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1.
Circular dichroism spectroscopy has been used to investigate the binding of valinomycin to bacteriorhodopsin in purple membrane suspensions. Addition of valinomycin to purple membrane suspensions obtained from Halobacterium halobium causes the circular dichroism spectrum to shift from an aggregate spectrum to one resembling a monomer spectrum, indicating a loss of chromophore-chromophore interactions. By observing the spectral change upon titration of valinomycin, an apparent dissociation constant of 30–40 M for valinomycin binding was determined. Kinetics of dark adaptation for valinomycin-treated purple membrane are comparable to those for monomeric bacteriorhodopsin. Centrifugation studies demonstrate that valinomycin-treated purple membrane sediments the same as untreated purple membrane suspensions. These results are consistent with a model in which valinomycin binds specifically to bacteriorhodopsin without disrupting the purple membrane fragments.Abbreviations BR
bacteriorhodopsin
- CD
circular dichroism
- Tricine
N-[tris-(hydroxymethyl) methyl] glycine 相似文献
2.
The contribution of two components (pH and E) of the proton motive force to photosynthesis of C. reinhardtii was studied. Valinomycin, a photophosphorylation uncoupler, decreased significantly the fast phase (related mainly to the membrane electric potential) of millisecond delayed light emission (ms-DLE) of C. reinhardtii. Nigericin, another photophosphorylation uncoupler, decreased the slow phase (related mainly to the proton gradient) and partly also the fast phase of ms-DLE. Both valinomycin and nigericin decreased the net ATP content and photosynthetic rate of C. reinhardtii, but the inhibition by nigericin was stronger than that by valinomycin. Hence both components of the proton motive force contribute to photosynthesis and although the contribution of pH is larger than that of E, the latter is not negligible in photosynthesis of C. reinhardtii. 相似文献
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Hong Shan Zhang Thomas V. McDonald Herbert B. Tanowitz Murray Wittner Louis M. Weiss John P. Bilezikian Stephen A. Morris 《The Journal of eukaryotic microbiology》1998,45(1):80-86
ABSTRACT Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca2+ concentration([Ca2+]i) of 64 ± 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca2+]o (preloaded cells) increased [Ca2+]i < 20 nM whereas total cell Ca2+ increased by 1.5 to 2.0 pmole/cell. This amount of Ca2+ would be expected to increase [Ca2+]i to > 10 μM suggesting active sequestration of Ca2+. We tested the hypothesis that maintenance of [Ca2+]i involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca2+]i through well characterized pathways in higher eukaryotic cells were employed. [Ca2+], responses in the presence or absence of [Ca2+]o were measured to asses the relative contribution of sequestration or extrusion processes in [Ca2+]i homeostasis. In the presence of 0.5 mM [Ca2+]o, the ability of several agents to increase [Ca2+]i was magnified in the order ionomycin ? nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca2+], observed only in response to monensin. Manoalide, an inhibitor of phospholipase A2, enhanced the accumulation of [Ca2+]i due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca2+]i involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca2+ may involve phospholipase A2 activity. A Na+/Ca2+ exchange mechanism did not appear to contribute to Ca2+ homeostasis. 相似文献
4.
Vibrational absorption and vibrational circular dichroism (VCD) spectra of valinomycin are measured, in different solvents, in the ester and amide carbonyl stretching regions. The influence of cations, namely Li(+), Na(+), K(+), and Cs(+), in methanol-d(4) solvent is also investigated. Ab initio quantum mechanical calculations using density functional theory and 6-31G* basis set are used to predict the absorption and VCD spectra. A bracelet-type structure for valinomycin that reproduces the experimental absorption and VCD spectra in inert solvents is identified. For the structure of valinomycin in polar solvents, a propeller-type structure was optimized, but further investigations are required to confirm this structure. A symmetric octahedral environment for the ester carbonyl groups in the valinomycin-K(+) complex is supported by the experimental VCD spectra. The results obtained in the present study demonstrate that even for large macrocyclic peptides, such as valinomycin, VCD can be used as an independent structural tool for the study of conformations in solution. 相似文献
5.
Mutations found in PTEN-induced putative kinase 1 (PINK1), a putative mitochondrial serine/threonine kinase of unknown function, have been linked to autosomal recessive Parkinson's disease. It is suggested that mutations can cause a loss of PINK1 kinase activity and eventually lead to mitochondrial dysfunction. In this report, we examined the subcellular localization of PINK1 and the dynamic kinetics of PINK1 processing and degradation. We also identified cytosolic chaperone heat-shock protein 90 (Hsp90) as an interacting protein of PINK1 by PINK1 co-immunoprecipitation. Immunofluorescence of PINK1 protein and mitochondrial isolation show that the precursor form of PINK1 translocates to the mitochondria and is processed into two cleaved forms of PINK1, which in turn localize more to the cytosolic than mitochondrial fraction. The cleavage does not occur and the uncleaved precursor stays associated with the mitochondria when the mitochondrial membrane potential is disrupted. Metabolic labeling analyses show that the PINK1 processing is rapid and the levels of cleaved forms are tightly regulated. Furthermore, cleaved forms of PINK1 are stabilized by Hsp90 interaction as the loss of Hsp90 activity decreases PINK1 level after mitochondrial processing. Lastly, we also find that cleaved forms of PINK1 are degraded by the proteasome, which is uncommon for mitochondrial proteins. Our findings support a dual subcellular localization, implying that PINK1 can reside in the mitochondria and the cytosol. This raises intriguing functional roles that bridge these two cellular compartments. 相似文献
6.
Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Δψ) generated by the Schizosaccharomyces pombe plasma membrane H+-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Δψ because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Δψ at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H+-ATPase generates Δψ of about 160 mV on the vesicle membrane. The generated Δψ was stable at least over tens of minutes. An influence of the H+ membrane permeability on the Δψ buildup was demonstrated by manipulating the H+ permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H+-ATPase-mediated Δψ buildup. 相似文献
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9.
Poul Bennekou 《The Journal of membrane biology》1988,102(3):225-234
Summary A transport model for translocation of the protonophore CCCP across the red cell membrane has been established and cellular CCCP binding parameters have been determined. The time course of the CCCP redistribution across the red cell membrane, following a jump in membrane potential induced by valinomycin addition, has been characterized by fitting values of preequilibrium extracellular pHvs. time to the transport model. It is demonstrated, that even in the presence of valinomycin, the CCCP-anion is well behaved, in that the translocation can be described by simple electrodiffusion. The translocation kinetics conform to an Eyring transport model, with a single activation energy barrier, contrary to translocation across lipid bilayers, that is reported to follow a transport model with a plateau in the activation energy barrier. The CCCP anion permeability across the red cell membrane has been calculated to be close to 2.0×10–4 cm/sec at 37°C with small variations between donors. Thus the permeability of CCCP in the human red cell membrane deviates from that found in black lipid membranes, in which the permeability is found to be a factor of 10 higher. 相似文献
10.
V. Pakhomova 《Biologia Plantarum》1996,38(4):495-499
Root excision fromTriticum vulgare L. var.muticum seedlings induced a membrane potential drop, homeostasis disturbances, and loss of absorbing capacity in roots. During subsequent
3 - 4 h incubation the initial physiological properties of the roots were restored. At that period K+absorption could be blocked by tetraethylammonium (TEA) without changing pH of the incubation medium. After 5 - 6 h of incubation
the membrane hyperpolarization and the enhancement of the absorbing capacity were observed. At that period K+ influx, at presence or absence of valinomycin in incubation medium, was coupled with acidification of the external medium
and was not blocked by TEA. 相似文献