Two highly conserved amino acid residues, an arginine and a glutamine, located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen bonding with residues in an anionic loop on one of the three catalytic beta subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. The catch is considered to play a critical role in the binding change mechanism whereby binding of ATP to one catalytic site releases the catch and induces a partial rotation of the gamma subunit. This role is supported by the observation that mutation of the equivalent arginine and glutamine residues in the Escherichia coli F1 gamma subunit drastically reduced all ATP-dependent catalytic activities of the enzyme [Greene, M. D., and Frasch, W. D. (2003) J. Biol. Chem. 278, 5194-5198]. In this study, we show that simultaneous substitution of the equivalent residues in the chloroplast F1 gamma subunit, arginine 304 and glutamine 305, with alanine decreased the level of proton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATP hydrolysis activities increased by more than 3-fold as a result of these mutations; however, the sulfite-stimulated activity decreased by more than 60%. The Mg2+-dependent, but not the Ca2+-dependent, ATPase activity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicating selective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catch residues are required for efficient proton coupling and for activation of multisite catalysis when MgATP is the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore, for rotation of the gamma subunit.

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591–598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory ε subunit (CF1-ε) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-ε or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-ε from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the α/β heterohexamer.

Researchers in the field of bioinformatics have developed a number of analytical programs and databases that are increasingly important for advancing biological research. Because bioinformatics programs are used to analyze, visualize, and/or compare biological data, it is likely that the use of these programs will have a positive impact on biology education. Over the past years, we have been working to help biology instructors introduce bioinformatics activities into their curricula by providing them with instructional materials that use bioinformatics programs and databases as educational tools. In this study, we measured the impact of a set of these materials on student learning. The activities in these materials asked students to use the molecular structure visualization program Cn3D to locate, identify, or analyze diverse features in DNA structures. Both the experimental groups of college and high school students showed significant increases in learning relative to control groups. Further, learning gains by the college students were correlated with the number of activities assigned. We conclude that working with Cn3D was important for improving student understanding of DNA structure. This study is one example of how a bioinformatics program for visualization can be used to support student learning.

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the γ subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the γ subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the ε subunit. Our results suggest that overcoming inhibition by the ε subunit costs energy.

The green alga Chlamydomonas reinhardtii is a model organism for the study of photosynthesis. The chloroplast ATP synthase is responsible for the synthesis of ATP during photosynthesis. Using genetic engineering and biolistic transformation, a string of eight histidine residues has been inserted into the amino-terminal end of the beta subunit of this enzyme in C. reinhardtii. The incorporation of these amino acids did not impact the function of the ATP synthase either in vivo or in vitro and the resulting strain of C. reinhardtii showed normal growth. The addition of these amino acids can be seen through altered gel mobility of the beta subunit and the binding of a polyhistidine-specific dye to the subunit. The purified his-tagged CF1 has normal Mg(2+)-ATPase activity, which can be stimulated by alcohol and detergents and the enzyme remains active while bound to a nickel-coated surface. Potential uses for this tagged enzyme as a biochemical tool are discussed.

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.

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.

Proton Motive Force R. E. McCarty, R. E. McCarty Johns Hopkins University, Baltimore, MDSearch for more papers by this author R. E. McCarty, R. E. McCarty Johns Hopkins University, Baltimore, MDSearch for more papers by this author First published: 15 January 2002 https://doi.org/10.1002/047120918X.emb1249 Read the full textAbout ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Encyclopedia of Molecular BiologyBrowse other articles of this reference work:BROWSE BY TOPICBROWSE A-Z RelatedInformation

The gene encoding the E subunit (atpf?) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia COK The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol to obtain E that i s as biologically active as E purified from chloroplastcoupling factor 1 (CF,). Recombinant E folded in this manner inhibits the ATPase activity of soluble and membrane-bound CF, deficient in e and restores proton impermeability to thylakoid membranes reconstituted with CF, deficient i n E . Site-directed mutagenesis was used to generate truncations and single amino acid substitutions i n the primary structure of E . I n the five mutants tested, alterations that weaken ATPase inhibition by recombinant E affect i ts ability t o restore proton impermeability to a similar extent, wi th one exception. Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeabilty. As in the case of E. coli, it appears that N-terminal truncations of the 4 subunit have more profound effects than C-terminal deletions on the function of E. Recombinant E with six amino acids deleted from the C terminus, which i s the only region of significant mismatch between the E of spinach and the E of Pisum sativum, inhibits ATPase activity with a reduced potency similar t o that of purified pea E . Four of the six amino acids are serine or threonine. These hydroxylated amino acids may be important in E-CF, interactions.

The epsilon subunit of the ATP synthases from chloroplasts and Escherichia coli regulates the activity of the enzyme and is required for ATP synthesis. The epsilon subunit is not required for the binding of the catalytic portion of the chloroplast ATP synthase (CF1) to the membrane-embedded part (CFo). Thylakoid membranes reconstituted with CF1 lacking its epsilon subunit (CF1-epsilon) have high ATPase activity and no ATP synthesis activity, at least in part because the membranes are very leaky to protons. Either native or recombinant epsilon subunit inhibits ATPase activity and restores low proton permeability and ATP synthesis. In this paper we show that recombinant epsilon subunit from which 45 amino acids were deleted from the C-terminus is as active as full-length epsilon subunit in restoring ATP synthesis to membranes containing CF1-epsilon. However, the truncated form of the epsilon subunit was significantly less effective as an inhibitor of the ATPase activity of CF1-epsilon, both in solution and bound to thylakoid membranes. Thus, the C-terminus of the epsilon subunit is more involved in regulation of activity, by inhibiting ATP hydrolysis, than in ATP synthesis.

A chloroplast ATP synthase complex (CFl [chloroplast-coupling factor l]-CFo [membrane-spanning portion of chloroplast ATP synthase]) depleted of all CF, subunits except subunit 111 (also known as the proteolipid subunit) was purified to study the interaction between CFl and subunit 111. Subunit 111 has a putative role in proton translocation across the thylakoid membrane during photophosphorylation; therefore, an accurate model of subunit interactions involving subunit 111 will be valuable for elucidating the mechanism and regulation of energy coupling. Purification of the complex from a crude CFl-CFo preparation from spinach (Spinacia oleracea) thylakoids was accomplished by detergent treatment during anion-exchange chromatography. Subunit 111 in the complex was positively identified by amino acid analysis and N-terminal sequencing. l h e association of subunit 111 with CF, was verified by linear sucrose gradient centrifugation, immunoprecipitation, and incorporation of the complex into asolectin liposomes. After incorporation into liposomes, CF, was removed from the CFl-III complex by ethylenediaminetetracetate treatment. l h e subunit Ill-proteoliposomes were competent to rebind purified CFl. lhese results indicate that subunit 111 directly interacts with CFl in spinach thylakoids.

ATPase R. E. McCarty, R. E. McCarty Johns Hopkins University, Baltimore, MDSearch for more papers by this author R. E. McCarty, R. E. McCarty Johns Hopkins University, Baltimore, MDSearch for more papers by this author First published: 15 January 2002 https://doi.org/10.1002/047120918X.emb0117 Read the full textAbout ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Encyclopedia of Molecular BiologyBrowse other articles of this reference work:BROWSE BY TOPICBROWSE A-Z RelatedInformation

The ionic environment of chloroplasts is important for photosynthesis, nitrogen metabolism and sulfur metabolism, yet how this internal environment is regulated is still not known. Isolated chloroplast inner envelope membranes are competent for transport studies. These membranes can be manipulated to form vesicles of largely right side-out and inside-out orientation and both the intravesicular and extravesicular buffer contents can be controlled. The vesicles can be loaded with an ion-sensitive fluorophore to measure the initial rates of ion transport across the membranes using spectrofluorometric methods. We have made measurements on a proton-pumping ATPase which may be involved in forming a pH gradient to assist in the transport other ions across the chloroplast inner envelope. Several ions such a sulfate, glycolate, phosphate, iron and calcium have enhanced rates of transport when an inward-directed pH gradient is imposed. Calcium may actually move as a result of the potential gradient which is formed across the inner envelope. In addition both nitrite and bicarbonate movement is aided by the presence of a pH gradient, nitrite moving as nitrous acid and bicarbonate moving as carbon dioxide. Movement of the latter is greatly accelerated when carbonic anhydrase is present on both sides of the membrane. A summary of our results on the study of ion transport across the chloroplast inner envelope will be presented with respect to rates of transport, effectors, and inhibitors.

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 μmol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the γ and ε subunits of CF1 play key roles in both regulation of activity and proton translocation.

Electron transport and the electrochemical proton gradient across the thylakoid membrane are two fundamental parameters of photosynthesis. A combination of the electron acceptor, ferricyanide and the D pH indicator, 9-aminoacridine, was used to measure simultaneously electron transport rates and D pH solely by changes in the fluorescence of 9-aminoacridine. This method yields values for the rate of electron transport that are comparable with those obtained by established methods. Using this method a relationship between the rate of electron transport and D pH at various uncoupler concentrations or light intensities was obtained. In addition, the method was used to study the effect of reducing the disulfide bridge in the g -subunit of the chloroplast ATP synthase on the relation of electron transport to D pH. When the ATP synthase is reduced and alkylated, the threshold D pH at which the ATP synthase becomes leaky to protons is lower compared with the oxidized enzyme. Proton flow through the enzyme at a lower D pH may be a key step in initiation of ATP synthesis in the reduced enzyme and may be the way by which reduction of the disulfide bridge in the g -subunit enables high rates of ATP synthesis at low D pH values.

In contrast to the well-characterized spinach ( Spinacea oleracea) chloroplast ATP synthase (CF1–CFo), the properties of the chloroplast ATP synthase from pea (Pisum sativum ) have not been as intensively studied. Preliminary data suggested that the regulatory properties of the two enzymes differ. In the absence of activating treatments the ATPase activity of pea thylakoids in the dark was higher than that in spinach thylakoids. When assayed in the presence of sulfite, the MgATPase activity of pea thylakoids was inhibited to a maximum of 67% by tentoxin, indicating that the dark ATPase activity is in part catalyzed by CF1–CFo. The ATPase activity of purified pea CF1 was also higher than that of spinach CF1 in the absence of activating treatments. These differences could result from the different regulatory properties of the pea ∈ or γ subunit or both. The pea ∈ subunit was less effective in binding to or inhibiting the ATPase activity of pea o r spinach CF1 deficient in ∈ (CF1-∈). Spinach ∈ inhibited the ATPase activity of pea CF1-∈ at lower concentrations than pea ∈. The gene encoding the pea ∈ subunit was cloned and over-expressed. Recombinant pea ∈ did not restore low proton permeability to spinach thylakoid membranes reconstitituted with spinach CF1-∈, although pea ∈ was effective when tested with pea thylakoids reconstitituted with pea CF1-∈. These results confirm earlier suggestions that the C-terminal region of ∈ is important in ∈-CF1 and ∈-CFo interactions.