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.

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.

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.

The dissociation of nuclear proteins from superhelical DNA

1978 ◽  
Vol 29 (1) ◽  
pp. 103-116
Author(s):  
J.M. Levin ◽  
E. Jost ◽  
P.R. Cook
Keyword(s):  
Molecular Weight ◽  
Free Energy ◽  
Binding Energy ◽  
Apparent Molecular Weight ◽  
Morphological Features ◽  
Nuclear Proteins ◽  
Double Labelling ◽  
Ionic Detergents ◽  
Labelling Procedure ◽  
High Concentrations

Structures retaining many of the morphological features of nuclei may be released by gently lysing human cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain superhelical DNA. Using a double-labelling procedure we have compared, at different salt concentrations, the amounts and types of protein associated with human nucleoides containings superhelical or relaxed DNA. We find that the slightly lysine-rich histones (H2A and H2B) but not the arginine-rich histones (H3 and H4) dissociate more slowly from nucleoids containing superhelical DNA than from those containing relaxed DNA. A protein of apparent molecular weight of 22000 also binds more tightly to superhelical DNA. We conclude that this protein and the slightly lysine-rich histones transmute free energy of supercoiling into binding energy when they bind to superhelical DNA.

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.

Effects of 3,5-dibromo-4-hydroxybenzaldehyde O-(2,4-dinitrophenyl)oxime on Reactions of Mitochondria and Chloroplasts

Weed Science ◽  
1970 ◽  
Vol 18 (3) ◽  
pp. 419-426 ◽  
Author(s):  
D. E. Moreland ◽  
W. J. Blackmon
Keyword(s):  
Electron Transport ◽  
Oxygen Uptake ◽  
Electron Donor ◽  
Spinacia Oleracea ◽  
Herbicidal Activity ◽  
Oxygen Utilization ◽  
White Potato ◽  
Ferricyanide Reduction ◽  
Cyclic Photophosphorylation ◽  
Limited Oxygen

The effects of 3,5-dibromo-4-hydroxybenzaldehyde O-(2,4-dinitrophenyl)oxime (hereinafter referred to as C-9122), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-dibromo-4-hydroxybenzaldoxime (hereinafter referred to as bromoxime), and 2,4-dinitrophenol (hereinafter referred to as DNP) on phosphorylation and electron transport were measured in mitochondria isolated from white potato tubers (Solarium tuberosum L.) and in chloroplasts from spinach leaves (Spinacia oleracea L.). Mitochondrial oxygen utilization was monitored polarographically. All four chemicals stimulated ADP-limited oxygen utilization, inhibited non-ADP-limited oxygen uptake, and relieved oligomycin-inhibited oxygen uptake. C-9122 produced responses at lower molar concentrations than did bromoxynil, bromoxime, and DNP. The I50 value for inhibition of state 3 respiration by C-9122 was 2.7 × 10−6 M.In chloroplasts, C-9122, bromoxime, and DNP inhibited photoreduction and coupled photophosphorylation with water as the electron donor, and with ferricyanide and NADP as electron acceptors. Cyclic photophosphorylation, with phenazine methosulfate as the electron mediator under an argon gas phase, also was inhibited. With ascorbate-2,6-dichlorophenolindophenol (hereinafter referred to as DPIP) as the electron donor, phosphorylation coupled to NADP reduction was inhibited, but not the reduction of NADP. C-9122 was the strongest inhibitor, and bromoxime was the weakest inhibitor of the several reactions. The I50 value for inhibition of the coupled ferricyanide reduction was 4.6 × 10−6 M for C-9122. C-9122 appeared to act in two different ways by (a) inhibiting electron transport at or near photosystem II and the oxygen evolution pathway, and (b) interfering with energy transfer and the generation of ATP. Bromoxynil inhibited photoreduction and photophosphorylation reactions in which water served as the electron donor; but it was a very poor inhibitor of both cyclic photophosphorylation, and photophosphorylation coupled to NADP reduction with ascorbate-DPIP serving as the electron donor. Because of the pivotal role of ATP in cellular metabolism, it is conceivable that interference with ATP generation could be a major (but not necessarily the only) mechanism through which the herbicidal activity of C-9122 is expressed.

The Effect of an Antiserum to Plastocyanin on Various Chloroplast Preparations

1975 ◽  
Vol 30 (3-4) ◽  
pp. 201-212 ◽  
Author(s):  
Georg Schmid ◽  
Alfons Radunz ◽  
Wilhelm Menke
Keyword(s):  
Electron Transport ◽  
Thylakoid Membrane ◽  
Electron Flow ◽  
Linear Electron ◽  
Wild Type ◽  
Lamellar System ◽  
Spinach Chloroplasts ◽  
Monospecific Antiserum ◽  
Cyclic Photophosphorylation ◽  
Aurea Mutant

Abstract A monospecific antiserum to tobacco plastocyanin agglutinates strom a-free sw ellable chloroplasts from wild type tobacco, (Nicotia na tabacum var. John William’s Broadleaf) from the tobacco aurea mutant Su/su2, (Nicotiana tabacum var. Su/su2) from Antirrhinum majus and spinach (Spi-nacia oleracea). In this condition the antiserum inhibits linear photosynthetic electron flow in tobacco and spinach chloroplasts. This inhibition of electron transport as well as the agglutination are not observed if the chloroplasts have been sonicated prior to antiserum addition. This is due to the fact that plastocyanin is removed by ultrasonication. The antiserum stimulates a number of photophosphorylation reactions in tobacco chloroplasts. This stimulation is always larger in the aurea mutant chloroplasts and in chloroplasts from yellow leaf patches of a variegated tobacco mutant (N . tabacum , var. NC95) than in the green type chloroplasts. The stimulation appears to be a consequence of the inhibition of linear electron transport. The antiserum does not affect PMS-mediated cyclic photophosphorylation in tobacco chloroplasts from the wild type whereas the reaction appears stimulated in the tobacco mutant chloroplasts. However, menadione-mediated cyclic photo­ phosphorylation is inhibited upon addition of the antiserum. The same is true for noncyclic photo­ phosphorylation coupled to electron transport in the aerobic system diaminodurene/ascorbate → methylviologen in the presence of N-tetraphenyl-p-phenylenediamine in spinach chloroplasts. If the lamellar system of Antirrhinum and spinach has lost its swellability neither agglutination nor inhibition of electron transport is observed. However, also in this state antibodies to plasto­ cyanin are specifically adsorbed onto the surface of the thylakoid membrane. This state which is characterized by a morphologically well preserved lamellar system is realized in chloroplast prepa­ rations from Antirrhinum and spinach and is termed stroma-freed, chloroplasts. In both states of the molecular structure of the thylakoid membrane, plastocyanin is located in the outer surface of the thylakoid. However, it cannot be excluded that functioning plastocyanin is also located in the interior 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.

Interference by Herbicides with the Transmembrane Potential of Thylakoids

1987 ◽  
Vol 42 (6) ◽  
pp. 718-726 ◽  
Author(s):  
Donald E. Moreland ◽  
William P. Novitzky
Keyword(s):  
Electron Transport ◽  
Thylakoid Membrane ◽  
Transmembrane Potential ◽  
Proton Motive Force ◽  
Cyclic Electron Transport ◽  
Low Concentrations ◽  
High Concentrations ◽  
Proton Shuttling ◽  
Electrical Components ◽  
Spinach Thylakoids

Interferences expressed by herbicides classified as inhibitory uncouplers were measured on the induction and maintenance of ΔpH and ΔΨ, the chemical and electrical components, respectively. of the proton motive force (pmf) generated by light-induced cyclic electron transport in spinach thylakoids. Maintenance of the pmf is required for the synthesis of ATP. The inhibitory uncouplers arc known to inhibit photophosphorylation, but the mechanisms involved remain to be identified. The dinoseb types (dinitrophenols. benzimidazoles. benzonitriles. bromophenoxim. perfluidone. thiadiazoles) of inhibitory uncouplers, most of which contain dissociable protons, were found to discharge ΔpΗ at low concentrations and to collapse ΔΨ at high concentrations. Collapse of ΔpΗ can be attributed to the protonophoric (proton shuttling) action of the herbicides. However, collapse of ΔΨ can be caused by alterations induced to the integrity and loss of semipermeability of the thylakoid membrane. As a result the membrane bccomes permeable to protons and other cations, and the electrical charges across the membrane are neutralized. The non-ionic dicryl types of inhibitory uncouplers (acylanilides. dinitroanilines. diphenylethcrs. bis-carbamates) collapsed ΔΨ at concentrations that were somewhat lower than those required for the collapse of ΔpΗ. These herbicides appear only to alter the integrity and permeability of the thylakoid membrane. Inhibition of photophosphorylation by the inhibitory uncouplcrs correlated with their ability to dissipate the pmf.

Zum Mechanismus des photosynthetischen Elektronentransportes in isolierten Chloroplasten

1965 ◽  
Vol 20 (2) ◽  
pp. 143-147 ◽  
Author(s):  
Achim Trebst ◽  
Elfriede Pistorius
Keyword(s):  
Electron Transport ◽  
Electron Transport Chain ◽  
Phosphorylation Site ◽  
Atp Formation ◽  
Point Of Entry ◽  
Transport Chain ◽  
Endogenous Compounds ◽  
Cyclic Photophosphorylation ◽  
Photosynthetic Reactions ◽  
High Concentrations

The behavior of 2,3,5,6-tetramethyl-p-phenylendiamine (DAD) in photosynthetic reactions of isolated chloroplast fragments was compared with that of N-tetramethyl-p-phenylendiamine (TMPD). Both reverse the DCMU-inhibition of photosynthetic NADP-reduction. The DAD-system (at high concentrations of DAD), is coupled to a stoichiometric ATP-formation, whereas the TMPD-system is not. This shows that p-phenylendiamines, depending on their constitution, may react with components of the electron transport chain of chloroplasts before or after the phosphorylation site, and locates the phosphorylation step of photosynthetic phosphorylation between two endogenous compounds in that part of the electron transport chain, which connects the two light reactions of photosynthesis.At lower concentrations of DAD the diminished NADP-reduction is no longer coupled to ATP-formation, indicating a second point of entry of electrons from DAD into the electron transport chain. DAD, furthermore, is a cofactor of cyclic photophosphorylation. It therefore behaves like DCPIP, but the rates of the DAD-system are higher.

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