Accumulation of alkaloids in plant vacuoles does not involve an ion-trap mechanism

Alkaloid uptake into vacuoles isolated from a Fumaria capreolata L. cell suspension culture was investigated. The uptake is carrier-mediated as shown by its substrate saturation, its sensitivity to metabolic inhibitors and especially by its exclusive preference for the (S)-forms of reticuline and scoulerine while the (R)-enantiomers which do not occur in this plant species were strictly discriminated. The carrier has a high affinity for (S)-reticuline with a K _m=0.3 M. The rate of alkaloid uptake was 6 pmol·h^-1·l^-1 vacuole, and 0.03 mg alkaloid·mg^-1 vacuolar protein were taken up. Transport was stimulated five-to seven-fold by ATP and was inhibited by the ATPase inhibitors N,N-dicyclohexylcarbodiimide and 4-4-diisothiocyanatostilbene-2,2 disulfonic acid, as well as by the protonophore carbonyl cyanide m-chlorophenylhydrazone. A number of alkaloids did not compete with labelled (S)-reticuline for uptake into vacuoles. The uptake system is absolutely specific for alkaloids indigenous to the plant from which the vacuoles were isolated. Slight modifications of the topography of an alkaloid molecule even with full retention of its electrical charge results in its exclusion. Alkaloid efflux was also shown to be mediated by a highly specific energy-dependent carrier. These results contradict the previously proposed ion-trap mechanism for alkaloid accumulation in vacuoles. A highly specific carrier-mediated and energy-dependent proton antiport system for alkaloid uptake and release is postulated.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4-4-diisothiocyanatostilbene-2,2 disulfonic acid Dedicated to Professor Harry Beevers, Santa Cruz, on the occasion of his 60^th birthday     

Membrane-potential changes in vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

The membrane potential in vacuoles isolated from storage roots of red beet (Beta vulgaris L.) has been studied by following changes in the fluorescence of the dye 3,3-diethylthiodicarbocyanine iodide, and by determining the uptake of the lipophilic triphenylmethylphosphonium cation. The vacuoles have a membrane potential, internal negative, which is estimated to be around-60 mV. These potentials become less negative by nearly 10 mV on addition of ATP. This ATP-dependent depolarisation is inhibited by the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and by the ATPase inhibitors, N,N-dicyclohexylcarbodiimide and trimethyltin chloride, but it is largely insensitive to sodium orthovanadate. Fusicoccin had no significant effect on the isolated vacuoles, but its addition to excised tissue caused a hyperpolarisation of the cells measured using a microelectrode.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DiS-C₂-(5) 3,3-diethylthiodicarbocyanine iodide - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Studies on the molecular basis of H+ translocation by cytochromec oxidase

We report here studies which characterize further the interaction ofN,N-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H⁺ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - NCCD N-(2, 2, 6, 6-tetramethylpiperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide - Hepes 2-(N-2-hydroxyethylpiperazin-N-yl) ethane sulfonate - TMPD N,N,N,N-tetramethylphenylenediamine     

Sodium efflux from perfused giant algal cells

Internodal cells of the giant alga Chara corallina were perfused internally to replace the native cytoplasm, tonoplast and vacuole with artificial cytoplasm. Sodium efflux from perfused cells, measured by including ²²Na in the perfusion media, was increased by increasing the internal sodium concentration and by decreasing the external pH, and was inhibited by external application of the renal diuretic amiloride. The sodium efflux was markedly ATP-dependent, with a 50-fold decrease in efflux observed after perfusion with media lacking ATP. Efflux in the presence of ATP was reduced by 33% by inclusion of 10 M N,N-dicyclohexylcarbodiimide in the perfusion medium. The membrane potential of the perfused cells approximated that of intact cells from the same culture. It is suggested that sodium efflux in perfused Chara cells proceeds via a secondary antiporter with protons, regulated by ATP in a catalytic role and with the proton motive force acting as the energy source.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino)ethanesulphonic acid - Mops 3(N-morpholino)propanesulphonic acid - Taps tris(hydroxymethyl)methylaminopropanesulphonic acid     

A highly selective alkaloid uptake system in vacuoles of higher plants

Vacuoles were isolated from different plant cell cultures and the transport mechanism for alkaloid uptake at the tonoplast membrane, as well as the compartmentation of enzymes and products inside the cells were investigated. While serpentine, the major alkaloid of Catharanthus roseus cells, is definitely located inside the vacuole, two key enzymes of the indole-alkaloid pathway, strictosidine synthase and a specific glucosidase, are located in the cytosol. Transport of alkaloids across the tonoplast into the vacuolar space has been characterized as an active, engergy-requiring mechanism, which is sensitive to the temperature and pH of the surrounding medium, stimulated by K⁺ and Mg²⁺, and inhibited by N,N-dicyclohexylcarbodiimid and Cu²⁺. The alkaloids accumulate inside the vacuoles against a concentration gradient, and the uptake system is specific for alkaloids indigenous to the plant from which the vacuoles have been isolated.Abbreviation DCCD N,N-dicyclohexylcarbodiimid Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Cation-activated ATPase activity of plasmalemma-enriched membrane preparations from maize coleoptiles

The ATPase activity present in plasmalemma-enriched preparations from maize coleoptiles shows an optimum at pH 6, a strong dependence on Mg²⁺, and is stimulated by K⁺ and other monovalent cations, both organic and inorganic. The activation of ATPase by K⁺ obeys Michaelis Menten kinetics, saturation being reached at 50 mM K⁺ concentration. K⁺, Mg²⁺-stimulated ATPase activity is strongly inhibited by N,N-dicyclohexylcarbodiimide and by diethylstilbestrol and, to a lesser extent, by octylguanidine.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DTE dithioerythritol - Ellmans r 5-5 dithiobis (2 nitrobenzoic) acid - FC fusicoccin - NPA naphthylphthalamic acid - OG octylguanidine - PCMBS p-chloromercuribenzensulphonate     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole     

The vacuolar ATPase ofNeurospora crassa

The filamentous fungusNeurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8–10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V₁ and an integral membrane complex called V₀. Genes encoding three of the subunits have been isolated.vma-1 andvma-2 encode polypeptides homologous to the and subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis.vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.     

DCCD induced sodium uptake by Anacystis nidulans

The Na level inside cells of Anacystis nidulans is lower than in the external medium reflecting an effective Na extrusion. Na efflux is an active process and is driven by a Na⁺/H⁺-antiport system. The necessary H⁺-gradient is generated by a proton translocating ATPase in the plasmalemma. This ATPase (electrogenic proton pump) also produces the membrane potential (about -110 mV) responsible for K accumulation. N,N-dicyclohexylcarbodiimide (DCCD) inhibits the ATPase and the H⁺-gradient completely, but the membrane potential is only reduced (<-70 mV), since K efflux initiated by DCCD maintains the potential partly by diffusion potential.With DCCD, active Na efflux is inhibited thus revealing Na uptake and leading by equilibration to the membrane potential to a 5–20 fold accumulation. Na uptake depends on the DCCD concentration with an optimum at (1–2)×10^-4 M DCCD. Pretreatment with DCCD for a few minutes followed by replacement of the medium suffices to induce Na uptake.DCCD induced Na influx is about 5 times faster in light than in darkness, and the steady state is reached much earlier in light; a 5 fold increase by light was also found for Rb uptake with untreated cells. Valinomycin stimulates the influx of Rb to about the same rate in light and dark. Therefore light may unspecifically increase the permeability of the plasma-lemma probably via the ATP level. Similarly to DCCD also 3×10^-3 M N-ethylmaleimide induces Na uptake.Abbreviations Used DCCD N,N-dicyclohexylcarbodiimide - NEM N-ethylmaleimide - CCCP carbonylcyanide m-chlorophenylhydrazone - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

11 S seed storage proteins fromHelianthus species (Compositae): Biochemical,size and charge heterogeneity

The seeds of 19 sunflower species were compared on the basis of their protein contents and the relative proportions of their protein fractions. The globulin content varied from 50% to about 70% and the albumin content from 18% to 35% according to the species. The level of intermediateMr polypeptides showed a great variability (9.6 to 24.3%). Comparative studies onMr polymorphism were carried out by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of non reduced and/or reduced samples using both mono- and bidimensional procedures. Polypeptide constituents of helianthinin were compared including both number and molecular size (cultivatedH. annuus was used as a standard). Studies focused on differences observed between the major two (Mr 38 000), (Mr 32 000) and (Mr 25 500), (21 000) polypeptides families constituting the main A, B, and C subunits. and polypeptides analyses permit to discriminate easilyH. petiolaris from the other species. Charge polymorphism was studied using isoelectric focusing (IEF) and IEF-PAGE in mono and bidimensional procedures in the presence or absence of 2-mercaptoethanol (2-ME). Only a specific ₄ polypeptide enables an easy discrimination betweenH. petiolaris and all the other species. Detailed nomenclature of the , and , polypeptides constituting the different helianthinin globulin subunits is given via the results of pI andMr analyses. Monodimensional IEF patterns of the more basic albumins (pI > 8.0) appear to provide a more valuable approach to identifying specific protein markers.     

Vacuolar H+-translocating ATPases from plants: Structure,function, and isoforms

The vacuolar H⁺-translocating ATPase (V-type ATPase) plays a central role in the growth and development of plant cells. In a mature cell, the vacuole is the largest intracellular compartment, occupying about 90% of the cell volume. The proton electrochemical gradient (acid inside) formed by the vacuolar ATPase provides the primary driving force for the transport of numerous ions and metabolites against their electrochemical gradients. The uptake and release of solutes across the vacuolar membrane is fundamental to many cellular processes, such as osmoregulation, signal transduction, and metabolic regulation. Vacuolar ATPases may also reside on endomembranes, such as Golgi and coated vesicles, and thus may participate in intracellular membrane traffic, sorting, and secretion.Plant vacuolar ATPases are large complexes (400–650 kDa) composed of 7–10 different subunits. The peripheral sector of 5–6 subunits includes the nucleotide-binding catalytic and regulatory subunits of 70 and 60 kDa, respectively. Six copies of the 16-kDa proteolipid together with 1–3 other subunits make up the integral sector that forms the H⁺ conducting pathway. Isoforms of plant vacuolar ATPases are suggested by the variations in subunit composition observed among and within plant species, and by the presence of a small multigene family encoding the 16-kDa and 70-kDa subunits. Multiple genes may encode isoforms with specific properties required to serve the diverse functions of vacuoles and endomembrane compartments.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - CAM Crassulacean acid metabolism - Nbd-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Bz-ATP 3-O-(4-benzoyl)benzolyadenosine 5-triphosphate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NEM N-ethylmaleimide - IP₃ inositol-1,4,5-triphosphate - H⁺-PPase H⁺-translocating pyrophosphatase - V-type vacuolar-type - P-type phosphorylated intermediate- or plasma membrane-type - F-type F₁F_o-type - V-ATPase vacuolar-type H⁺-ATPase     

Nuclear skeleton structures in some normal and tumor cells

Nuclear protein fractions, described earlier, were identified as constituents of the nuclear sap (the globulin fraction), that of the nucleoli and ribonucleoprotein network (the acidic protein), and of the nuclear envelope (the residual protein). The latter two fractions compose the protein skeleton of the cell nucleus.An essential difference between electrophoretic profiles of nuclear skeleton structures in experimental tumors and those of normal tissues was revealed. Tumor preparations contained more high molecular weight polypeptides and, in earlier stages of growth, low molecular weight components as well. Fractionation of the nuclear matrix proteins showed that the bulk of them are soluble in diluted alkali. The alkali-insoluble fraction retains the shape of the nucleus and appears in the electron microscope as a spongy nuclear skeleton. A finely dispersed fraction sedimenting from the alkaline suspension is enriched with the pore complexes. The fractions obtained differ in protein composition and probably contain protein components which are similar in molecular weights but non-identical.Abbreviations KD kilodaltons - NM nuclear matrix - PAAG polyacrylamide gel - PC pore complex - RP residual protein     

Hydrolysis and synthesis of ATP by membrane-bound ATPase from a motile Streptococcus

ATPase was detected in the membranes of a motile Streptococcus. Maximal enzymic activity was observed at pH 8 and ATP/Mg²⁺ ratio of 2. Mn²⁺ and Ca²⁺ could replace Mg²⁺ to some extent. Besides ATP, GTP and ITP were substrates. The enzyme was inhibited by N,N-dicyclohexylcarbodiimide but not by sodium azide, uncouplers or bathophenanthroline.An electrochemical gradient of protons, which was artificially imposed across the membranes of Streptococcus cells by manipulation of either the K⁺ diffusion potential or the transmembrane pH gradient, led to ATP synthesis. ATP synthesis was abolished by proton conductors, an inhibitor of the ATPase or an increase in the extracellular K⁺ concentration. A comparison between the phosphate potential and the electrochemical proton gradient showed that the data found are in agreement with a stoichiometry of 2 protons translocated per molecule ATP synthesized.Abbreviations electrochemical gradient of protons - DMO 5,5-dimethyl-2,4-oxazolidinedione - CCCP carbonylcyanide m-chlorophenylhydrazone - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DNP 2,4-dimitrophenol     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Oxidative phosphorylation in membrane vesicles of a gram-positive methylotroph

Membrane preparations, capable of high rates of respiration-linked ATP synthesis, have been obtained from a gram-positive methylotrophic bacterium Bacillus sp. MGA3. NADH, succinate, reduced TMPD and methanol were shown to be suitable substrates for the oxidative phosphorylation. Esterification of orthophosphate was dependent on electron transfer, as evidenced by the requirement for both substrate and oxygen. Phosphorylation was also dependent on ADP and was destroyed by boiling the membrane preparation. The phosphorylation was markedly uncoupled by carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone (CCCP) and was inhibited by N,N-dicyclohexylcarbodiimide (DCCD). KCN caused strong inhibition of substrate oxidation as well as phosphorylation for all substrates tested. Rotenone, amytal and antimycin A caused inhibition when NADH or methanol were used as substrates. Antimycin A inhibited respiration and ATP synthesis with succinate as substrate and had no effect on ascorbate —N,N,N,N-tetramethyl-p-phenylenediimide (TMPD) oxidation by membrane preparations of Bacillus sp. MGA3. P/O ratios determined were 2.4 with NADH, 1.7 with succinate and 0.8 with reduced TMPD. The measured P/O ratio with methanol-oxidizing system was similar to that with NADH (about 2.4).Abbreviations CCCP Carbonyl cyanide p-(trichloromethoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - TMPD N,N,N,N-tetramethyl-p-phenylenediimide - Q ubiquinone Q     

The proton-translocating nicotinamide adenine dinucleotide transhydrogenase

H⁺-transhydrogenase couples the reversible transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. There are separate sites on the enzyme for the binding of NAD(H) and of NADP(H). There are some indications of the position of the binding sites in the primary sequence of the enzymes from mitochondria andEscherichia coli. Transfer of hydride ion equivalents only proceeds when a reduced and an oxidized nucleotide are simultaneously bound to the enzyme. When p=0 the rate of interconversion of the ternary complexes of enzyme and nucleotide substrates is probably limiting. An increase in p accelerates the rate of interconversion in the direction of NADH NADP⁺ until another kinetic component, possibly product release, becomes limiting. The available data are consistent with either direct or indirect mechanisms of energy coupling.Abbreviations DCCD N N¹-dicyclohexylcarbodiimide - FSBA 5¹-[p-(fluorosulfonyl)benzoyl] adenosine - FCCP carbonylcyanide-p-fluoromethoxyphenylhydrazone - H⁺-Thase H⁺-transhydrogenase - thio-NADP⁺ thionicotinamide adenine dinucleotide phosphate - AcPdAd⁺ 3-acetylpyridine adenine dinucleotide - p proton electrochemical gradient - membane potential - pH pH difference across the membrane     

Membrane-bound NADPH dehydrogenase- and ferredoxin: NADP oxidoreductase activity involved in electron transport during acetate oxidation to CO2 in Desulfobacter postgatei

Desulfobacter postgatei grows on acetate and sulfate as energy source. The oxidation of acetate to 2 CO₂ proceeds via the citric acid cycle involving membrane-bound succinate dehydrogenase and membrane-bound malate dehydrogenase. We report here that the organism contains membrane-bound NADPH dehydrogenase and ferredoxin: NADP oxidoreductase for the reoxidation of NADPH and reduced ferredoxin generated during isocitrate- and 2-oxoglutarate oxidation, respectively. The presence of proton translocating ATPase activity is also described.NADPH dehydrogenase and succinate dehydrogenase were found to be electrically connected within the membrane and electron transfer between these two enzymes was shown to be coupled with proton translocation. The membrane fraction catalyzed the oxidation of NADPH with fumarate and the reduction of NADP with succinate. NADPH oxidation with fumarate was stimulated by protonophores and inhibited by the proton translocating ATPase inhibitor dicyclohexylcarbodiimide (DCCD) and by heptylhydroxyquinoline-N-oxide (HQNO); inhibition by DCCD was relieved by protonophores. NADP reduction with succinate was dependent on ATP and inhibited by protonophores, DCCD, and HQNO. The membrane fraction also mediated the oxidation of NADPH with the water soluble menaquinone analogue dimethylnaphthoquinone (DMN) and the reduction of fumarate with DMNH₂. Only the former reaction was stimulated by protonophores and only the latter reaction was inhibited by HQNO. This suggests that the NADPH dehydrogenase reaction is the site of energy conservation and the succinate dehydrogenase is the site of HQNO inhibition.Non-standard abbreviations APS Adenosine 5-phosphosulfate - DCCD N,N-dicyclohexylcarbodiimide - DCPIP 2,6-dichloroindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - HQNO 2(n-heptyl)-4-hydroxyquinoline-N-oxide - TCS 3,5,3,4-tetrachlorosalicylanilide - Tricine N-tris-(hydroxymethyl)methylglycine - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - SF-6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile     

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K⁺ up to 3.6 M. This creates a very large K⁺ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K⁺ but the residual K⁺ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K⁺ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K _m of between 1 to 2 mM. The driving force for K⁺ transport has been explored. Internal K⁺ concentration is not in equilibrium with m suggesting that K⁺ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K⁺ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K⁺ accumulation has not yet been clearly demonstrated. K⁺ transport in H. volcanii can be compared with K⁺ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Freezing of isolated thylakoid membranes in complex media

Chloroplast thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw treatment in a buffered medium containing 70 mM KCl, 30 mM NaNO₃ and 20 mM K₂SO₄ in different combinations. In the presence of the three predominant inorganic electrolytes, inactivation of photophosphorylation was mainly caused by a decrease in the capacity of the photosynthetic electron transport; release of proteins from the membranes was not manifest and light-induced H⁺ gradient and proton permeability were largely unaffected. Omission of nitrate from the medium had little effect. When either sulfate or chloride or both were omitted prior to freezing, inactivation of photophosphorylation was correlated with stimulation of the phosphorylating electron flow, marked increase in H⁺ permeability and loss of the ability of the thylakoids to accumulate protons in the light. In the absence of sulfate, uncoupling was mainly a consequence of the dissociation of chloroplast coupling factor (CF₁). Partial restoration of proton impermeability and pH gradient occurred upon the addition of N,N-dicyclohexylcarbodiimide (DCCD). When sulfate was present but chloride omitted, CF₁ remained attached to the membranes and the addition of DCCD had no effect, indicating that the increase in proton efflux was caused by a different mechanism. It is concluded that sulfate stabilizes the CF₁ and prevents its release from the membranes, but KCl is also necessary for maintaining the low permeability of the membranes to protons. The importance of complex media for investigations on isolated biomembrane systems is stressed.Abbreviations CF₁ chloroplast coupling factor - DCCD N,N-dicyclohexylcarbodiimide - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid I=Santarius 1986 b