Biomedical subjects

G Garab

Publications and source records attributed to G Garab.

30 records · Page 2Linked to original sources

PubMed1997

Isolation of lamellar aggregates of the light-harvesting chlorophyll a/b protein complex of photosystem II with long-range chiral order and structural flexibility.

Isolation of LHCII, the light-harvesting chlorophyll a/b complex of photosystem II, based on the procedure described by Krupa et al. (1987, Plant Physiol. 84, 19-24), was optimized for obtaining purified lamellar aggregates with long-range chiral order and structural flexibility (the capability of undergoing light-induced reversible structural changes). By varying the concentration of the detergent Triton X-100 for the solubilization of thylakoid membranes, we obtained four types of LHCII aggregates: (i) With low detergent concentration, < or = 0.6% (v/v), the aggregates contained lipids in high amount. These preparations with Chl a/b ratios of about 1.4 contained minor antenna complexes with a fingerprint of an additional CD band at (+) 505 nm; they formed disordered lamellae and exhibited no or weak psi-type CD bands (psi, polymerization- or salt-induced), which did not possess the ability to undergo light-induced changes (deltaCD). (ii) At the optimal concentration, around 0.7 +/- 0.1% (v/v), the detergent removed some lipids and most of the minor complexes, and the Chl a/b ratio dropped to 1.0-1.1. LHCII formed loosely stacked two-dimensional lamellae which exhibited psi-type CD bands and large light-induced reversible structural changes (deltaCD). (iii) At detergent concentration above the optimum, around 0.8-1% (v/v), the lipid content of LHCII decreased and minor complexes could not be detected. LHCII formed disordered aggregates and showed neither psi-type CD nor deltaCD. (iv) High concentrations (> or = 1.1% (v/v)) Triton X-100 led to very pure but largely delipidated samples assembled into tightly stacked three-dimensional lamellar structures with intense psi-type CD but no deltaCD.

Circular Dichroism↗

PubMed1996

Structural flexibility of chiral macroaggregates of light-harvesting chlorophyll a/b pigment-protein complexes. Light-induced reversible structural changes associated with energy dissipation.

In this paper, we show that stacked lamellar aggregates of the purified chlorophyll a/b light-harvesting antenna complexes (LHCII) and granal thylakoid membranes are capable of undergoing light-induced reversible changes in the chiral macroorganization of the chromophores as well as in the photophysical pathways. In granal thylakoids, the light-induced reversible structural changes, detected by circular dichroism (CD) measurements, are accompanied by reversible changes in the fluorescence yield that indicate an increased dissipation of the excitation energy. These changes become gradually more significant in excess light compared to nonsaturating light intensities, and can be eliminated by suspending the membranes in hypotonic, low-salt medium in which the chiral macroaggregates are absent. In lamellar aggregates of LHCII, the light-induced reversible changes of the main, nonexcitonic CD bands are also accompanied by reversible changes in the fluorescence yield. In small aggregates and trimers, no light-induced delta CD occurs, and the fluorescence changes are largely irreversible. It is proposed that the structural changes are induced by thermal effects due to the excess light energy absorbed by the pigments. Our data strongly suggest that the structure and function of the antenna system of chloroplasts can be regulated by the absorption of excess light energy with a mechanism independent of the operation of the photochemical apparatus.

Circular Dichroism↗

PubMed1996

Acridones: a chemically new group of protonophores.

Although the interaction of proton-conducting ionophores (protonophores) with photosynthetic electron transport has been extensively studied during the past decade, the mode of action of protonophores remained uncertain. For a better understanding of the molecular mechanism of the action of protonophores, the introduction of chemically new types of molecules will be required. In this work, we demonstrate that acridones (9-azaanthracene-10-ones) completely fulfill this requirement. At low concentrations of acridones, the thermoluminescence bands at +20 degrees C and +10 degrees C were strongly inhibited, while normal electron transport activity was retained. This indicates that the concentrations of S2 and S3 states involved in the generation of these bands are reduced. At higher concentrations, an increased activity of electron transport was observed, which is attributed to the typical uncoupler effect of protonophores. Indeed, acridones accelerate the decay of the electrochromic absorbance change at 515 nm and also inhibit the generation of the transmembrane proton gradient, measured as an absorbance transient of neutral red. Variable fluorescence induction was quenched even at low concentrations of acridones but was restored by either a long-term illumination or high light intensity. Acridones, similarly to other protonophores, promoted the autooxidation of the high-potential form of cytochrome b559 and partially converted it to lower potential forms. These results suggest that acridones, acting as typical protonophores, uncouple electron transport, accelerate the deactivation of the S2 and S3 states on the donor side, and facilitate the oxidation of cytochrome b559 on the acceptor side of photosystem II.

Journal Article↗

PubMed1995

Nonlinear polarization spectroscopy in the frequency domain of light-harvesting complex II: absorption band substructure and exciton dynamics.

Spectral substructure and ultrafast excitation dynamics have been investigated in the chlorophyll (Chl) a and b Qy region of isolated plant light-harvesting complex II (LHC II). We demonstrate the feasibility of Nonlinear Polarization Spectroscopy in the frequency domain, a novel photosynthesis research laser spectroscopic technique, to determine not only ultrafast population relaxation (T1) and dephasing (T2) times, but also to reveal the complex spectral substructure in the Qy band as well as the mode(s) of absorption band broadening at room temperature (RT). The study gives further direct evidence for the existence of up to now hypothetical "Chl forms". Of particular interest is the differentiated participation of the Chl forms in energy transfer in trimeric and aggregated LHC II. Limits for T2 are given in the range of a few ten fs. Inhomogeneous broadening does not exceed the homogeneous widths of the subbands at RT. The implications of the results for the energy transfer mechanisms in the antenna are discussed.

Circular Dichroism↗

PubMed1994

Size dependency of circular dichroism in macroaggregates of photosynthetic pigment-protein complexes.

Large molecular aggregates, condensed biological macromolecules, intact membrane systems, and cell organelles often exhibit intense anomalous circular dichroism (CD) bands which are absent in systems of lower structural complexity. Theory predicts that in dense, chirally organized macroaggregates the size of the aggregate controls the magnitude of the anomalous CD bands [Keller, D., & Bustamante, C. (1986) J. Chem. Phys. 84, 2972-2979]. Photosynthetic pigment-protein complexes in their native thylakoid membranes and in vitro were used to provide direct experimental evidence of the size dependency of CD in macroaggregates.

Chlorophyll↗

PubMed1994

Trapping magnetically oriented chloroplast thylakoid membranes in gels for electric measurements.

Electrochromic absorbance change and light gradient photovoltage measurements were carried out in chloroplast thylakoid membranes embedded in different compositions of gels. The goal was to find a system suitable for determining the dependence of the amplitude of the anomalous light gradient photovoltage signal, with opposite sign with respect to the ordinary signal, on the alignment of membranes. We found that polyacrylamide gel drastically increased the permeability of membranes which rendered electric measurements impossible in this gel. Agarose gel was successfully used in electric measurements. We will show that the light gradient photovoltage induced by a 580 nm laser flash depends strongly on the alignment of membranes with respect to the direction of the excitation beam. With face-aligned membranes the amplitude of the anomalous (negative) signal was drastically diminished, and the ordinary (positive) signal became clearly discernible. The observed differences in the decay of the two signals are tentatively explained by differences in the rates of ionic equilibration between different subcompartments of the inner aqueous phase of the thylakoid membrane system.

Acrylamides↗

PubMed1991

Involvement of superoxide dismutase in heat-induced stimulation of photosystem I-mediated oxygen uptake.

We have investigated the effect of heat-treatment of chloroplast thylakoid membranes on photosystem I-mediated electron transport. Spectroscopic techniques, oxidation of dichlorophenolindophenol (donor side) and reduction of NADP or methyl purple (acceptor side), showed no indication of an increased activity of photosystem I electron transport. Enhancement of oxygen uptake in the heat-treated (40 degrees C-48 degrees C) samples could largely be accounted for by decline in the activity of superoxide dismutase.

Chloroplasts↗

PubMed1991

Diurnal Fluctuations in the Content and Functional Properties of the Light Harvesting Chlorophyll a/b Complex in Thylakoid Membranes : Correlation with the Diurnal Rhythm of the mRNA Level.

Diurnal fluctuations were observed in the content and some structural and functional properties of the light-harvesting chlorophyll (Chl) a/b pigment-protein complex of photosystem II (LHCII) in young developing wheat (Triticum aestivum) leaves grown under 16 hours light/8 hours dark illumination regime. The fluctuations could be correlated with the diurnal oscillation in the level of mRNA for LHCII. The most pronounced changes occurred in the basal segments of the leaves. They were weaker or hardly discernible in the middle and tip segments. As judged from the diurnal variations of the Chl-a/Chl-b molar ratio, the LHCII content of the thylakoid membranes peaked around 2 pm. This can be accounted for by the cumulative effect of the elevated level of mRNA in the morning and early afternoon. In the basal segment, the extent of the fluctuation in the LHCII content was approximately 25%, as determined from gel electrophoresis ("green gels"). The amplitude of the principal bands of the circular dichroism (CD) spectra of isolated chloroplasts paralleled the changes in the LHCII content. Our circular dichroism data suggest that the newly synthesized LHCII complexes are incorporated into the existing helically organized macrodomains of the pigment-protein complexes or themselves form such macrodomains in the thylakoid membranes. Chl-a fluorescence induction kinetics also showed diurnal variations especially in the basal segments of the leaves. This most likely indicates fluctuations in the ability of membranes to undergo "state transitions." These observations suggest a physiological role of diurnal rhythm of mRNA for LHCII in young developing leaves.

Journal Article↗

PubMed1989

Direct observation of large chiral domains in chloroplast thylakoid membranes by differential polarization microscopy.

Long-range chiral organization of the pigment-protein complexes in mature granal chloroplasts has been established by differential polarization imaging and local circular dichroism spectra. Linear and circular dichroism images of oriented chloroplasts were obtained in a confocal differential polarization microscope. The circular dichroism images display signals of opposite signs emerging from discrete regions with local dichroic values much larger than anticipated, indicating domains in the thylakoid membranes having long-range chiral organization. These domains are associated with positive and negative circular dichroism bands obtained at specific locations on the chloroplasts. Surprisingly, the local circular dichroism spectra do not display the excitonic shape of spectra obtained for macroscopic suspensions, but the latter can be produced by superposition of two local spectra of opposite sign. These data are evidence for the existence of long-range chiral order of the pigment-protein complexes in thylakoid membranes. The possible role of the long-range chiral domains in the efficiency of energy delocalization through the thylakoid membranes is discussed.

Chloroplasts↗

PubMed1988

Helically organized macroaggregates of pigment-protein complexes in chloroplasts: evidence from circular intensity differential scattering.

Angle dependence of circular intensity differential scattering (CIDS) and of nonpolarized scattering was determined in isolated spinach chloroplasts at 514.5 nm. CIDS between 0 degrees and 170 degrees was independent of the nonpolarized scattering and showed intense lobes of alternating signs, exhibiting the negative and positive maxima around 15 degrees and 70 degrees, respectively. These results provide experimental evidence for the existence of large helically organized macroaggregates of pigment-protein complexes in thylakoid membranes. Modeling of the CIDS data by a simple helical array of uniaxial polarizable groups suggests that the chiral structure is left-handed with pitch and radius of the order of 385 nm.

Chloroplasts↗

PubMed1984

Effect of dibromothymoquinone (DBMIB) on reduction rates of Photosystem I donors in intact chloroplasts.

Dual effect of dibromothymoquinone ( DBMIB ), inhibitor and reducing agent at the donor side of Photosystem I, was investigated in isolated intact chloroplasts by flash-induced absorbance changes at 820 and 515 nm. We show that in the absence of other electron donors, rereduction of P700+ by DBMIB proceeds at a very low rate (half-time of approximately 10 s) Dual effect of DBMIB explains that the initial rise of electrochromic absorbance change induced by repetitive flashes is usually not diminished while the slow rise is fully inhibited by this compound.

Chloroplasts↗

PubMed1983

Is antimycin A a specific inhibitor of the slow rise of the electrochromic absorbance change in intact chloroplasts?

The inhibitory effect of antimycin A on the slow rise of the flash-induced electrochromic absorbance change was reinvestigated in intact chloroplasts isolated from pea leaves. It is shown that in the absence of nigericin and +K at low repetition rates (less than 0.5 s-1) of the excitation flashes not only the slow (approximately 10 ms) rise but also the initial (much less than 1 ms) rise generated by photosystem 1 is inhibited by antimycin A.

Antimycin A↗