckenius

Publications and source records attributed to W Stoeckenius.

At least 37 records · Page 2Linked to original sources
PubMed1986

Color discrimination in halobacteria: spectroscopic characterization of a second sensory receptor covering the blue-green region of the spectrum.

Halobacterium halobium is attracted by green and red light and repelled by blue-green and shorter wavelength light. a photochromic, rhodopsin-like protein in the cell membrane, sensory rhodopsin sR587, has been identified as the receptor for the long-wavelength and near-UV stimuli. Discrepancies between the action spectrum for the repellent effect of blue light and the absorption spectrum of sR587 and its photocycle intermediate S373 strongly suggest the existence of an additional photoreceptor for the blue region of the spectrum. Transient light-induced absorbance changes in intact cells and cell membranes show, in addition to sR587, the presence of a second photoactive pigment with maximal absorption near 480 nm. It undergoes a cyclic photoreaction with a half-time of 150 msec. One intermediate state with maximal absorption near 360 nm has been resolved. The spectral properties of the new pigment are consistent with a function as the postulated photoreceptor for the repellent effect of blue light. The phototactic reactions and both pigments are absent when retinal synthesis is blocked; both can be restored by the addition of retinal. These results confirm and extend similar observations by Takahashi et al. [Takahashi, T., Tomioka, H., Kamo, N. & Kobatake, Y. (1985) FEMS Microbiol. Lett. 28, 161-164]. The archaeobacterium H. halobium thus uses two different mechanisms for color discrimination; it uses two rhodopsin-like receptors with different spectral sensitivities and also the photochromicity of at least one of these receptors to distinguish between three regions covering the visible and near-UV spectrum.

Bacteriorhodopsins↗
PubMed1986

Photoactive retinal pigments in haloalkaliphilic bacteria.

Light-induced fast transient absorbance changes were detected by time-resolved spectroscopy in 38 of 51 haloalkaliphilic isolates from alkaline salt lakes in Kenya and the Wadi Natrun in Egypt. They indicate the presence of two retinal pigments, Pf and Ps, which undergo cyclic photoreactions with half-times of 2 ms and 500 ms respectively. Pf absorbs maximally near 580 nm and Ps near 500 nm. The pigments differ in their sensitivity to hydroxylamine and detergent bleaching and the photoreactions of Pf are strongly dependent on chloride concentration. Of the 38 pigment-containing strains, 29 possess both Pf and Ps, 9 possess only Ps. Inhibition of retinal synthesis with nicotine blocks pigment formation and addition of retinal restores it. Hydroxylamine-bleached pigments can be reconstituted with retinal or retinal analogues. Their similarity to the retinal pigments of Halobacterium halobium strongly suggests that they are also rhodopsin-like retinyledene proteins. Pf in all properties tested is almost identical to halorhodopsin, the light-driven chloride pump of H. halobium, and may serve the same function in the haloalkaliphiles. Ps has photocycle kinetics similar to sensory rhodopsin and a far-blue-shifted long-lived photocycle intermediate, but its ground state absorption maximum is near 500 nm instead of 587 nm. We have not found a bacteriorhodopsin-like pigment in the haloalkaliphiles.

Bacteriorhodopsins↗
PubMed1985

Effects of tyrosine-26 and tyrosine-64 nitration on the photoreactions of bacteriorhodopsin.

In the dark, all titratable tyrosine residues of bacteriorhodopsin have pK's of greater than 11.0, which may be caused by the hydrophobic environment for buried residues and by high negative charge density for surface residues [Scherrer, P., & Stoeckenius, W. (1984) Biochemistry 23, 6195-6202]. Under illumination, deprotonation of only one tyrosine is observed in the micro- and millisecond time ranges of the photocycle; this is Tyr-64. Nitration of Tyr-64 decreases the chromophore absorbance, shifts the absorption maximum to 535 nm, and affects photocycle kinetics. However, restoring its native pK by reduction after nitration has no effect on the changes in photocycle kinetics or absorbance of the chromophore. Nitration of Tyr-64 apparently causes a conformational change in bR, which is independent of the pK of its phenolic group. These observations contradict earlier conclusions that in the photocycle a tyrosine residue directly interacts with the Schiff base during its deprotonation or reprotonation. The protonation state of Tyr-26 and the alkaline chromophore transition are correlated, as shown earlier (Scherrer & Stoeckenius, 1984). Lowering the pK of Tyr-26 by nitration decreases the M-decay rate, and this effect is partially reversed by reduction of the nitro group. We conclude that Tyr-26 may be located close to the chromophore and interact with it; but its protonation state does not change at physiological pH and in the microsecond time range of the photocycle. Tyr-64 is apparently located at or close to the external surface; its modification strongly affects the chromophore but apparently indirectly and not through its protonation changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Transient proton inflows during illumination of anaerobic Halobacterium halobium cells.

In Halobacterium halobium strain R1 containing both bacteriorhodopsin (bR) and halorhodopsin (hR), the light-driven proton uptake has been experimentally resolved into three transient inflows which are superimposed on the larger proton outflow. Under anaerobic conditions the early proton uptake consists of two components: (i) an inflow which can be blocked using the ATPase inhibitor, Dio-9, and (ii) an inflow which can be abolished by low concentrations (less than 125 nM) of triphenyltin chloride (TPT) with no inhibition of ATP synthesis. At pH 6 these two inflows are approximately equal in magnitude and duration. Measurements of buffering capacity and internal pH indicate that Dio-9 does not alter the passive proton-hydroxyl permeability of the cell membrane and that TPT at these low concentrations slightly decreases it. At later times of illumination (iii) another transient light-driven proton inflow occurs. This inflow is most evident during the first illumination after cells have been stored for extended times in the dark. The internal potassium concentration is not changed by storage, but apparently sodium is taken up, and we attribute the third inflow to sodium extrusion in exchange for protons. These results demonstrate the existence of three distinct triggered secondary proton inflows through the cell membrane. The proton inflow, which can be inhibited by Dio-9, correlates with proton-dependent ATP synthesis. The second inflow, which disappears in the presence of low TPT concentrations, is a passive proton uptake through an otherwise unidentified channel in response to electrogenic chloride pumping by bacteriorhodopsin and/or halorhodopsin. The third system correlates with the Na+/H+ antiporter function that has been demonstrated in H. halobium cell envelope vesicles. In contrast to observations on hR-containing vesicles, which can develop substantial Cl- gradients, the electroneutral OH-/Cl- exchange function can be demonstrated in intact cells only at TPT concentrations greater than 500 nM.

Adenosine Triphosphate↗
PubMed1985

Photoconversion from the light-adapted to the dark-adapted state of bacteriorhodopsin.

Dark and light adaptation of bacteriorhodopsin in purple membrane multilayers at less than 100% relative humidity differs from that seen in suspensions. Equilibrium between the two bacteriorhodopsin isomers (bR cis 550 and bR trans 570) in the light-adapted state becomes dependent on the wavelength of actinic light. Excitation at the red edge of the visible absorption band causes dark adaptation in a light-adapted sample. Using polarized actinic and measuring light, we show that acceleration of the dark adaptation through heating by actinic light cannot explain this observation. A light-driven bR trans 570 to bR cis 550 reaction that competes with the well-known 13 cis-to-all-trans light adaptation reaction must exist under our experimental conditions. Trans-to-cis conversion is a one-photon process distinct from the two photon process observed by others in purple membrane suspensions (Sperling, W., C. N. Rafferty, K. D. Kohl, and N. A. Dencher, 1978, FEBS (Fed. Eur. Biochem. Soc.) Lett. 97:129-132). Its quantum efficiency increases monotonously on reducing the hydration level, and is paralleled by an increase in the lifetime of the M410 intermediate of the trans photocycle. We suggest that at this point a branch leads from the all-trans into the 13-cis photocycle. It is probably the same reaction that causes the reduced light adaptation in monomeric bacteriorhodopsin (Casadio, R., H. Gutowitz, P. Mowery, M. Taylor, and W. Stoeckenius, 1980, Biochim. Biophys. Acta. 590:13-23; Casadio, R., and W. Stoeckenius, 1980, Biochemistry. 19:3374-3381).

Bacteriorhodopsins↗
PubMed1985

Coupling between the bacteriorhodopsin photocycle and the protonmotive force in Halobacterium halobium cell envelope vesicles. III. Time-resolved increase in the transmembrane electric potential and modeling of the associated ion fluxes.

Bacteriorhodopsin functions as an electrogenic, light-driven proton pump in Halobacterium halobium. In cell envelope vesicles, its photocycle kinetics can be correlated with membrane potential. The initial decay rate of the M photocycle intermediate(s) decreases with increasing membrane potential, allowing the construction of a calibration curve. The laser (592.5 nm) was flashed at various time delays following the start of background illumination (592 +/- 25 nm) and transient absorbance changes at 418 nm monitored in cell envelope vesicles. The vesicles were loaded with and suspended in either 3 M NaCl or 3 M KCl buffered with 50 mM HEPES at pH 7.5 and the membrane permeability to protons modified by pretreatment with N,N'-dicyclohexylcarbodiimide. In each case the membrane potential rose with a halftime of approximately 75 ms. The steady-state potential achieved depends on the cation present and the proton permeability of the membrane, i.e., higher potentials are developed in dicyclohexylcarbodiimide treated vesicles or in NaCl media as compared with KCl media. The results are modeled using an irreversible thermodynamics formulation, which assumes a constant driving reaction affinity (Ach) and a variable reaction rate (Jr) for the proton-pumping cycle of bacteriorhodopsin. Additionally, the model includes a voltage-gated, electrogenic Na+/H+ antiporter that is active when vesicles are suspended in NaCl. Estimates for the linear phenomenological coefficients describing the overall proton-pumping cycle (Lr = 3.5 X 10(-11)/mol2/J X g X s), passive cation permeabilities (LHu = 2 X 10(-10), LKu = 2.2 X 10(-10), LNau = 1 X 10(-11)), and the Na+/H+ exchange via the antiporter (Lex = 5 X 10(-11)) have been obtained.

Bacteriorhodopsins↗
PubMed1984

Selective nitration of tyrosines-26 and -64 in bacteriorhodopsin with tetranitromethane.

Nitration of tyrosine-26 at pH 9.0 in bacteriorhodopsin does not change its absorption spectrum but lowers the apparent pK of the alkaline transition to a blue-shifted chromophore from about pH 12.0 to 10.6. This effect is reversed by reducing the nitrotyrosine-26 to aminotyrosine which demonstrates that the protonation state of tyrosine-26 and the alkaline chromophore transition are correlated. Nitration of tyrosine-64 resulted in a shift of the purple complex from 570 to 535 nm at neutral pH. The alkaline transition pK of such a nitrated membrane was below 10 but was clearly independent of the protonation state of tyrosine-64 because it is not reversed by reduction of the nitrotyrosine. Nitrotyrosine-26 showed spectral properties similar to L-nitrotyrosine in aqueous environment while nitrotyrosine-64 showed only a 360-nm absorbance in the apomembrane but not in the retinal-containing membrane. Both tyrosines are accessible to water-soluble reagents.

Bacteriorhodopsins↗
PubMed1984

Coupling between the bacteriorhodopsin photocycle and the protonmotive force in Halobacterium halobium cell envelope vesicles. II. Quantitation and preliminary modeling of the M----bR reactions.

The cell membrane of Halobacterium halobium (H. halobium) contains the proton-pump bacteriorhodopsin, which generates a light-driven transmembrane protonmotive force. The interaction of the bacteriorhodopsin photocycle with the electric potential component of the protonmotive force has been investigated. H. halobium cell envelope vesicles have been prepared by sonication and further purified by ultracentrifugation on Ficoll/NaCl/CsCl density gradients. Under continuous illumination (550 +/- 50 nm) varied from 0 to 40 mW cm-2, the vesicles maintain a membrane potential of 0 to -100 mV. The membrane potential was measured by flow dialysis of 3H-TPMP+ uptake and could be abolished by the uncoupler carbonylcyanide-m-chlorophenylhydrazone. Time-resolved absorption spectroscopy was used to measure the decay kinetics of the M photocycle intermediate, which was initiated by a weak laser flash (588 nm), while the vesicles were continuously illuminated as above. The M decay kinetics were fitted with two exponential decays by a computer deconvolution program. The faster decaying form decreases in amplitude (70 to 10% of the total) and the slower decaying form increases in amplitude and lifetime (23 to 42 ms) as the background light intensity increases. Although any correlation between the membrane potential and the bacteriorhodopsin photocycle M-forms is complex, the present data will allow specific tests of the physical mechanism for this interaction to be designed and conducted.

Bacteriorhodopsins↗
PubMed1984

Reconstitution of purified halorhodopsin.

Asolectin lipid vesicles containing halorhodopsin show light-induced acidification in the presence of proton ionophores. This effect is abolished by triphenyltin chloride, a chloride/hydroxyl antiporter, and is greatly diminished by valinomycin in the presence of potassium ions, which collapse the membrane potential. This indicates that halorhodopsin orients in the lipid vesicles preferentially inside out, pumping chloride into the extravesicular compartment. The absorption maximum of halorhodopsin in asolectin vesicles in 3 M NaCl is at 567 nm, and the action spectrum for the light-induced pH changes followed closely the absorption spectrum. Replacement of chloride by acetate or sulfate causes a shift in the absorption maximum to approximately equal to 559 nm and renders the pump inactive. The different photocycles of the two forms were used to show that 80% of the molecules have the extracellular side exposed to the vesicle interior and that the halide-binding site(s) associated with the spectral transition is accessible from the extracellular side of the molecule. The data presented demonstrate that the purified chromoprotein is the light-driven chloride pump in Halobacterium halobium.

Bacteriorhodopsins↗
PubMed1984

Retinal migration during dark reduction of bacteriorhodopsin.

When the retinal Schiff base in chymotryptically cleaved bacteriorhodopsin is reduced to a secondary retinylamine by prolonged exposure to 10% (wt/vol) sodium cyanoborohydride, at pH 10, in the absence of light, approximately 45% of the retinal is found linked to Lys-41 and 22% to Lys-40, and the remainder is scattered over various sites on the large chymotryptic fragment, including the physiological site at Lys-216. The retinal-binding site is destroyed or blocked by the reduction conditions, but the bacteriorhodopsin lattice remains intact. The results demonstrate that artifactual linkage to Lys-40/41 is possible under special conditions. Under these conditions, the epsilon-amino groups of Lys-40/41 show an enhanced ability to form retinylidene linkages with the retinal released by the physiological linkage site at Lys-216, due to some combination of close proximity to the normal linkage site, and increased reactivity with respect to other lysine epsilon-amino groups. The results are of interest for the characterization of the two newly discovered rhodopsin-like proteins, halorhodopsin and slow rhodopsin.

Amino Acids↗
PubMed1984

Bacteriorhodopsin.

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Bacteriorhodopsins↗
PubMed1982

Box-shaped halophilic bacteria.

Three morphologically similar strains of halophilic, box-shaped procaryotes have been isolated from brines collected in the Sinai, Baja California (Mexico), and southern California (United States). Although the isolates in their morphology resemble Walsby's square bacteria, which are a dominant morphological type in the Red Sea and Baja California brines, they are probably not identical to them. The cells show the general characteristics of extreme halophiles and archaebacteria. They contain pigments similar to bacteriorhodopsin which apparently mediate light-driven ion translocation and photophosphorylation.

Adenosine Triphosphate↗
PubMed1981

Photoisomerization of the chromophore in bacteriorhodopsin during the proton pumping photocycle.

The configurations of the 13,14 double bond of the retinylidene chromophore in bacteriorhodopsin (bR) were investigated during the photocycle. The cycle was monitored by measuring the extent of formation of the M intermediate absorbing at 400-420 nm in the light and its rate of decay in the dark. It was inhibited by forming purple membrane films on glass and by using the synergistic ionophores beauvericin and valinomycin, in an equimolar ratio with bR, at 7 degrees C. These systems afforded enrichment in the M intermediate and a rate of decay that could be conveniently studied. The configuration was analyzed by extracting the chromophore with methylene chloride and measuring the ratio of 13-cis-to all-trans-retinals by high-performance liquid chromatography. The relationship between the percent bR cycling and percent 13-cis-retinal extracted was measured for the films by varying the light intensity; the molar ratio of bR cycling to 13-cis-retinal extracted was 1.11. This confirms previous studies which showed photoisomerization of the chromophore during the pumping cycle. The decay kinetics of the blue-shifted intermediate absorbing at 400-420 nm and of the 13-cis-retinal extracted were also measured for the purple membrane films and purple membrane suspensions containing the ionophores. The decay of the 13-cis-retinal extracted followed first-order kinetics with t 1/2 = 0.6 times the decay of the fastest measured component of the 400-420-nm decay. We conclude that the proton pumping photocycle of bR contains a photoisomerization about the 13,14 double bond of the chromophore from the all-trans to the 13-cis configuration and that the thermal rearrangement back to the all-trans form may occur more rapidly than the decay of the M photointermediate.

Anti-Bacterial Agents↗
PubMed1981

Attachment site(s) of retinal in bacteriorhodopsin.

After chemical reduction of the retinylidene-lysine Schiff base linkage in bacteriorhodopsin, the retinyl residue is covalently attached to Lys-216 (with a possible minor fraction on Lys-172) or to both Lys-216(172) and Lys-40/41. The linkage site (up to 100% on Lys-216; up to 70% on Lys-40/41) depends on whether the sample is reduced in the light or dark, whether the sample is light or dark adapted, and on temperature. Absorbance and circular dichroism spectra indicate that the retinyl residue is in its original binding site after reduction in the light. Thus, the different attachment sites may reflect changes that occur during the photoreaction cycle or during light/dark adaptation, or the reduction of accidental physiologically irrelevant Schiff base linkages to lysines close to the normal linkage in the structure of bacteriorhodopsin. In either case, the retinal does not leave its binding site. This last point severely limits the possible arrangements of the amino acid sequence in the bacteriorhodopsin tertiary structure and clearly distinguishes two models that are consistent with all criteria.

Bacteriorhodopsins↗