Modifications of the gamma subunit of chloroplast coupling factor 1 alter interactions with the inhibitory epsilon subunit.

The treatment of chloroplast coupling factor 1 (CF1) with dithiothreitol or with trypsin modifies the gamma subunit. Reduction of the gamma subunit disulfide bond in CF1 in solution with dithiothreitol enhances the dissociation of epsilon (Duhe, R. J., and Selman, B. R. (1990) Biochim. Biophys. Acta 1017, 70-78). The Ca(2+)-ATPase activity of either oxidized or reduced CF1 increases as the enzyme is diluted. Added epsilon subunit inhibits the Ca(2+)-ATPase activity of both forms of the diluted CF1, suggesting that epsilon dissociation is the cause of activation by dilution. Half-maximal activation occurred at much higher concentrations of the reduced CF1, indicating that reduction decreases the affinity for epsilon about 20-fold. Immunoblotting techniques show that there is only one epsilon subunit/CF1 in intact chloroplasts, in thylakoid membranes, and in solution. No epsilon is released from CF1 in thylakoids under conditions of ATP synthesis. The gamma subunit of CF1 in illuminated thylakoids is specifically cleaved by trypsin. CF1 purified from thylakoids treated with trypsin in the light is deficient in epsilon subunit, and has a high rate of ATP hydrolysis. Added epsilon neither inhibits the ATPase activity of, nor binds tightly to the cleaved enzyme.     

Role of a disulfide bond in the gamma subunit in activation of the ATPase of chloroplast coupling factor 1

The relationship between activation of the latent ATPase activity of isolated chloroplast coupling factor 1 (CF1) and reduction of a disulfide in the gamma subunit has been assessed. The sulfhydryl residues involved in the disulfide bond are distinct from residues normally accessible to maleimide modification during incubation of thylakoids in the dark or the light. Dithiothreitol-induced activation is time dependent, and correlates with reduction of the disulfide. Sulfhydryl residues exposed during activation can be reoxidized to disulfide by incubation with iodosobenzoate , with a concomitant loss of ATPase activity. Activation and deactivation are reversible, but deactivation is prevented by treatment of the reduced enzyme with N-ethylmaleimide. Heat activation does not reduce the disulfide bond unless dithiothreitol is present during activation. Prior heating of CF1, which partially activates the enzyme, renders the disulfide more susceptible to subsequent dithiol reduction. The activity obtained when heat and dithiothreitol are used together is approximately equal to the sum of the partial activations obtained with heat or dithiothreitol alone. Iodosobenzoate has no effect on heat-activated CF1. Enzyme activated by heating in the presence of dithiothreitol can be partially deactivated, consistent with reversal of the activity attributable to the dithiol effect. Fluorescence polarization of anilinonaphthylmaleimide bound to the reduced enzyme indicates that the sulfhydryl residues involved in the disulfide are in a less rigid environment than the other two sulfhydryl residues in the gamma subunit. Polarization of anilinonaphthylmaleimide bound to these sulfhydryls is reduced by heat treatment of CF1. The increased susceptibility of the disulfide to reduction upon heat treatment, and the activation of ATPase activity with or without disulfide bond cleavage are indicative of conformational changes within the gamma subunit that occur during the conversion of CF1 from a latent to an active ATPase. In addition the results are consistent with at least two distinct conformational forms of CF1 that can hydrolyze ATP.     

Regulatory role of the C-terminus of the epsilon subunit from the chloroplast ATP synthase

The ATP synthases from chloroplasts and Escherichia coli are regulated by several factors, one of which is the epsilon subunit. This small subunit is also required for ATP synthesis. Thylakoid membranes reconstituted with CF1 lacking the epsilon subunit (CF1-epsilon) exhibit no ATP synthesis and very high ATP hydrolysis. Either native or recombinant epsilon restores ATP synthesis and inhibits ATP hydrolysis. Previously, we showed that truncated epsilon, lacking the last 45 C-terminal amino acids, restored ATP synthesis to membranes reconstituted with CF1-epsilon but was not an efficient inhibitor of ATP hydrolysis. In this paper, we show that this truncated epsilon is unable to inhibit ATP hydrolysis when Mg(2+) is the divalent cation present, both for the enzyme in solution and on the thylakoid membrane. In addition, the rate of reduction of the disulfide bond of the gamma subunit by dithiothreitol is not decreased by truncated epsilon, although full-length epsilon greatly impedes reduction. Thylakoid membranes can synthesize ATP at the expense of proton gradients generated by pH transitions in the dark. Our reconstituted membranes are able to produce a limited amount of ATP under these "acid-bath" conditions, with approximately equal amounts produced by the membranes containing wild-type epsilon and those containing truncated epsilon. However, the membranes containing truncated epsilon exhibit much higher background ATP hydrolysis under the same acid-bath conditions, leading to the conclusion that, without the C-terminus of epsilon, the CF1CFo is unable to check unwanted ATP hydrolysis.     

Participation of three distinct active states of chloroplast ATPase complex CF0 X CF1 in the activation by light and dithiothreitol

Chloroplast ATPase complex (CF0 X CF1) in thylakoids is activated by illumination in the presence or absence of dithiothreitol or by incubation with both dithiothreitol and Pi in the post-illumination dark. The activation by dithiothreitol and Pi is inhibited by ADP and decreases with increasing time interval between the end of illumination and the addition of dithiothreitol and Pi. The dithiothreitol/Pi-activated ATP hydrolysis is highly sensitive to pH. The ATP hydrolysis activated by illumination in the presence of dithiothreitol decreases its sensitivity to stimulation by NH4Cl and increases its sensitivity to pH, with increasing time interval between the end of illumination and the addition of ATP. Its pH dependence approaches that of the dithiothreitol/Pi-activated ATP hydrolysis. These results suggest that in the post-illumination dark, the light/dithiothreitol-activated CFo X CF1 converts its state from the one (E2) which is sensitive to an uncoupler and relatively insensitive to pH to the one (E2i) which is insensitive to an uncoupler and highly sensitive to pH. In the post-illumination dark, the presence of both dithiothreitol and Pi brings about the activation of CFo X CF1 to E2i.     

The Decay of the ATPase Activity of Light Plus Thiol-Activated Thylakoid Membranes in the Dark

Oxidized ATP synthase of spinach thylakoid membranes catalyzes high rates of ATP synthesis in the light, but very low rates of ATP hydrolysis in the dark. Reduction of the disulfide bond in the γ subunit of the ATP synthase in the light enhances the rate of Mg²⁺-ATP hydrolysis in the dark. The light plus thiol-activated state decays in a few minutes in the dark after illumination in Tris buffer, but not when Tricine was used in place of Tris. In this paper, it is shown that Tris in the assay mixture is an inhibitor of the light plus thiol-activated ATPase activity of thylakoids, but only after the activated membranes had incubated in the dark. Aminopropanediols and diethanolamine, also selectively inhibited ATPase activity of activated membranes after storage in the dark, whereas NH₄Cl and imidazole inhibit the ATPase activity of activated thylakoids almost equally whether they are added directly after the illumination or several minutes later. The fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA) is quenched by the establishment of proton gradients by ATP-dependent proton uptake. Addition of ATP to activated membranes results in rapid quenching of ACMA fluorescence. If the activated membranes were incubated in the dark prior to ATP addition, a lag in the ATP-dependent ACMA fluorescence quenching as well as a similar lag in the rate ATP hydrolysis were seen. It is concluded that ADP rebinds to CF1 in the dark following illumination and inhibits the activity of the ATP synthase. Reactivation of the ATP synthase in the dark can occur by the slow generation of proton gradients by ATP hydrolysis in the dark. This reactivation takes place in Tricine buffer, but not in Tris because of its uncoupling action. Whether ADP binding plays a role in the regulation of the activity of the ATP synthase in situ remains to be established.     

Photophosphorylation and the chemiosmotic perspective

Photophosphorylation was discovered in chloroplasts by D. Arnon and coworkers, and in bacterial ‘chromatophores’ (intercytoplasmic membranes) by A. Frenkel. Initial low rates were amplified by adding electron-carrying compounds such as FMN, later shown to support the ‘pseudocyclic’ electron flow. ATP synthesis, and coupling to electron flow, was detected accompanying linear electron flow from H₂O to either NADP⁺ or ferricyanide. Another pattern of electron flow supporting photophosphorylation was that of a cycle around Photosystem I (PS I). Isolation and analysis of the ATP synthase showed, as with mitochondrial and bacterial analogues, an intrinsic membrane complex (CF₀) and an extrinsic complex (CF₁). CF₁ is a latent ATPase, activated additively by the high-energy state of the thylakoids, and by reduction of a disulfide bond on the gamma subunit. Once reduced, ATP synthesis occurs at lower energy levels. The search for an ‘intermediate’ linking electron flow and ATP synthesis led to the discovery of post-illumination ATP synthesis by thylakoids, where turnover occurs in the dark. Once interpreted by P.Mitchell's chemiosmotic hypothesis, this led to the discovery of light-driven proton uptake into the thylakoid lumen, with accompanying Cl⁻ intake and Mg²⁺ and K⁺ output. Chemiosmosis was confirmed in several ways, including ATP synthesis in the dark due to an acid-to-base transition of thylakoids, and photophosphorylation accomplished in artificial lipid vesicles containing both the proton-pumping bacterial rhodopsin and a mitochondrial ATPase complex. The now generally accepted chemiosmotic interpretation is able to clarify some other aspects of photosynthesis as well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sulphydryl groups in photosynthetic energy conservation. V. Localization of the new disulfide bridges formed by o-iodosobenzoate in coupling factor of spinach chloroplasts.

1. Chemical modification by o-iodosobenzoate of soluble chloroplast coupling factor 1 (CF1) during heat activation resulted in inhibition of its Ca-ATPase activity and in the formation of two new intrapeptide disulfide bridges as suggested by: (a) the disappearance of three out of four accessible thiol groups, two from gamma and one from a beta subunit as a consequence of CF1 modification by o-iodosobenzoate; (b) the total free sulphydryl groups of CF1 were reduced from 8 to 4 after modification of CF1 by o-iodosobenzoate. Two groups disappeared from beta and two from gamma subunits; (c) a second heating step of CF1 in the presence of 10 mM dithioerythritol reversed the inhibition of the ATPase and reduced both the newly formed disulfide bridges and those present in native CF1. 2. Modification of chloroplasts in the light with o-iodosobenzoate resulted in the inhibition of photophosphorylation and ATPase. CF1 isolated and purified from these chloroplasts had its Ca-ATPase activity inhibited and two new disulfide bridges. The total number of free sulphydryl groups was reduced from 8 to 4 and three accessible groups disappeared from beta and gamma subunits.     

Reversible uncoupling of photophosphorylation by a new bifunctional maleimide.

A new bifunctional maleimide that contains a disulfide bond has been synthesized. This maleimide, dithiobis-N-ethylmaleimide (DTEM), like another bifunctional maleimide, o-phenylenebismaleimide, is about 500-fold more effective as an inhibitor of photophosphorylation than N-ethylmaleimide. Thylakoids must be illuminated in the presence of DTEM before the assay of phosphorylation for the inhibition to occur. Phosphoryalation in thylakoids treated with DTEM in the light is uncoupled and proton permeability of the treated thylakoids is enhanced. This uncoupling of photophosphorylation in thylakoids treated with DTEM can be reversed by thiol compounds. The addition of 50 mM dithiothreitol restores H+ uptake in thylakoids treated with DTEM in the light to control levels and partially reverses the inhibition of phosphorylation. Evidence is provided to show that DTEM cross-links groups within the gamma subunit of the coupling factor 1, and that the cross-link is broken by high concentrations of thiols. These results suggest that cross-linking is the cause for the increased proton permeability in thylakoids treated with bifunctional maleimides in the light.     

叶绿体atpE基因表达产物的生化特性

在细菌中表达的叶绿体ａｔｐＥ基因产物ε亚基蛋白对不同方式激活的叶绿体ＡＴ－Ｐａｓｅ均有抑制作用,而其抗血清则促进ＡＴ－Ｐａｓｅ活力。Ｅ．ｃｏｌｉ中表达的ε亚基蛋白在光合磷酸化反应中对循环和非循环光合磷酸化都有促进作用,其抗血清对循环光合磷酸化有抑制作用,而对非循环光合磷酸化则起促进作用。     

Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes

Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo.     

Selective modification of coupling factor 1 in spinach chloroplast thylakoids by a fluorescent maleimide

N-(1-Anilinonaphthyl-4)maleimide (ANM) has been used to modify coupling factor 1 (CF1), the terminal coupling factor of photophosphorylation in chloroplasts. As with other monofunctional maleimides, incubation of thylakoids with ANM in the light, but not in the dark, causes energy transfer inhibition of photophosphorylation. In the dark, sites on both the gamma and epsilon subunits of CF1 are modified. The light-accessible site is also on the gamma subunit. Trypsin digestion of the enzyme after dithiothreitol activation reveals that the dark-and light-accessible sites on the gamma subunit are different amino acid residues. Fluorescence of ANM bound at the dark-and light-accessible sites has been measured after isolation of CF1 from thylakoids. The fluorescence emission maximum of ANM at the light-accessible site is blue-shifted and the quantum yield is increased 2-fold relative to ANM bound at dark-accessible sites. On the soluble enzyme, fluorescence polarization is high and equivalent for ANM bound at both dark-and light-accessible sites. Fluorescence energy transfer from a tryptophan in a hydrophilic region of the epsilon subunit to ANM bound to the epsilon subunit but not to the gamma subunit has been observed. The significance of these observations is discussed with respect to the structure of the gamma subunit and its role in conformational transitions within CF1 that occur during energization of the membrane.     

Light-dependent cleavage of the gamma subunit of coupling factor 1 by trypsin causes activation of Mg2+-ATPase activity and uncoupling of photophosphorylation in spinach chloroplasts

Trypsin treatment of spinach chloroplast thylakoids in the light but not in the dark, results in a highly active Mg2+-ATPase and an uncoupling of photophosphorylation. These light-dependent effects are due to a modification of coupling factor 1 (CF1). CF1 purified from thylakoids treated with trypsin in the light contained a clipped beta subunit and a partially clipped gamma subunit, whereas that from thylakoids treated in the dark with trypsin contained only the clipped beta subunit. CF1 containing this modified gamma subunit also retained a high level of Ca2+-ATPase activity in solution. These results suggest that the gamma subunit becomes highly sensitive to trypsin only when the CF1 is in an active conformation. A similar hypersensitivity to proteases of the gamma subunit in highly purified CF1 is seen only after the enzyme is activated (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). The conversion of the enzyme to its active form, both on the membrane and in solution, therefore, seems to involve conformational changes that expose the gamma subunit to proteolysis.     

Characteristics of the Mg-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg²⁺-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na₂SO₃-dependent Mg²⁺-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg²⁺-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg²⁺-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg²⁺-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t_½ of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one.     

Partial proteolysis as a probe of the conformation of the gamma subunit in activated soluble and membrane-bound chloroplast coupling factor 1

Treatments that enhance the latent ATPase activity of the chloroplast coupling factor (CF1) also induce hypersensitivity of the gamma subunit toward trypsin. A number of different gamma subunit cleavage products are formed (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). We have compared the gamma cleavage products of membrane-bound and isolated CF1, activated either by reduction of the gamma disulfide bond or by removal of the epsilon subunit. The gamma subunit of isolated CF1 lacking the epsilon subunit was cleaved to a 27,000-Da species. The same cleavage site became exposed following energy-dependent conformational changes in the membrane-bound enzyme. Activation by reduction of the gamma disulfide bond also exposed this site. However, the gamma subunit of reduced CF1 was cleaved rapidly at an additional site and trypsin treatment gave rise to a 25,000-Da gamma species. The small peptide generated by the second cleavage contains one of the cysteinyl residues of the reduced disulfide bridge of gamma. This peptide dissociates from the enzyme and can be isolated by gel filtration. The close proximity of the trypsin cleavage sites to the disulfide bond of gamma is discussed with respect to the effects of tryptic cleavage on the ATPase activity of CF1. The data indicate that structural changes in a limited region of the gamma subunit strongly influence the catalytic properties of both soluble and membrane-bound CF1.     

ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity

The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.     

Formation and properties of hybrid photosynthetic F1-ATPases. Demonstration of different structural requirements for stimulation and inhibition by tentoxin.

A hybrid ATPase composed of cloned chloroplast ATP synthase beta and gamma subunits (betaC and gammaC) and the cloned alpha subunit from the Rhodospirillum rubrum ATP synthase (alphaR) was assembled using solubilized inclusion bodies and a simple single-step folding procedure. The catalytic properties of the assembled alpha3Rbeta3CgammaC were compared to those of the core alpha3Cbeta3CgammaC complex of the native chloroplast coupling factor 1 (CF1) and to another recently described hybrid enzyme containing R. rubrum alpha and beta subunits and the CF1 gamma subunit (alpha3Rbeta3RgammaC). All three enzymes were similarly stimulated by dithiothreitol and inhibited by copper chloride in response to reduction and oxidation, respectively, of the disulfide bond in the chloroplast gamma subunit. In addition, all three enzymes exhibited the same concentration dependence for inhibition by the CF1 epsilon subunit. Thus the CF1 gamma subunit conferred full redox regulation and normal epsilon binding to the two hybrid enzymes. Only the native CF1 alpha3Cbeta3CgammaC complex was inhibited by tentoxin, confirming the requirement for both CF1 alpha and beta subunits for tentoxin inhibition. However, the alpha3Rbeta3CgammaC complex, like the alpha3Cbeta3CgammaC complex, was stimulated by tentoxin at concentrations in excess of 10 microm. In addition, replacement of the aspartate at position 83 in betaC with leucine resulted in the loss of stimulation in the alpha3Rbeta3CgammaC hybrid. The results indicate that both inhibition and stimulation by tentoxin require a similar structural contribution from the beta subunit, but differ in their requirements for alpha subunit structure.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

N-Ethylmaleimide inhibition of the catalytic activities of the Dunaliella salina coupling factor 1 (CF1) and the restoration of the inhibition of the CF1 ATPase activity by N-ethylmaleimide

The sensitivity of the catalytic activities of the D. salina chloroplast coupling factor 1 (CF1) to chemical modification by N-ethylmaleimide has been investigated. When D. salina thylakoid membranes are treated with N-ethylmaleimide, both photophosphorylation and the inducible CF1 ATPase activity are partially (approx. 60%) inhibited. The inhibition of both activities does not require the presence of a proton-motive force, and the inhibition of photophosphorylation is directly related to the N-ethylmaleimide-covalent modification of CF1 as shown by the time-course for the inhibition and the maximal extent of inhibition. Treatment of the purified, latent, D. salina CF1 with low concentrations of N-ethylmaleimide also results in the partial (approx. 60%) inhibition of the inducible ATPase activity (I50 approximately 50 microM). The inhibition does not require the presence of the chemical modifier during the activation of the enzyme. N-ethylmaleimide-induced inhibition of the ATPase activity of either membrane-bound or solubilized CF1 is partially reversed by either prolonged incubation at low concentrations of N-ethylmaleimide or short incubation times at high concentrations of N-ethylmaleimide. The results are interpreted as indicating multiple binding sites on the D. salina CF1 that have different rates of reactivity with N-ethylmaleimide. Those sites (or site) that react rapidly with N-ethylmaleimide cause(s) an inhibition of both ATP synthase and ATPase activities, whereas those sites (or site) that react more slowly partially restore(s) the original ATPase activity. The effects of N-ethylmaleimide on the catalytic activity of D. salina CF1 are probably mediated by N-ethylmaleimide-induced conformational changes of the enzyme.     

Kinetics of electron transport, proton transfer and photophosphorylation in chloroplasts and their relation to temperature-induced structural changes in the thylakoid membrane

Effects of various temperatures on the rates of electron transport between two photosystems, the light-induced uptake of protons, kinetics of proton efflux from the chloroplasts in the dark and photophosphorylation were studied in isolated chloroplasts. There are correlations between the physical state of thylakoid membrane and the rates of electron- and proton transport processes. The temperature dependence of "structural" parameter (fluidity of lipids in membrane) as well as the rates of electron- and proton transport processes reveal the breaks under the same temperatures. Stimulation of photophosphorylation by temperature increasing correlates with the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the protein part of CF0-CF1 complex. The rate of photophosphorylation also correlates with the physical state of membrane lipids. Thermoinduced "melting" of the thylakoid membrane inhibits the ATP formation because of a decrease in photosystem 2 photochemical activity and stimulation of membrane conductivity for protons.     

Effect of proteolytic digestion on the Ca2+-ATPase activity and subunits of latent and thiol-activated chloroplast coupling factor 1

The activation by proteases of the Ca2+-dependent ATPase of chloroplast coupling factor 1 (CF1) has been investigated. Using low concentrations of papain and trypsin, the increase in ATPase activity and the degradation of the five subunits of CF1 were compared. Sodium dodecyl sulfate-gel electrophoresis of protease-treated CF1 revealed that the delta subunit was very rapidly degraded and that the alpha and beta subunits were clipped. The gamma and epsilon subunits were more resistant to digestion. The modification of the alpha subunit of latent CF1 most closely correlated with the activation of Ca2+-ATPase activity. Trypsin treatment of dithiothreitol-activated CF1 resulted in a very rapid increase in Ca2+-ATPase activity and a corresponding rapid cleavage of the gamma subunit to a 25,000-dalton species. With more prolonged treatment, the 25,000-dalton species was cleaved to fragments of 14,000 and 11,000-daltons. Dithiothreitol treatment did not alter the rate of attack on the other subunits. The gamma subunit of heat-activated CF1 was also more susceptible to protease digestion. The increased protease sensitivity of the gamma subunit of soluble CF1 after treatment with dithiothreitol or heat mimics the increased protease sensitivity of the gamma subunit of bound CF1 when thylakoids are treated with trypsin during illumination (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5915-5920). These results suggest that the conformational changes that occur when purified CF1 is exposed to dithiothreitol are similar to those that CF1 bound to thylakoid membranes undergoes under illumination.