Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

Winget, G.Douglas; Kanner, N.; Racker, Efraim

doi:10.1016/0005-2728(77)90087-1

Cited by 79 publications

(24 citation statements)

References 16 publications

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“…of Winget et al (50), resuspended in STM buffer at 24 mg Chi/ ml, and either used directly or stored at -80°C for up to 4 weeks until use. Such storage resulted in less than a 20%1o decrease in rates of 02 evolution and no change in behaviors of extraction/reconstitution.…”

Section: Methodsmentioning

confidence: 99%

Studies on the Reconstitution of O₂-Evolution of Chloroplasts

Sayre

Cheniae²

1982

Plant Physiol.

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Extraction of spinach (Spinacia oleracea L.) chloroplasts with cholateasolectin in the absence of Mge+ results in the rapid and selective inactivation of 02 evolution and a partial (30 to 40%) loss of photosystem II (PSII) donor activity without extraction of thylakoid bound Mn (-5 to 6 Mn per 400 Chlorophyll). Inclusion of ethylene glycol in the extractions inhibits loss of 02 evolution and results in quantitative and qualitative differences in proteins solubilized but does not significantly inhibit the partial loss of PSII donor activity. Similarly, in two stage experiments (extraction followed by adtion oforganic solvent and solubilized thylakold protein), 02 evolution (V and V..) of extracted chlorplasts is enhanced approximately 2.5-to 8-fold. However, PSII donor activity remains unaffected. This reversal of cholate inactivation of 02 evolution can be induced by solvents including ethanol, methanol, 2-propanol, and dinethyl sulfoxide.Such enhancements of 02 evolution specificaly required cholate-solubilized proteins, which are insensitive to NH2OH and are only moderately heat-labile. NH2OH extraction of chloroplasts prior to cholate-asolectin extraction abolishes reconstitutablilty of 02 evolution. Thus, the protein(s) affecting reconstitution is unlike those of the 02 .M enzyme. eThe specific activity of the protein fraction effecting reconstitution of 03 evolution is greatest in fractions depleted of the reported Mn-containg, 65-kilodalton, and the Fe-heme, 232-kilodalton (58-kilWalton monomer), proteins. Divalent (-3 millimlar) and monovalent (-30 illimhr) cations do not affect reconstitution of PSII donor activity but do affect reconstitution of 02 evolution by decreasing the protein(s) concentration required for reconstitution of 02 evolution in nonfractionated, cholate-asolectin extractions. The data indicate a reconstitution of the PSII segment inkig the PSII secondary donor(s) to 0revolving centers.Considerable information is available concerning the kinetic aspects of 02 evolution (37) and the intermediates linking the Sstates of 02 evolution to P60 (6). Additionally, much evidence has accumulated which strongly suggests that thylakoid-bound Mn is essential in the 02-evolving reactions (37) and that such Mn undergoes oxidation-reduction during cycling of the S-states (14,40,51).By and large, however, this segment of the electron transport sequence of photosynthesis, in contrast to PSI, has been rather intractable to biochemical approaches aimed at the isolation and characterization of the proteinaceous catalysts involved (37). Such information is vital to the basic understanding of this biologically unique sequence of reactions, namely, the photosynthetic production of 02-Recently, two different approaches have been used in attempts to gain such insights: analyses of polypeptide composition and thylakoid Mn abundance of mutants blocked specifically on the oxidizing side of PSII (32, 33, 41); and detergent extraction and reconstitution analyses of chloroplasts with the use of artificial lipo...

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Section: Methodsmentioning

confidence: 99%

Studies on the Reconstitution of O₂-Evolution of Chloroplasts

Sayre

Cheniae²

1982

Plant Physiol.

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“…Chloroplasts were isolated from fresh market spinach and extracted with cholate (6). Briefly, specific chloroplast lipids and proteins were solubilized by using 0.05 M sodium cholate and 0.4 M ammonium sulfate at pH 8.0 with stirring for 15 min on ice.…”

Section: Methodsmentioning

confidence: 99%

Purification of a manganese-containing protein involved in photosynthetic oxygen evolution and its use in reconstituting an active membrane

Spector

Winget

1980

Proc. Natl. Acad. Sci. U.S.A.

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Extraction of thylakoid membranes with cholate in the presence of ammonium sulfate inactivated oxygen evolution and liberated a manganese-containing protein. This protein could be combined with preformed liposomes containing the depleted thylakoid membranes to restore 85% of the original oxygen-evolution activity. The protein did not affect the primary photochemical events of photosystem I or photosystem II, and it was required only for electron transport in which water was the electron donor. The protein has been purified to homogeneity and has an apparent molecular weight of 65,000 (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate). Atomic absorption revealed two atoms of manganese bound to each 65,000-dalton protein molecule. Treatment with alkaline Tris removed the bound manganese and rendered the protein incapable of restoring oxygen evolution; however, Tris treatment of the depleted membranes before reconstitution had no effect. Thus, this manganese protein is probably the site of Tris action in isolated chloroplasts and is at least part of the water-oxidation enzyme system. The chemical events leading to the photosynthetic evolution of oxygen are still largely a matter of speculation. Many aspects of the kinetics of oxygen evolution have been elucidated. A model involving the storage of oxidizing equivalents on an unknown enzyme (the "S-states") has been proposed and seems to have gained general acceptance (1). The photoactivation of an uncharacterized manganese-containing protein may be essential for oxygen evolution (2, 3). In addition, the specific requirement for manganese in the oxygen-evolving reaction has been demonstrated by inducing deficiency during growth or by depleting manganese by extraction, in both algae and higher plants (4, 5).A subchloroplast system in which depletion and restoration of oxygen evolution can be accomplished by the extraction and replacement of individual thylakoid components would be helpful in furthering our understanding of photosynthetic oxygen evolution. We present here a method for extracting a manganese-containing protein and using it to reconstitute an active system; our results provide evidence that this protein is required for photosynthetic oxygen evolution. METHODS AND MATERIALSChloroplasts were isolated from fresh market spinach and extracted with cholate (6). Briefly, specific chloroplast lipids and proteins were solubilized by using 0.05 M sodium cholate and 0.4 M ammonium sulfate at pH 8.0 with stirring for 15 min on ice. The suspension was then centrifuged at 144,000 X g for 90 min, and the supernatant was collected; the pellet was set aside. Saturated ammonium sulfate (pH 8.0) was then added to the supernatant to increase the concentration to 1.2 M. After incubation for 20 min on ice the precipitate was collected by centrifugation (10,000 X g, 10 min). This pellet was dissolved in about 3 ml of 0.2 M sucrose/0.02 M N-[tris(hydroxymethyl)methyllglycine (Tricine)-NaOH (pH 8.0)/S mM MgCl2, then adjusted to 0.84 M with sat...

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“…Band 8 protein was a proteolipid that reacted specifically with dicyclohexylcarbodiimide and seemed to play a central role in H+ conduction through the membrane. Accumulating evidence has indicated that biomembranes that synthesize ATP contain a reversible H+-translocating ATPase complex, named Fo-Fl, as an energy transducing coupling device (1)(2)(3)(4)(5)(6). The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase complex purified from the thermophilic bacterium PS3 (TFo-Fl), when reconstituted into liposomes, was actually able to translocate protons coupled to ATP hydrolysis (7,8).…”

Section: Somes (Tfo Vesicles) the Binding Of Tf1 To Tfo Present In Ves-mentioning

confidence: 99%

Resolution of the membrane moiety of the H+-ATPase complex into two kinds of subunits.

Sone¹,

Yoshida²,

Hirata³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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The HW-translocating ATPase complex from the thermophilic bacterium PS3 (TFo.F1) is composed of a water-soluble part with ATP-hydrolyzing activity (TF1) and a membrane moiety with H+-conducting activity (TFo). TFo was obtained by treating TFo0F1 with urea and removing contaminations on a carboxymethyl-cellulose column. This TFo contained only two kinds of subunits, band 6 protein (13,500 daltons) and band 8 protein (5400 daltons), and it was active in H+ conduction and TF1 binding when reconstituted into proteoliposomes (TFo vesicles Accumulating evidence has indicated that biomembranes that synthesize ATP contain a reversible H+-translocating ATPase complex, named Fo-Fl, as an energy transducing coupling device (1-6). The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase complex purified from the thermophilic bacterium PS3 (TFo-Fl), when reconstituted into liposomes, was actually able to translocate protons coupled to ATP hydrolysis (7,8). The electrochemical proton gradient (A7gH+) formed (inside acidic and positive) by ATP hydrolysis was as much as 300 mV (7), and ATP synthesis took place when a L\IH+ of over 200 mV was imposed across the liposomal membranes (6, 9).It seems both interesting and important to determine the subunit structure of Fo-Fl, especially for clarifying the method by which this transducer works. The catalytic and peripheral moiety of the transducer is Fl-ATPase, which is composed of five subunits and has a molecular weight of about 3.8 X 105.Recently these five subunits (a, f3, y, 6, and c) were each purified in reconstitutively active forms from TF1 (thermophilic F1) and their roles in TFo-F1 were proposed (10,11 (19), with slight modifications: a solution (20 ml) of purified TF0-Fj (40-100 mg of protein) in 7 M urea/0.2 M sucrose/5 mM EDTA/5 mM dithiothreitol/50 mM Tris-H2SO4, pH 8.0 was stirred for 2.5-3 hr at 50, diluted with distilled water (6 ml) to reduce the concentration of urea, and centrifuged at 200,000 X g for 40 min. This process was repeated once more and the resultant precipitate (TFo) was suspended in 10 mM N-[tris(hydroxymethyl)methyl]glycine (Tricine)/NaOH (pH 8.0) containing 2 mM EDTA and stored at -80°until use. About one-fifth of TFo-F1 was recovered as TFo. The method for reconstitution of TFo or TF0-F1 into vesicles was as described (8,19) except for the protein concentration of TFo used (0.15-0.3 mg/ml). TFo treated with CM-cellulose was prepared as follows: TFo (3 mg of protein) was incubated in 2 ml of 4 M urea containing 0.25% Triton X-100 and 10 mM phosphate buffer (pH 7.0) for 1 hr at 500 and then applied to a column of CMcellulose (1 X 6 cm) equilibrated with the same solution. The unabsorbed material was collected and precipitated with ammonium sulfate (45% saturation) in the presence of 2% cholate (18).Assay of Proton Permeability of Vesicles. An aliquot (0.2 ml) of reconstituted vesicles was incubated with 0.5 M KCI (2 ml), 0.1 M EDTA (0.1 ml), and 0.5 M dithiothreitol (0.02 ml) for 30 min at 55°. Then the mixture was cooled and centrifuged a...

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Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

Cited by 79 publications

References 16 publications

Studies on the Reconstitution of O₂-Evolution of Chloroplasts

Studies on the Reconstitution of O₂-Evolution of Chloroplasts

Purification of a manganese-containing protein involved in photosynthetic oxygen evolution and its use in reconstituting an active membrane

Resolution of the membrane moiety of the H+-ATPase complex into two kinds of subunits.

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Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

Cited by 79 publications

References 16 publications

Studies on the Reconstitution of O2-Evolution of Chloroplasts

Studies on the Reconstitution of O2-Evolution of Chloroplasts

Purification of a manganese-containing protein involved in photosynthetic oxygen evolution and its use in reconstituting an active membrane

Resolution of the membrane moiety of the H+-ATPase complex into two kinds of subunits.

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Studies on the Reconstitution of O₂-Evolution of Chloroplasts

Studies on the Reconstitution of O₂-Evolution of Chloroplasts