Membrane-Bound Bacteriophytochrome-Like Complex of Phototrophic Purple Non-Sulfur Bacterium Rhodopseudomonas palustris.

A pigment-protein complex of yellow color with absorption maxima at 682 and 776 nm, characteristic for bacteriophytochromes, was isolated from the photosynthetic membranes of the purple bacterium Rhodopseudomonas palustris. Zinc-induced fluorescence of the complex indicated the presence of the biliverdin chromophore covalently bound to the protein. The parameters of low-temperature fluorescence (λ excitation at 680 nm, λ emission at 695 nm) indicated the ability of the complex to undergo photoconversion. These data, as well as the kinetics of accumulation of the red (Pr)-form on far red light, allowed the complex to be classified as a bacteriophytochrome-like complex with its localization in the photosynthetic membranes of Rps. palustris.

[Fluorescence characteristics and photophysical parameters of aggregates of light-harvesting chlorophyll a/b complexes].

Changes in the fluorescence characteristics and molecular photophysical parameters induced by disaggregation of light-harvesting chlorophyll a/b complexes isolated from pea were studied. Disaggregation was achieved by increasing the concentration of the detergent triton X-100 (concentration range 0-230 microM, chlorophyll concentration 2.45 microg/ml). Laser fluorimetry methods were used to measure the molecular photophysical parameters. It was shown that the decrease in aggregate size is accompanied by a decrease in fluorescence at 700 nm at 77 K, a fall of the singlet-singlet annihilation rate (by more than two orders), and the growth of fluorescence life time (from 160 ps to 3.2 ns).

Heterogeneous origin of carbonic anhydrase activity of thylakoid membranes.

Carbonic anhydrase activities of pea thylakoids as well as thylakoid fragments enriched either in Photosystem 1 (PS1-membranes) or Photosystem 2 (PS2-membranes) were studied. The activity of PS1-membranes if calculated on chlorophyll basis was much higher than the activity of PS2-membranes. Acetazolamide, a non-permeable inhibitor of carbonic anhydrases, increased carbonic anhydrase activity of PS2-membranes at concentrations lower than 10(-6) M and suppressed this activity only at higher concentrations. A lipophilic inhibitor of carbonic anhydrases, ethoxyzolamide, effectively suppressed the carbonic anhydrase activity of PS2-membranes (I50 = 10(-9) M). Carbonic anhydrase activity of PS1-membranes was suppressed alike by both inhibitors (I50 = 10(-6) M). In the course of the electrophoresis of PS2-membranes treated with n-dodecyl-beta-maltoside "high-molecular-mass" carbonic anhydrase activity was revealed in the region corresponding to core-complex of this photosystem. Besides, carbonic anhydrase activity in the region of low-molecular-mass proteins was discovered in the course of such an electrophoresis of both PS2- and PS1-membranes. These low-molecular-mass carbonic anhydrases eluted from corresponding gels differed in sensitivity to specific carbonic anhydrase inhibitors just the same as PS1-membranes versus PS2-membranes. The results are considered as evidence for the presence in the thylakoid membranes of three carriers of carbonic anhydrase activity.

Photosystem II associated carbonic anhydrase activity in higher plants is situated in core complex.

The thylakoid membrane containing photosystem II (PSII membranes) from pea and wheat leaves catalyzed the reaction of CO2 hydration with low rate, which increased after their incubation either with Triton X-100, up to Triton/chlorophyll ratio 1:1, or 1 M CaCl2. The presence of the inhibitor of CAs, p-aminomethylbenzensulfonamide (mafenide), at the start line in the course of electrophoresis of PSII membranes solubilized by n-dodecyl-beta-maltoside (DM) decreased the amount of PSII core complex in the gel. The elution of PSII core complex from the column with immobilized mafenide occurred only either by mafenide or another inhibitor of CAs, ethoxyzolamide. The above results led to a conclusion that membrane-bound CA activity associated with PSII is situated in the core complex.

Carbonic anhydrase activities in pea thylakoids.

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 micromol H(+) (mg Chl)(-1) h(-1) at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal beta-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO(3) (-) dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 micromol H(+) (mg Chl)(-1) h(-1)) were observed in the presence of detergents (Triton X-100 or n-octyl-beta-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 micromol H(+) (mg Chl)(-1) h(-1) which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.

[Effect of DNAase on fluorescent properties of pigment-protein complexes, contained in photosystem II]. / Deistvie DNKazy na fluorestsentnye svoistva pigment-belkovykh kompleksov, vkhodiashchikh v sostav fotosistemy II.

It was found that chlorophyll fluorescence spectra and spectra of fluorescence excitation of pigment-protein complexes of photosystem II are affected by treatment with DNase. Pigment-protein complexes were isolated from pea thylakoid membranes. Spectra were measured at room temperature. It was shown that the treatment with DNase leads to a 30% increase in fluorescence yield at excitation in chlorophyll absorption bands in the fraction containing CP47, CP43, and CP29, and also in the fraction containing reaction center complexes with minor contaminations of light-harvesting complexes. Upon excitation at 260-300 nm and in the region of 500 nm, a diminishing of fluorescence yield takes place. These results suggest that pigments and/or pigment-protein complexes are bound to nucleic acids. This association, by influencing the pigment properties, can participate in the photoregulation of biochemical reactions through changes in the thermal dissipation of excited chlorophyll molecules.

[After effect of heat shock on induction of fluorescence and low temperature fluorescence spectra of wheat leaves]. / Posledeistvie teplovogo shoka na induktsiiu fluoretsentsii i nizkotemperaturnye spektry fluorestsentsii list'ev pshenitsy.

The dynamics of restoration of variable and millisecond delayed fluorescence of chlorophyll a, as well as low-temperature fluorescence spectra at 77 K in leaves of 11-day-old wheat seedlings subjected to heat shock (41.5 degrees C, 20 min) and cultivated for 4 days under white light of different intensity was investigated. A comparative analysis of changes in variable, delayed, and low-temperature fluorescence of chlorophyll a depending on light intensity and the proteolitic activity in leaf homogenates during the restoration of the seedlings was carried out. It follows from the data that the restoration of fluorescence includes several phases and is stimulated by light. A possible mechanism of restoration of fluorescence and photochemical activity of photosystem II is discussed.

Extraction and stability of the pigment-protein complexes of the photosystem 2 from membranes of the unicellular alga Dunaliella salina: effect of glycerol.

Effects of glycerol on the extraction efficiency and the stability of pigment-protein complexes (PPC) of photosystem 2 (PS2) from membranes of the green unicellular alga Dunaliella salina were studied. PS2 membranes were extracted with the detergent n-dodecyl-beta-maltoside (DM) in the presence of 4 M glycerol and 1 M NaCl at a DM:chlorophyll ratio of 15:1 (w/w). The extracts were purified on a DEAE fractogel 650S column. To assess the effect of glycerol on the PPC stability, PS2 membranes were illuminated with white light of high intensity. Then PPCs were extracted and studied spectroscopically and using PAG electrophoresis under denaturing and native conditions. It was found that addition of 4 M glycerol and 1 M NaCl to the extraction medium affects the extraction of the PS2 PPCs but not of the light-harvesting chlorophyll a/b protein (LH2). It was shown that 4 M glycerol protects PS2 from the destructive effects of intense white light, high NaCl concentration, and the detergent Triton X-100. It is suggested that, apart from its osmotic action, glycerol performs a protective function and stabilizes PS2 PPCs in the cells of Dunaliella salina.

Extraction of functionally active photosystem 2 pigment-protein complexes from pea thylakoids and their purification on Sepharose DEAE 6B.

A method for selective extraction of photosystem 2 (PS 2) pigment-protein complexes (PPC) from pea thylakoids and their purification from Sepharose DEAE 6B was developed. It was shown that extraction of thylakoids (3 mg chlorophyll/ml) with Triton X-100 (Triton X-100:chlorophyll = 15-20:1 w/w) in the presence of 1 M sucrose, 1 M NaCl, 50 mM MES in the solubilization medium (pH 5.0), tenfold dilution with 50 mM MES buffer (pH 5.0), and centrifugation at 16,000 g for 10 min provided selective extraction of PS 2 PPC. Spectral, electrophoretic and functional properties of the isolated PPC were determined. This method allowed us to isolate native pigment-protein oxygen-evolving complexes (core complexes) and D1-D2-cytochrome b559 complexes of the reaction centre (RC). The core complex immobilized on a Sepharose DEAE 6B column was treated with 2 mM hydroxylamine or 1 M CaCl2. In the former case, modification by hydroxylamine led to the removal of 4 atoms of Mn, but not of the 33-kDa protein; in the latter case, modification by CaCl2 led to the removal of the 33 kDa protein. The modified core complexes were unstable upon native electrophoresis in the presence of n-dodecyl-beta-d-maltoside and deriphat-160. We suggest that the structural organization of the Mn cluster and 33 kDa protein stabilized the core complex and increased its stability upon the action of the detergents.

Expression of the barley psbA gene in Escherichia coli yields a functional in vitro photosystem II protein D1.

The barley chloroplast psbA gene encoding D1 protein, one of the main photosystem II components, has been over-expressed in E. coli cells. The existance of two in vivo expression products, a protein with M(r) about 33.5 kDa, corresponding to the full-length precursor of the 32 kDa D1 mature form, and a truncated 29 kDa polypeptide was revealed. A modified D1 protein containing six histidine residues at the carboxy-terminus was also obtained. After isolation and renaturation, the ability of the recombinant D1 protein to bind atrazine and pigments from barley thylakoids was demonstrated.

[The rapid electrophoresis of proteins on polyacrylamide gel]. / Ekspress-élektroforez belkov v poliakrilamidnom gele.

A system for express electrophoretic separation of proteins in a thin layer of polyacrylamide gel covalently attached to a glass base has been proposed. A technology of covalent attachment of gel to a glass base has been developed and a small-scale instrument has been constructed which allows to carry out the separation, staining and electrophoregram washing off for 60 min. The quality of the cleaved protein mixtures is not worse than the electrophoregrams obtained using the "Phase System T. M." instrument of "Pharmacia".

[Interaction between immobilized plastocyanin and cytochrome f and the photosystem I reaction center in a reconstituted system]. / Issledovanie vzaimodeistviia immobilizovannogo plastotsianina s tsitokhromom f i reaktsionnym tsentrom fotosistemy I v rekonstruirovannoi sisteme.

The effects of redox conversions of plastocyanin copper chromophore on the formation of plastocyanin complexes with cytochrome f and the reaction center of photosystem I from pea chloroplasts were studied. In order to investigate the complex formation plastocyanin and cytochrome f were immobilized on Sephadex G-200. The cytochrome f and reaction center assembly takes place on the immobilized plastocyanin, which is necessary for cytochrome f photooxidation. It was found that in a reconstituted system the reduced plastocyanin forms more stable complexes with the proteins than the oxidized one, which is due to its lower pI value.

[Protein carriers of the electron transport chain of non-photosynthesizing mutants of Chlamydomonas reinhardii]. / Izuchenie belkovykh perenoschikov elektrontransportnoi tsepi khloroplastov u nefotosinteziruiushchikh mutantov Chlamydomon as reinhardii.

The amounts of ferredoxin, plastocyanin, ferredoxin-NADP-reductase were determined by electrophoresis and differential spectroscopy. The cytochrome levels in the chloroplasts of non-photosynthesizing mutants Chlamydomonas reinhardii were determined both in active and inactive photosystems. It was shown that the loss of PS-1 activity did not affect the amount and activity of the electron carriers. The disturbances of the donor side of PS-2 in the mutants were accompanied by a loss of the reaction center activity and by a decrease of cytochrome b599. The amounts of other protein components in the mutants with inactive PS-2 remained unchanged. The disturbances in the cytochrome c553 content presumably blocked the electron transfer between the photosystems but did not affect the activity of the reaction centers of PS and the levels of other carriers of the chloroplast electron transport chain.

[Immobilization of ferredoxin]. / Immobilizatsiia ferredoksina

The method of pea ferredoxin (Fd) immobilization has been worked out. Under binding Fd with sepharose 4B activated by cyanogen bromide, a destruction took place in the Fd active center and apoprotein was bound with the matrix. The native immobilized Fd has been obtained when using P-300 biogel as a matrix. The gel was activated by glutaraldehyde and protein was linked with gel through a hexamethylenediamine spacer.

[Cytochrome c immobilized on Sepharose 4B and its participation in photochemical reactions of chloroplasts]. / Immobilizovannyi na sefaroze 4v tsitokhrom i ego uchastie v fotokhimicheskikh reaktsiiakh khloroplastov

Cytochrome c immobilized on cyanogen bromide-activated Sepharose 4B may be used to study photochemical reactions in chloroplasts. Chloroplast reduction of both immobilized and soluble forms of the cytochrome occurs along the exogenous and endogenous pathways which results in a weaker reduction of the immobilized protein as compared to that of the soluble one. The time of the reduced immobilized cytochrome c oxidation in the dark is two orders of magnitude greater than that of the soluble one. This fact may be interpreted in terms of spatial dissociation of reductase and oxidase centers of chloroplasts with reference to the cytochrome. The optimal ionic strength for cytochrome reduction, i.e. ionic strength causing an in vitro destruction of the ferredoxin-NADP-reductase complex was found to equal to 0.2 M.

[Two forms of pea leaf ferredoxin]. / Dve formy ferredoksina list'ev gorokha

Some causative factors resulting in the appearance of two components of ferredoxin (Fd) isolated from the pea leaves preparations have been studied by 15% PAG electrophoresis. It has been shown that the composition of the buffer systems (tris-glycine buffer, pH 8,3; veronal buffer, pH 7.0) does not cause Fd separation into two components. Study of the effects of the modifying agents on the active centre and apoprotein of Fd. Showed that the destruction of the active centre does not affect Fd separation and modification of the Fd protein part results in greater amount of the bands on the electrophoregrams. It has been concluded that heterogeneity of Fd is possibly of the genetic nature. The following facts can be regarded as an evidence for existence of both forms of Fd in pea leaves: occurrence of two forms of Fd in chloroplasts isolated in organic media, differing in Fd content during pea ontogenesis and upon varying illumination conditions during their growth.