Binding of Antibodies onto the Thylakoid Membrane III. Proteins in the Outer Surface of the Thylakoid Membrane

1978 ◽  
Vol 33 (9-10) ◽  
pp. 731-734 ◽  
Author(s):  
Alfons Radunz
Keyword(s):  
Molecular Weight ◽  
Electron Transport ◽  
Outer Surface ◽  
Thylakoid Membrane ◽  
Apparent Molecular Weight ◽  
Photosynthetic Electron Transport ◽  
Coupling Factor ◽  
Cytochrome F ◽  
Threefold Increase ◽  
Close Relationship

Abstract The maximal binding of antibodies to ferredoxin-NADP+ -reductase, cytochrome f, plastocyanin, coupling factor of photophosphorylation, carboxydismutase and to a polypeptide with the apparent molecular weight 24 000 onto stroma-freed chloroplasts of Antirrhinum majus was determined. The three proteins involved in photosynthetic electron transport bind approximately 0.05 to 0.07 g antibodies per g chloroplasts. The chloroplast preparation itself binds maximally about 1 gantibodies. From an antiserum to carboxydismutase and to a membrane polypeptide with the apparent molecular weight 24 000 approximately double the amount of antibodies namely 0.1 to 0.14 g antibodies per g chloroplasts are bound. Extraction of stroma-freed chloroplasts with 0.02 ᴍ Tris buffer pH 7.8 containing 0.7 mᴍ EDTA caused a threefold increase of the amount of bound anti­ bodies in the case of the membrane protein. 40% of the amount of antibodies which can be maxi­mally bound by this chloroplast preparation is adsorbed out of an antiserum to the coupling factor.Out of an antiserum which contains equal concentrations of antibodies to ferredoxin-NADP+ -reductase, cytochrome f and plastocyanin the same amount of antibodies is bound as out of an antiserum directed to only one of these components. This shows that the proteins involved in electron transport are located in a very close relationship to each other in the outer surface of the thylakoid membrane.

Localization and Function of Cytochrome f in the Thylakoid Membrane

1977 ◽  
Vol 32 (3-4) ◽  
pp. 271-280 ◽  
Author(s):  
Georg H. Schmid ◽  
Alfons Radunz ◽  
Wilhelm Menke
Keyword(s):  
Electron Transport ◽  
Outer Surface ◽  
Thylakoid Membrane ◽  
Time Lag ◽  
Photosynthetic Electron Transport ◽  
Cytochrome F ◽  
Antigenic Determinants ◽  
Light Reaction ◽  
Transport Reaction ◽  
Cyclic Photophosphorylation

Abstract A monospecific antiserum to cytochrome f agglutinates stroma-free swellable chloroplasts from tobacco and Antirrhinum. Consequently, antigenic determinants towards which the antiserum is directed are located in the outer surface of the thylakoid membrane. The antiserum inhibits linear photosynthetic electron transport. Just as described earlier for the antiserum to polypeptide 11000 this inhibition develops in the course of the light reaction. Ultrasonication in the presence of anti­ serum abolishes the light requirement and the maximal inhibition of the electron transport reaction is immediately observed. Electron transport in chloroplasts from a tobacco mutant which ex­ hibits only photosystem I-reactions is also inhibited by the antiserum. No time lag in the light for the onset of inhibition is observed with these chloroplasts. As chloroplasts of this mutant have only single unfolded thylakoids it appears that light might preponderantly open up partitions. If the light effect is interpreted in this way, cytochrome f should be located in the partition regions but nevertheless in the outer surface of the thylakoid membrane. However, a rearrangement of molecules in the membrane in the light by which the accessibility of cytochrome f is changed can­ not be excluded. The inhibition of linear electron transport by the antiserum is approximately 50 per cent and can only be increased to 75% upon the addition of antibodies to plastocyanin. The inhibition by the antiserum to cytochrome f as well as the combined inhibition by the antisera to cytochrome f and plastocyanin can be by-passed by DCPiP. It appears that cytochrome f and plastocyanin cannot be connected in series in the electron transport chain but are both closely associated in the thylakoid membrane. PMS-mediated cyclic photophosphorylation in chloroplasts from wild type tobacco and the tobacco mutant NC95 is only inhibited if the chloroplasts are sonicated in the presence of anti­ serum. If one disregards, that ultrasonication might cause reaction artifacts, it is thinkable that the cytochrome f, involved in the PMS-mediated cyclic photophosphorylation reaction, might be located inside the membrane.

Localization and Functional Characterization of Three Thylakoid Membrane Polypeptides of the Molecular Weight 66000

1977 ◽  
Vol 32 (9-10) ◽  
pp. 817-827 ◽  
Author(s):  
Friederike Koenig ◽  
Wilhelm Menke ◽  
Alfons Radunz ◽  
Georg H. Schmid
Keyword(s):  
Molecular Weight ◽  
Electron Transport ◽  
Outer Surface ◽  
Thylakoid Membrane ◽  
Functional Characterization ◽  
Apparent Molecular Weight ◽  
Photochemical Reactions ◽  
Antigenic Determinants ◽  
Reaction Centre

Abstract Three polypeptide fractions with the apparent molecular weight 66 000 were isolated from stroma-freed Antirrhinum chloroplasts which were solubilized with dodecyl sulfate. Antisera to these fractions affect electron transport in distinctly different ways. For the characterization of the three antisera photochemical reactions of chloroplast preparations with artificial electron donors and acceptors as well the analysis of fluorescence rise curves were used. Antiserum 66 000 PSI-96 inhibits electron transport apparently on the acceptor side of photosystem I, provided the antibodies are adsorbed onto the outer surface of the thylakoid membrane. Antiserum 66 000 PSI-88 probably acts directly on the reaction centre I or on its immediate vicinity, if the antibodies are adsorbed at the inner surface of the thylakoid membrane. Antiserum 66 000 PSII-42 inhibits electron trans­ port in the region of photosystem II. The antigen towards which the antiserum is directed appears to belong to the reaction centre II, as also in the condition of high inhibition degrees, the fluorescence intensity remains unchanged. The antigenic determinants are located at the outer surface of the thylakoid membrane.

Polypeptides of the Thylakoid Membrane and Their Functional Characterization

1978 ◽  
Vol 33 (9-10) ◽  
pp. 723-730 ◽  
Author(s):  
Georg H. Schmid ◽  
Wilhelm Menke ◽  
Alfons Radunz ◽  
Friederike Koenig
Keyword(s):  
Molecular Weight ◽  
Electron Transport ◽  
Electron Donor ◽  
Photosystem I ◽  
Thylakoid Membrane ◽  
Apparent Molecular Weight ◽  
Variable Fluorescence ◽  
Phenazine Methosulfate ◽  
Cyclic Photophosphorylation ◽  
High Concentrations

Abstract From stroma-freed chloroplasts of Antirrhinum majus polypeptides with the apparent molecular weights 44 000, 26 000 and 20 000 were isolated.The antiserum to a polypeptide with the moleculair weight 44 000 inhibits the photoreduction of anthraquinone-2-sulfonate with dichlorophenol indophenol/ascorbate when the concentration of the electron donor dichlorophenol indophenol is low. The antiserum enhances the rate of phenazine methosulfate-mediated cyclic photophosphorylation. The variable fluorescence yield is increased by the antiserum . It is assumed that this polypeptide plays a role in electron transport between the two photosystems. From two polypeptides with the apparent molecular weight 26 000 one seems to belong to the reaction center of photosystem II as it inhibits the photooxidation of tetramethyl benzidine and diphenyl carbazide with suitable electron acceptors and inhibits electron transport between water and silicomolybdate. Variable fluorescence is not or not too strong decreased by the antiserum . The other polypeptide of the apparent molecular weight 26 000 inhibits the photoreduction of anthraquinone-2-sulfonate with high concentrations of dichlorophenol indophenol as the electron donor. Phenazine methosulfate-mediated cyclic photophosphorylation is also inhibited by the antiserum . Therefore, we should like to associate it with the reaction center of photosystem I. The antiserum to the polypeptide with the apparent molecular weight 20 000 inhibits the photoreduction of anthraquinone-2-sulfonate with low and high concentrations of the electron donor dichlorophenol indophenol. It enhances phenazine methosulfate-mediated cyclic photophosphorylation. The polypeptide, therefore, should be functionally involved on the acceptor side of photosystem I.The results obtained up-to-now on the function and localization of the polypeptides in the thylakoid membrane are summarized.

Photochemically Active Chlorophyll-Containing Proteins from Chloroplasts and their Localization in the Thylakoid Membrane

1972 ◽  
Vol 27 (10) ◽  
pp. 1225-1238 ◽  
Author(s):  
Friederike Koenig ◽  
Wilhelm Menke ◽  
Hans Craubner ◽  
Georg H. Schmid ◽  
Alfons Radunz
Keyword(s):  
Molecular Weight ◽  
Electron Transport ◽  
Electron Donor ◽  
Photosystem I ◽  
Outer Surface ◽  
Thylakoid Membrane ◽  
Gel Filtration ◽  
Lamellar System ◽  
Exciton Splitting ◽  
Chlorophyll Molecule

After solubilization of stroma-freed chloroplasts with deoxycholate, the lipids and the detergent used are separated from the proteins by gel filtration. In this way not denatured pigment-con-taining protein preparations were obtained. The particles in fraction 1 exhibited a molecular weight of 600 000 and contained an average of 25 chlorophyll molecules. The circular dichroism spectrum showed exciton splitting of the red band. The particles in fraction 2 contained 1 chlorophyll molecule and exhibited a molecular weight of 110 000. The particles in fraction 3 also contained only 1 chlorophyll molecule and had a molecular weight of between 80 000 and 100 000. Pure preparations of fraction 1 only carried out the methylviologen Mehler reaction with the dichlorophenol indophenol/ascorbate couple as electron donor. Fraction 3 only reduced ferricyanide with diphenylcarbazide as an electron donor in the light. Fraction 2 exhibited both the photosystem I reaction and the photosystem II reaction. An antiserum to extracted fraction 1 does not inhibit electron transport in the intact lamellar system. The photoreduction of methylviologen is only inhibited after disruption of the thylakoids. The antiserum to fraction 2 inhibits the photoreduction of methylviologen in the intact lamellar system. Consequently, one inhibition site for this photosystem I reaction must be located on the inner and another on the outer surface of the thylakoid membrane. In addition, antibodies to fraction 1 are specifically adsorbed onto the lamellar system without any effect on electron transport and without a concomitant agglutination. Antibodies to fraction 3 partially inhibit the photoreduction of ferricyanide with diphenylcarbazide as an electron donor in the intact lamellar system. Hence, the inhibition site of this system II reaction is located on the outer surface of the thylakoids. We have reason to believe that the inhibition sites not reacting are located in the partitions, which are not accessible to antibodies.

Inhibition of Electron Transport on the Oxygen-Evolving Side of Photosystem II by an Antiserum to a Polypeptide Isolated from the Thylakoid Membrane

1976 ◽  
Vol 31 (5-6) ◽  
pp. 304-311 ◽  
Author(s):  
Georg Schmid ◽  
Wilhelm Menke ◽  
Friederike Koenig ◽  
Alfons Radunz
Keyword(s):  
Electron Transport ◽  
Photosystem Ii ◽  
Water Splitting ◽  
Thylakoid Membrane ◽  
Inhibitory Action ◽  
Apparent Molecular Weight ◽  
Photosynthetic Electron Transport ◽  
Inhibitory Effect ◽  
Light Reaction ◽  
Tetramethyl Benzidine

Abstract A polypeptide fraction with the apparent molecular weight 11000 was isolated from stroma-freed chloroplasts from Anthirrhinum majus. An antiserum to this polypeptide fraction inhibits photosynthetic electron transport in chloroplasts from Nicotiana tabacum. The relative degree of inhibition is pH dependent and has its maximum at pH 7.4. The maximal inhibition observed was 93%. The dependence of the inhibition on the amount of antiserum yields a sigmoidal curve which hints at a cooperative effect. A calculation of the Hill interaction coefficient gave the value of 10. The inhibition occurs on the water splitting side of photosystem II between the sites of electron donation of tetramethyl benzidine and diphenylcarbazide. Tetramethyl benzidine donates its electrons before the site where diphenylcarbazide feeds in its electrons. Analysis of the steady state level of the variable fluorescence also indicates that the inhibition site is on the water splitting side of photosystem II. Tris-washed chloroplasts are equally inhibited by the antiserum and the inhibition is also observed in the presence of an inhibitor of photophosphorylation like dicyclo-hexyl carbodiimide and in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydra-zone (CCCP) which means that the inhibitory action is directed towards the electron transport chain. Valinomycin which is supposed to affect the cation permeability of the thylakoid membrane has no influence on the inhibitory action of the antiserum. The same is valid for gramicidin. Methylamine on the other hand can induce a state in the thylakoids in which the antiserum is not effective. If the antibodies are already adsorbed prior to the methylamine addition then the high inhibitory effect by the antiserum remains unchanged upon addition of methylamine. From the experiments it follows that a component from the vicinity of photosystem II is ac­cessible to antibodies that is, the component is located in the outer surface of the thylakoid mem­ brane. It appears that the inhibitory effect is produced in the course of the light reaction.

The Effect of Antibodies to Violaxanthin on Photosynthetic Electron Transport

1979 ◽  
Vol 34 (5-6) ◽  
pp. 427-430 ◽  
Author(s):  
Ursula Lehmann-Kirk ◽  
Georg H. Schmid ◽  
Alfons Radunz
Keyword(s):  
Electron Transport ◽  
Thylakoid Membrane ◽  
Specific Binding ◽  
Photosynthetic Electron Transport ◽  
Antigenic Determinants ◽  
Coombs Test ◽  
Diphenyl Carbazide ◽  
Photosynthetic Electron Transport Chain ◽  
Transport Chain ◽  
Oxygen Evolving

Abstract An antiserum to violaxanthin in hibits photosynthetic electron transport between water, iodide or tetramethylbenzidine and various electron acceptors in chloroplasts from green tobacco (Nicotian a tabacum var. John William’s Broadleaf). However, electron transport from manganese or diphenyl-carbazide to these acceptors is not impaired. The typical photosystem I reaction from DPIP / ascorbate to anthraquinone-2-sulfonate in the presence of DCMU shows no inhibition. From this it is concluded that the effect of violaxanthin on the photosynthetic electron transport chain lies on the oxygen-evolving side of photosystem II before the site from which diphenylcarbazide or manganese donate electrons.In the presence of DCMU after preillumination we find an effect of the antiserum on fluorescence.The reaction of the antibodies to violaxanthin with stroma-freed chloroplasts depends on the condition of the thylakoid membrane. Chloroplasts which are still swellable react in a bivalent manner and are agglutinated. Non swellable chloroplasts react only in a monovalent manner. This specific binding was demonstrated by means of the Coombs-test.From these reactions it follows that the antigenic determinants of violaxanthin are accessible to the antibodies, hence, they must be located in the outer surface of the thylakoid membrane.

Molecular Weight and the Dodecyl Sulphate Binding of a Thylakoid Membrane Polypeptide Involved in a Reaction on the Oxygen-Evolving Side of Photosystem II

1977 ◽  
Vol 32 (5-6) ◽  
pp. 384-391 ◽  
Author(s):  
Hans Craubner ◽  
Friederike Koenig
Keyword(s):  
Molecular Weight ◽  
Thylakoid Membrane ◽  
Phosphate Buffer ◽  
Equilibrium Dialysis ◽  
Sodium Dodecyl ◽  
Spherical Shape ◽  
Apparent Molecular Weight ◽  
Partial Specific Volume ◽  
Experimental Conditions ◽  
Sodium Phosphate Buffer

Abstract The molecular weight of a thylakoid membrane polypeptide with the apparent molecular weight 11 000 was determined by measurement of the sedimentation velocity, the diffusion and the ef­fective partial specific volume. The molecular weight was found to be 6300 and that of the poly-peptide-dodecyl sulphate micelle was found to be 11 200. The frictional ratio was 1.35. In ad­dition, we determined the binding of dodecyl sulphate onto the polypeptide by equilibrium dialysis. We found that 1 g polypeptide binds 0.77 g sodium dodecyl sulphate which corresponds to 17 molecules dodecyl sulphate bound per polypeptide chain. In the absence of dodecyl sulphate the polypeptide aggregates. The molecular weights of the aggregates are in 0.01 м sodium phosphate buffer pH 7.2 150 000 and in a 1 :1 mixture of 0.01 м phosphate buffer and 96% ethanol 365 000. The frictional ratios were 1.07 and 1.16 respectively which points at a spherical shape. The experimental conditions for the determination of the dodecyl sulphate binding were critically scrutinised.

Formation mechanisms of superoxide radical and hydrogen peroxide in chloroplasts, and factors determining the signalling by hydrogen peroxide

2018 ◽  
Vol 45 (2) ◽  
pp. 102 ◽  
Author(s):  
Boris N. Ivanov ◽  
Maria M. Borisova-Mubarakshina ◽  
Marina A. Kozuleva
Keyword(s):  
Hydrogen Peroxide ◽  
Electron Transport ◽  
Thylakoid Membrane ◽  
Electron Transport Chain ◽  
Superoxide Radical ◽  
Photosynthetic Electron Transport ◽  
Formation Mechanisms ◽  
H2o2 Production ◽  
Photosynthetic Electron Transport Chain ◽  
Transport Chain

Reduction of O2 molecule to superoxide radical, O2•−, in the photosynthetic electron transport chain is the first step of hydrogen peroxide, H2O2, production in chloroplasts in the light. The mechanisms of O2 reduction by ferredoxin, by the components of the plastoquinone pool, and by the electron transfer cofactors in PSI are analysed. The data indicating that O2•− and H2O2 can be produced both outside and within thylakoid membrane are presented. The H2O2 production in the chloroplast stroma is described as a result of either dismutation of O2•− or its reduction by stromal reductants. Formation of H2O2 within thylakoid membrane in the reaction of O2•− with plastohydroquinone is examined. The significance of both ways of H2O2 formation for specificity of the signal being sent by photosynthetic electron transport chain to cell adaptation systems is discussed.

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