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1.
Photosynth Res ; 147(1): 91-106, 2021 Jan.
Article in English | MEDLINE | ID: mdl-33280077

ABSTRACT

Most photosynthetic organisms are sensitive to very high light, although acclimation mechanisms enable them to deal with exposure to strong light up to a point. Here we show that cultures of wild-type Chlamydomonas reinhardtii strain cc124, when exposed to photosynthetic photon flux density 3000 µmol m-2 s-1 for a couple of days, are able to suddenly attain the ability to grow and thrive. We compared the phenotypes of control cells and cells acclimated to this extreme light (EL). The results suggest that genetic or epigenetic variation, developing during maintenance of the population in moderate light, contributes to the acclimation capability. EL acclimation was associated with a high carotenoid-to-chlorophyll ratio and slowed down PSII charge recombination reactions, probably by affecting the pre-exponential Arrhenius factor of the rate constant. In agreement with these findings, EL acclimated cells showed only one tenth of the 1O2 level of control cells. In spite of low 1O2 levels, the rate of the damaging reaction of PSII photoinhibition was similar in EL acclimated and control cells. Furthermore, EL acclimation was associated with slow PSII electron transfer to artificial quinone acceptors. The data show that ability to grow and thrive in extremely strong light is not restricted to photoinhibition-resistant organisms such as Chlorella ohadii or to high-light tolerant mutants, but a wild-type strain of a common model microalga has this ability as well.


Subject(s)
Acclimatization/radiation effects , Chlamydomonas reinhardtii/physiology , Photosynthesis/radiation effects , Photosystem I Protein Complex/radiation effects , Photosystem II Protein Complex/radiation effects , Carotenoids/analysis , Carotenoids/radiation effects , Chlamydomonas reinhardtii/growth & development , Chlamydomonas reinhardtii/radiation effects , Chlorophyll/analysis , Chlorophyll/radiation effects , Electron Transport/radiation effects , Oxygen/metabolism , Phenotype , Plastoquinone/analysis , Singlet Oxygen/metabolism , Thylakoids/metabolism
2.
Plant Cell Environ ; 38(12): 2698-706, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26013323

ABSTRACT

In the present study, we have identified new prenyllipid metabolites formed during high light stress in Arabidopsis thaliana, whose origin and function remained unknown so far. It was found that plastoquinone-C accumulates mainly in the reduced form under high light conditions, as well as during short-term excess light illumination both in the wild-type and tocopherol biosynthetic vte1 mutant, suggesting that plastoquinone-C, a singlet oxygen-derived prenyllipid, is reduced in chloroplasts by photosystem II or enzymatically, outside thylakoids. Plastoquinone-B, a fatty acid ester of plastoquinone-C, was identified for the first time in Arabidopsis in high light grown wild-type plants and during short-time, excess light illumination of the wild-type plants and the vte1 mutant. The gene expression analysis showed that vte2 gene is most pronouncedly up-regulated among the prenyllipid biosynthetic genes under high light and induction of its expression is mainly caused by an increased level of singlet oxygen, as was demonstrated in experiments with D2 O-treated plants under excess light conditions.


Subject(s)
Alkyl and Aryl Transferases/genetics , Arabidopsis Proteins/genetics , Arabidopsis/metabolism , Gene Expression Regulation, Enzymologic/radiation effects , Intramolecular Transferases/genetics , Plastoquinone/metabolism , Singlet Oxygen/metabolism , Alkyl and Aryl Transferases/metabolism , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/radiation effects , Arabidopsis Proteins/metabolism , Chloroplasts/metabolism , Gene Expression Regulation, Plant/radiation effects , Intramolecular Transferases/metabolism , Light , Mutation , Oxidative Stress , Photosystem II Protein Complex/metabolism , Plastoquinone/analysis , Thylakoids/metabolism , Tocopherols/metabolism
3.
Methods Mol Biol ; 775: 411-26, 2011.
Article in English | MEDLINE | ID: mdl-21863456

ABSTRACT

Chloroplasts of plants contain an intricate membrane system, the thylakoids, which harbor the complexes of the photosynthetic machinery. Chloroplasts are confined by two membranes, the inner and outer envelope. The major glycerolipids of chloroplasts are the glycolipids monogalactosyl diacylglycerol (MGD), digalactosyl diacylglycerol (DGD), and sulfoquinovosyl diacylglycerol (SQD). Furthermore, two phospholipids, phosphatidyl glycerol (PG) and phosphatidyl choline (PC), are found in chloroplast membranes. The photosystems and light-harvesting complexes in the thylakoids are rich in photosynthetic pigments (chlorophyll, carotenoids, and xanthophylls) and contain a unique set of prenylquinol lipids (tocochromanol/vitamin E, plastoquinol, and phylloquinol/vitamin K1). In this chapter, methods for the isolation and quantification of chloroplast and leaf glycerolipids and prenylquinol lipids are presented. Glycerolipids are separated by thin-layer chromatography prior to conversion of the fatty acids into methyl esters. Fatty acid methyl esters are subsequently quantified by gas chromatography. Prenylquinol lipids are separated by HPLC and quantified by UV absorption (plastoquinol) or fluorescence (tocochromanol, phylloquinol).


Subject(s)
Arabidopsis/cytology , Chemistry Techniques, Analytical/methods , Chloroplasts/chemistry , Glycerides/analysis , Chloroplasts/metabolism , Chromans/analysis , Chromatography, Gas , Chromatography, Thin Layer , Fatty Acids/biosynthesis , Fatty Acids/chemistry , Glycerides/biosynthesis , Glycerides/chemistry , Glycerides/isolation & purification , Plastoquinone/analogs & derivatives , Plastoquinone/analysis
4.
Plant Cell Physiol ; 51(5): 836-41, 2010 May.
Article in English | MEDLINE | ID: mdl-20375106

ABSTRACT

For a better understanding of the metabolic interaction between chloroplasts and mitochondria, it is important to analyze the in vivo redox states of the electron transport chains in both organelles at the same time. For this purpose, we devised an HPLC-based measurement system simultaneously analyzing plastoquinone (PQ) and ubiquinone (UQ) contents and redox states. Using this system, we discovered that, in addition to PQ, the reduction levels of UQ were elevated under high-light conditions. We also provide direct evidence that mitochondrial alternative oxidase contributes to alleviate UQ over-reduction under such conditions.


Subject(s)
Arabidopsis/chemistry , Chloroplasts/metabolism , Mitochondria/metabolism , Plastoquinone/analysis , Ubiquinone/analysis , Arabidopsis/enzymology , Chromatography, High Pressure Liquid , Light , Mitochondrial Proteins , Oxidation-Reduction , Oxidoreductases/metabolism , Plant Proteins
5.
Curr Microbiol ; 55(4): 334-8, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17849161

ABSTRACT

Growth, morphological variation, and liquid chromatography-photodiode array detection-mass spectrometric analysis of pigments have been studied in a diazotrophic cyanobacterium Anabaena cylindrica in response to NaCl stress. The chlorophyll and cellular protein contents increased initially in response to 50 mM: NaCl. Further increment in NaCl concentration, however, resulted in a significant decrease in both chlorophyll and cellular protein. A. cylindrica cells subjected to NaCl stress also showed morphological variations by having alteration in their size and volume. A. cylindrica cells subjected to NaCl stress also exhibited altered plastoquinone and chlorophyll-a (chl a) levels in comparison to its NaCl-untreated counterpart. Furthermore, a relative increase in plastoquinone level and a subsequent decrease in chl a level were recorded in NaCl adapted cells of A. cylindrica in response to NaCl stress. These results suggest that owing to adaptation various morphological, physiological, and biochemical changes occur in the cyanobacterium A. cylindrica in response to NaCl stress.


Subject(s)
Anabaena cylindrica/chemistry , Anabaena cylindrica/physiology , Sodium Chloride/pharmacology , Anabaena cylindrica/ultrastructure , Bacterial Proteins/analysis , Bacterial Proteins/metabolism , Chlorophyll/analysis , Chlorophyll/metabolism , Chlorophyll A , Chromatography, Liquid , Mass Spectrometry , Microscopy, Electron, Scanning , Nitrogen Fixation , Plastoquinone/analysis , Plastoquinone/metabolism
6.
FEBS Lett ; 581(7): 1342-6, 2007 Apr 03.
Article in English | MEDLINE | ID: mdl-17349633

ABSTRACT

The oxidation of the PQ-pool after illumination with 50 or 500 micromol quantam(-2)s(-1) was measured in isolated thylakoids as the increase in DeltaA(263), i.e., as the appearance of PQ. While it was not observed under anaerobic conditions, under aerobic conditions it was biphasic. The first faster phase constituted 26% or 44% of total reappearance of PQ, after weak or strong light respectively. The dependence on oxygen presence as well as the correlation with the rate of oxygen consumption led to conclusion that this phase represents the appearance of PQ from PQ(*-) produced in the course of PQH(2) oxidation by superoxide accumulated in the light within the membrane.


Subject(s)
Light , Oxygen/metabolism , Photosynthesis , Plastoquinone/metabolism , Thylakoids/metabolism , Electron Transport , Kinetics , Oxidation-Reduction , Pisum sativum/metabolism , Pisum sativum/radiation effects , Photic Stimulation , Plastoquinone/analogs & derivatives , Plastoquinone/analysis , Superoxides/metabolism , Thylakoids/radiation effects
7.
Environ Sci Technol ; 40(16): 5116-23, 2006 Aug 15.
Article in English | MEDLINE | ID: mdl-16955916

ABSTRACT

Statin pharmaceuticals, heavily prescribed in the treatment of hypercholesterolemia, are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR). In plants, these compounds also inhibit HMGR, which regulates cytosolic isoprenoid biosynthesis in the mevalonic acid (MVA) pathway. Phytotoxicity was evaluated in the higher aquatic plant Lemna gibba exposed to atorvastatin and lovastatin for 7-days by measuring the concentrations of sterols and ubiquinone; products downstream in the MVA pathway. The efficiency of the parallel and unaffected methylerythritol phosphate pathway (MEP) was also evaluated by measuring the end product, plastoquinone. Statin treatment caused an accumulation of plastoquinone, and unexpectedly, ubiquinone, an artifact likely due to metabolite sharing from the plastidial MEP pathway. Statins were, however, highly phytotoxic to L. gibba and HPLC-UV analysis of plant extracts showed significantly decreased concentrations of both stigmasterol and beta-sitosterol, which are critical components of plant membranes and regulate morphogenesis and development. EC10 values for atorvastatin and lovastatin were as small as 26.1 and 32.8 microg/L, respectively. However, hazard quotients indicated that statins present little risk to the model higher aquatic plant Lemna gibba at environmentally relevant concentrations, even though pathway-specific endpoints were 2-3 times more sensitive than traditional gross morphological endpoints typically used in risk assessment.


Subject(s)
Heptanoic Acids/pharmacology , Herbicides/pharmacology , Hydroxymethylglutaryl CoA Reductases/analysis , Lovastatin/pharmacology , Pharmaceutical Preparations/analysis , Plants/metabolism , Pyrroles/pharmacology , Atorvastatin , Chromatography, High Pressure Liquid , Mevalonic Acid/chemistry , Models, Chemical , Plastoquinone/analysis , Risk Assessment , Sitosterols/analysis , Sterols/analysis , Sterols/chemistry , Ubiquinone/analysis , Ultraviolet Rays
8.
Biochim Biophys Acta ; 1757(12): 1669-75, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16989770

ABSTRACT

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25+/-3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 micromol/m(2).s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.


Subject(s)
Arabidopsis/chemistry , Chloroplasts/chemistry , Plastoquinone/chemistry , Arabidopsis/metabolism , Chlorophyll/chemistry , Chlorophyll/metabolism , Chloroplasts/metabolism , Chromatography, High Pressure Liquid/methods , Oxidation-Reduction , Photochemistry , Plant Leaves/chemistry , Plastoquinone/analysis , Plastoquinone/metabolism , Thylakoids/chemistry , Thylakoids/metabolism
9.
Biochim Biophys Acta ; 1757(2): 106-14, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16472760

ABSTRACT

Binding of herbicides to photosystem II inhibits the electron transfer from Q(A) to Q(B) due to competition of herbicides with plastoquinone bound at the Q(B) site. We investigated herbicide binding to monomeric and dimeric photosystem II core complexes (PSIIcc) isolated from Thermosynechococcus elongatus by a combination of different methods (isothermal titration and differential scanning calorimetry, CD spectroscopy and measurements of the oxygen evolution) yielding binding constants, enthalpies and stoichiometries for various herbicides as well as information regarding stabilization/destabilization of the complex. Herbicide binding to detergent-solubilized PSIIcc can be described by a model of single independent binding sites present on this important membrane protein. Interestingly, binding stoichiometries herbicide:PSIIcc are lower than 1:1 and vary depending on the herbicide under study. Strong binding herbicides such as terbutryn stabilize PSIIcc in thermal unfolding experiments and endothermically binding herbicides like ioxynil probably cause large structural changes accompanied with the binding process as shown by differential scanning calorimetry experiments of the unfolding reaction of PSIIcc monomer in the presence of ioxynil. In addition we studied the occupancy of the Q(B) sites with plastoquinone (PQ9) by measuring flash induced fluorescence relaxation yielding a possible explanation for the deviations of herbicide binding from a 1:1 herbicide/binding site model.


Subject(s)
Herbicides/chemistry , Photosystem II Protein Complex/chemistry , Binding Sites , Calorimetry/methods , Cyanobacteria/chemistry , Fluorescence , Iodobenzenes/metabolism , Iodobenzenes/pharmacology , Kinetics , Nitriles/metabolism , Nitriles/pharmacology , Photosystem II Protein Complex/drug effects , Plastoquinone/analysis , Plastoquinone/metabolism , Thermodynamics , Triazines/metabolism , Triazines/pharmacology
10.
Gene ; 349: 15-24, 2005 Apr 11.
Article in English | MEDLINE | ID: mdl-15777727

ABSTRACT

Alternative oxidase (AOX) represents a non-energy conserving branch in mitochondrial electron transport while plastoquinol terminal oxidase (PTOX) represents a potential branch in photosynthetic electron transport. Using a metagenomics dataset, we have uncovered numerous and diverse AOX and PTOX genes from the Sargasso Sea. Sequence similarity, synteny and phylogenetic analyses indicate that the large majority of these genes are from prokaryotes. AOX appears to be widely distributed among marine Eubacteria while PTOX is widespread among strains of cyanobacteria closely related to the high-light adapted Prochlorococcus marinus MED4, as well as Synechococcus. The wide distribution of AOX and PTOX in marine prokaryotes may have important implications for productivity in the world's oceans.


Subject(s)
Oxidoreductases/analysis , Plastoquinone/analogs & derivatives , Plastoquinone/analysis , Prokaryotic Cells/enzymology , Seawater/microbiology , Amino Acid Motifs , Amino Acid Sequence , Bacteria/enzymology , Conserved Sequence , Cyanobacteria/enzymology , DNA, Bacterial/genetics , Evolution, Molecular , Mitochondrial Proteins , Molecular Sequence Data , Oceans and Seas , Phylogeny , Plant Proteins , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Synechococcus/enzymology , Synteny
11.
J Gen Appl Microbiol ; 48(1): 35-41, 2002 Feb.
Article in English | MEDLINE | ID: mdl-12469314

ABSTRACT

In this study, a quinone profiling method was applied to clarify the differences in community structure between suspended and sessile microorganisms in rivers. The compositions of microbial quinone of 6 sites for 4 rivers were analyzed. Ubiquinone (UQ)-8, UQ-10, menaquinone (MK)-7, and plastoquinone (PQ)-9 were observed in all samples of suspended and sessile microorganisms for the sites investigated. The dominant quinone species in suspended microorganisms was ubiquinone, and that in sessile microorganism was photosynthetic quinones (namely PQ-9 and vitamin K1). This indicated that aerobic bacteria were abundant in the suspended microorganisms, and photosynthetic microorganisms such as micro-algae and cyanobacteria dominated in the sessile microorganisms. The quinone concentration in the river waters tested, which reflects the concentration of suspended microorganisms, ranged from 0.045 to 1.813 nmol/L. The microbial diversities of suspended and sessile microorganisms calculated based on the composition of all quinones were in the range from 3.4 to 7.5, which was lower than those for activated sludge and soils. Moreover, the diversity of heterotrophic bacteria for sessile microorganisms in the rivers was higher than that for the suspended microorganisms.


Subject(s)
Fresh Water , Geologic Sediments/microbiology , Water Microbiology , Plastoquinone/analysis , Ubiquinone/analysis , Vitamin K 2/analysis , Water Pollutants
12.
Acta Biochim Pol ; 49(3): 775-80, 2002.
Article in English | MEDLINE | ID: mdl-12422246

ABSTRACT

The plant Solanum nigrum treated with the pathogen Phytophthora infestans-derived elicitor responded by elevated reactive oxygen species (ROS) production, lipid peroxidation and lipoxygenase (EC 1.13.11.12) activity in comparison with control plants indicating that oxidative stress took place. We demonstrate that these events are accompanied by a significant increase in plastoquinone (PQ) level. It is postulated that PQ may be associated with mechanisms maintaining a tightly controlled balance between the accumulation of ROS and antioxidant activity that determines the full expression of effective defence.


Subject(s)
Adaptation, Physiological/physiology , Plastoquinone/metabolism , Solanaceae/metabolism , Antioxidants/chemistry , Antioxidants/metabolism , Antioxidants/pharmacology , Lipid Peroxidation/physiology , Lipoxygenase/metabolism , Oxidation-Reduction , Phytophthora/pathogenicity , Plant Diseases/microbiology , Plant Leaves/cytology , Plant Leaves/metabolism , Plant Leaves/microbiology , Plastoquinone/analysis , Plastoquinone/pharmacology , Reactive Oxygen Species/metabolism , Solanaceae/cytology , Solanaceae/microbiology
13.
FEBS Lett ; 523(1-3): 177-81, 2002 Jul 17.
Article in English | MEDLINE | ID: mdl-12123828

ABSTRACT

The disruption of the Synechocystis open reading frame Deltaslr0090 encoding a gene with high homology to plant genes encoding 4-hydroxyphenylpyruvate dioxygenase results in an impairment of tocopherol biosynthesis without affecting levels of plastoquinone, carotenoids and chlorophyll as well as cell growth and photosynthesis. Our results indicate that in Synechocystis in contrast to the situation in higher plants the 4-hydroxyphenylpyruvate dioxygenase is not required for the synthesis of plastoquinone.


Subject(s)
4-Hydroxyphenylpyruvate Dioxygenase/metabolism , Cyanobacteria/enzymology , Photosynthetic Reaction Center Complex Proteins/metabolism , Plastoquinone/metabolism , 4-Hydroxyphenylpyruvate Dioxygenase/genetics , Cyanobacteria/genetics , Cyanobacteria/metabolism , Gene Deletion , Light-Harvesting Protein Complexes , Mutation , Plastoquinone/analysis , Tocopherols/analysis , Vitamin K 1/analysis
14.
Biochemistry ; 41(25): 8004-12, 2002 Jun 25.
Article in English | MEDLINE | ID: mdl-12069591

ABSTRACT

A highly active oxygen-evolving photosystem II (PSII) complex was purified from the HT-3 strain of the widely used cyanobacterium Synechocystis sp. PCC 6803, in which the CP47 polypeptide has been genetically engineered to contain a polyhistidine tag at its carboxyl terminus [Bricker, T. M., Morvant, J., Masri, N., Sutton, H. M., and Frankel, L. K. (1998) Biochim. Biophys. Acta 1409, 50-57]. These purified PSII centers had four manganese atoms, one calcium atom, and two cytochrome b(559) hemes each. Optical absorption and fluorescence emission spectroscopy as well as western immunoblot analysis demonstrated that the purified PSII preparation was devoid of any contamination with photosystem I and phycobiliproteins. A comprehensive proteomic analysis using a system designed to enhance resolution of low-molecular-weight polypeptides, followed by MALDI mass spectrometry and N-terminal amino acid sequencing, identified 31 distinct polypeptides in this PSII preparation. We propose a new nomenclature for the polypeptide components of PSII identified after PsbZ, which proceeds sequentially from Psb27. During this study, the polypeptides PsbJ, PsbM, PsbX, PsbY, PsbZ, Psb27, and Psb28 proteins were detected for the first time in a purified PSII complex from Synechocystis 6803. Five novel polypeptides were also identified in this preparation. They included the Sll1638 protein, which shares significant sequence similarity to PsbQ, a peripheral protein of PSII that was previously thought to be present only in chloroplasts. This work describes newly identified proteins in a highly purified cyanobacterial PSII preparation that is being widely used to investigate the structure, function, and biogenesis of this photosystem.


Subject(s)
Arabidopsis Proteins , Bacterial Proteins , Cyanobacteria/chemistry , Peptide Fragments/isolation & purification , Photosynthetic Reaction Center Complex Proteins/analysis , Photosystem II Protein Complex , Proteome/analysis , Benzoquinones/metabolism , Calcium/analysis , Chlorophyll/analysis , Cyanobacteria/enzymology , Cytochrome b Group/analysis , Cytochrome c Group/isolation & purification , Iron/analysis , Light-Harvesting Protein Complexes , Manganese/analysis , Molecular Weight , Oxygen/metabolism , Pheophytins/analysis , Photosynthetic Reaction Center Complex Proteins/isolation & purification , Photosystem I Protein Complex , Phycobilisomes , Plastoquinone/analysis , Proteins/isolation & purification , Spectrometry, Fluorescence , Thylakoids/chemistry , Xanthophylls , Zeaxanthins , beta Carotene/analogs & derivatives , beta Carotene/analysis
15.
Biochemistry ; 35(24): 7802-11, 1996 Jun 18.
Article in English | MEDLINE | ID: mdl-8672481

ABSTRACT

Photosystem II, the photosynthetic water-oxidizing complex, can be isolated from both plants and cyanobacteria. A variety of methods have been developed for purification of this enzyme, which can be isolated in several functional and structural forms. Knowledge of the pigment content of photosystem II preparations is important for precise spectroscopic, biochemical, and functional analysis. We have determined pigment stoichiometries in oxygen-evolving photosystem II preparations from plants and cyanobacteria. We have employed a solvent system for the isocratic elution of a reverse phase HPLC column in which we have determined the extinction coefficients of the relevant pigments. Pigments were extracted from four photosystem II preparations. These preparations included spinach photosystem II membranes [Berthold, D. A., Babcock, G. T., & Yocum, C. F. (1981) FEBS Lett. 134, 231-234], spinach photosystem II reaction center complexes [Ghanotakis, D. F., & Yocum, C. F. (1986) FEBS Lett. 197, 244-248], spinach photosystem II complexes [MacDonald, G. M., & Barry, B. A. (1992) Biochemistry 31, 9848-9856], and photosystem II particles isolated from the cyanobacterium, Synechocystis sp. PCC 6803 [Noren, G. H., Boerner, R. J., & Barry, B. A. (1991) Biochemistry 30, 3943-3950]. Pigment stoichiometries were determined using two different methods of data analysis and were based on the assumption that there are two pheophytin a molecules per photosystem II reaction center. The pigment stoichiometries obtained were comparable for the two methods of data analysis and agreed with previous biophysical and biochemical characterizations of the preparations. The average pigment stoichiometries (chlorophyll:plastoquinone-9 per 2 pheophytin a) determined using the two data analysis methods were as follows: photosystem II membranes, 274:3.2; photosystem II reaction center complexes, 78:2.5; Synechocystis PS II particles, 55:2.4; photosystem II complexes, 121:2.0.


Subject(s)
Chlorophyll/analysis , Cyanobacteria/metabolism , Photosynthetic Reaction Center Complex Proteins/chemistry , Spinacia oleracea/metabolism , Chlorophyll A , Chromatography, High Pressure Liquid/methods , Light-Harvesting Protein Complexes , Pheophytins/analysis , Photosystem II Protein Complex , Plastoquinone/analysis , Spectrophotometry
16.
Biochim Biophys Acta ; 1278(1): 89-97, 1996 Jan 12.
Article in English | MEDLINE | ID: mdl-8611612

ABSTRACT

Cyclodextrins (CDs) have been used in controlled lipid depletion of thylakoid membranes avoiding the use of either detergents or lipolytic enzymes. Spinach thylakoid membranes were first treated with different CDs under various conditions. After removal of the CDs by washing, the amounts of mono-- and digalactosyldiacylglycerol (MGDG and DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), protein, pigment and plastoquinone remaining in the membranes were determined. The main results, obtained with alpha-CD and heptakis-(2,6-di-O-methyl)-beta-CD (DM-beta-CD), were as follows. (1)Acyl lipids were removed from thylakoid membranes by both CDs (DM-beta-CD being more efficient than alpha-CD; the extent of removal depended on both CD and chlorophyll concentrations. (2) alpha-CD presented a higher selectivity towards lip classes than did DM-beta-CD, but in both cases the removal order was SQDG > PG > MGDG > DGDG. (3) alpha-CD showed a preference for those lipids containing saturated 16-carbon acyl chains whereas DM-beta-CD was essentially insensitive to the fatty acid composition of the lipids. (4) The protein, chlorophyll and carotenoid contents of thylakoids were not affected by CD treatments. (5) Plastoquinones were removable but in small amounts only and with a low efficiency (DM-beta-CD > alpha-CD). (6) For all lipid classes, the extent of lipid removal was higher at 0 degrees than at 20 degrees C. (7) The presence of MgCl(2) reduced the removal of PG and SQDG but not affect galactolipid depletion levels. (8) Staple lipid depletion levels in thylakoid membranes were reached after 5-10 min of CD treatment at 0 degrees C. (9) Of the four CDs tested, only three (alpha-CD, beta-CD, and DM-beta-CD) promoted lipid depletion whereas one (hydroxypropyl-beta-CD) failed completely to do so. It is concluded that CD-mediated lipid removal provides a valuable and versatile tool to achieve controlled and specific lipid depletions in biological membranes. A few examples of the consequences of a CD-induced lipid depletion on fluorescence and electron transport properties of thylakoids are given to show the usefulness of CDs in the investigation of structure-function relationship in photosynthetic membranes.


Subject(s)
Chloroplasts/chemistry , Cyclodextrins/pharmacology , Intracellular Membranes/chemistry , Membrane Lipids/analysis , alpha-Cyclodextrins , beta-Cyclodextrins , Carotenoids/analysis , Chlorophyll/analysis , Cyclodextrins/chemistry , Fatty Acids/analysis , Glycerol/analogs & derivatives , Glycerol/analysis , Glycolipids/analysis , Intracellular Membranes/drug effects , Kinetics , Membrane Proteins/analysis , Plastoquinone/analysis , Spectrometry, Fluorescence , Spinacia oleracea , Temperature
17.
J Biol Chem ; 264(3): 1387-92, 1989 Jan 25.
Article in English | MEDLINE | ID: mdl-2912961

ABSTRACT

An azidoquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone (azido-Q), was used to study the plastoquinone-protein interaction and to identify the plastoquinone-binding protein in the cytochrome b6-f complex from spinach chloroplasts. When the lipid- and plastoquinone-deficient cytochrome b6-f complex is incubated with varying concentrations of azido-Q and illuminated with long wavelength UV light for 7 min at 2 degrees C, the enzymatic activity, assayed after reconstitution with lipid, decreases as the concentration of azido-Q increases. Maximum inactivation (45%) is observed when 30 mol of azido-Q is used per mol of cytochrome f. The extent of the decrease in activity upon illumination correlates with the amount of azido-Q incorporated into the protein. The 50% inactivation is in good agreement with that expected based on the amount of plastoquinone deficiency of the isolated enzyme complex. When the photolyzed, [3H]azido-Q-treated sample is extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity is found primarily in the Mr = 17,000 subunit. When the enzyme is pretreated with the electron transfer inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, significantly less radioactive label is observed in the Mr = 17,000 protein, suggesting that the action sites of these inhibitors are the same or near the plastoquinone-binding site. When the deficient complex is reconstituted with glycolipid prior to the addition of azido-Q, less than 5% inactivation is observed upon photolysis, and the amount of radioactive label on the Mr = 17,000 protein decreases greatly, suggesting that the plastoquinone-binding site is easily masked by glycolipid when endogenous plastoquinone is absent. Plastoquinol-2 apparently competes with azido-Q for the plastoquinone-binding site since it decreases the radioactive label on the Mr = 17,000 protein.


Subject(s)
Carrier Proteins/analysis , Chloroplasts/enzymology , Cytochrome b Group/analysis , Plastoquinone/analysis , Quinones/analysis , Azides/pharmacokinetics , Cytochrome b6f Complex , Electron Transport , Electrophoresis, Polyacrylamide Gel , Glycolipids/pharmacology , Molecular Weight , Ubiquinone/analogs & derivatives , Ubiquinone/pharmacokinetics
19.
Biochim Biophys Acta ; 641(1): 99-105, 1981 Feb 20.
Article in English | MEDLINE | ID: mdl-7213720

ABSTRACT

The isolated and purified chloroplast envelope of spinach leaves contains, besides carotenoids, several prenylquinones as basic constituents: plastoquinone-9, phylloquinone K1, alpha-tocoquinone and the chromanol, alpha-tocopherol. The relative quinone and carotenoid composition of the envelope differs distinctively from that of the thylakoid membranes. The possible role of prenylquinones in metabolic envelope activities and the mediator function of the envelope in prenylquinone biosynthesis are discussed.


Subject(s)
Chloroplasts/analysis , Plants/analysis , Quinones/analysis , Intracellular Membranes/analysis , Plastoquinone/analysis , Vitamin E/analogs & derivatives , Vitamin E/analysis , Vitamin K 1/analysis
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