Dicyclohexylcarbodiimide Treatment Research Articles

Dicyclohexylcarbodiimide and disodium succinate regulate the pulp softening and breakdown in fresh longan by modulating the metabolism of cell wall polysaccharides

Cell wall polysaccharides (CWP) are an important component of cell structure, and its content variation is a primary factor affecting the texture of fresh longan. The influences of dicyclohexylcarbodiimide (DCC) and disodium succinate (DS) treatments on the pulp softening and pulp breakdown, and their relation to CWP metabolism in pulp of longan fruit were explored. Compared with the control longan, DCC-treated fruit showed lower value of pulp firmness, higher index of pulp breakdown, higher expression levels of cell wall disassembly enzymes (CWDEs)-related genes (DlPME1, DlPME3, DlPG1, Dlβ-Gal1, Dlβ-Gal16, DlCx7, DlXET3, DlXET27), and higher activities of CWDEs including pectin methyl esterase (PME), polygalacturonase (PG), β-galactosidase (β-Gal), cellulase (Cx), xyloglucan endotransglycosylase (XET), lower values of cell wall materials, and lower levels of CWP including ionic-soluble pectin, covalent-soluble pectin, cellulose, and hemicellulose. Whereas, the opposite effects were exhibited in DS-treated fruit. The above data indicate that DCC-hastened longan pulp softening and pulp breakdown was due to the up-regulating expressions of CWDEs-related genes, the activating activities of CWDEs, and the accelerating CWP disassembly by DCC treatment. Whereas, DS repressed longan pulp softening and pulp breakdown through down-regulating the expressions of CWDEs-related genes, inhibiting CWDEs activities, and reducing CWP degradation.

Adaptive responses of Bacillus cereus ATCC14579 cells upon exposure to acid conditions involve ATPase activity to maintain their internal pH.

This study examined the involvement of ATPase activity in the acid tolerance response (ATR) of Bacillus cereus ATCC14579 strain. In the current work, B. cereus cells were grown in anaerobic chemostat culture at external pH (pHe) 7.0 or 5.5 and at a growth rate of 0.2 h−1. Population reduction and internal pH (pHi) after acid shock at pH 4.0 was examined either with or without ATPase inhibitor N,N’-dicyclohexylcarbodiimide (DCCD) and ionophores valinomycin and nigericin. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted cells) compared with cells grown at pH 7.0 (unadapted cells), indicating that B. cereus cells grown at low pHe were able to induce a significant ATR and Exercise-induced increase in ATPase activity. However, DCCD and ionophores had a negative effect on the ability of B. cereus cells to survive and maintain their pHi during acid shock. When acid shock was achieved after DCCD treatment, pHi was markedly dropped in unadapted and acid-adapted cells. The ATPase activity was also significantly inhibited by DCCD and ionophores in acid-adapted cells. Furthermore, transcriptional analysis revealed that atpB (ATP beta chain) transcripts was increased in acid-adapted cells compared to unadapted cells before and after acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. These adaptations depend on the ATPase activity induction and pHi homeostasis. Our data demonstrate that the ATPase enzyme can be implicated in the cytoplasmic pH regulation and in acid tolerance of B. cereus acid-adapted cells.

Enhancement of ginsenoside biosynthesis in cell cultures of Panax ginseng by N,N′-dicyclohexylcarbodiimide elicitation

In this work, the effect of N,N′-dicyclohexylcarbodiimide (DCCD) on ginsenoside biosynthesis in suspension cultures of Panax ginseng cells was investigated. The optimal concentration and timing of DCCD addition were found to be 10μM and on day 4 of cultivation. Under this condition, the maximal content of total ginsenosides increased to 3.0-fold that of untreated control, and the contents of Rg-group (Rg1 and Re) ginsenosides and Rb1 were 2.5- and 8.9-fold higher, respectively, which coincided with elevated activities of protopanaxatriol biosynthetic enzyme protopanaxadiol 6-hydroxylase and UDPG-ginsenoside Rd glucosyltransferase that converts Rd to Rb1. In addition, DCCD treatment induced the activity of defense response enzyme, phenylalanine ammonia lyase. To gain a better understanding of the molecular processes underlying the elicitation, we examined nitric oxide (NO) content and expression levels of the triterpene biosynthetic genes encoding squalene synthase (sqs), squalene epoxidase (se), and dammarenediol-II synthase (ds). It was found that DCCD up-regulated NO generation and transcription levels of sqs, se and ds. Interestingly, these effects of DCCD were compromised by an NO biosynthetic inhibitor, while an NO donor alone recapitulated the elicitation effect of DCCD on ginsenoside biosynthesis. These results suggest that DCCD may induce the ginsenoside biosynthesis via NO signaling in the P. ginseng cells. The information obtained might also be helpful to hyperproduction of valuable secondary metabolites in other plant cell cultures.

Rotation of the Proteolipid Ring in the V-ATPase

V0V1-ATPase is a proton-translocating ATPase responsible for acidification of eukaryotic intracellular compartments and for ATP synthesis in archaea and some eubacteria. We demonstrated recently the rotation of the central stalk subunits in V1, a catalytic sector of V0V1-ATPase (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315), but the rotation of the proteolipid ring, a predicted counterpart rotor in the membrane V0 sector, has remained to be proven. V0V1-ATPase that retained sensitivity to N',N'-dicyclohexylcarbodiimide was isolated from Thermus thermophilus, immobilized onto a glass surface through the N termini of the A subunits of V1, and decorated with a bead attached to a proteolipid subunit of V0. Rotation of beads was observed in the presence of ATP, and direction of rotation was always counterclockwise viewed from the membrane side. The rotation proceeded at approximately 3.0 rev/s in average at 4 mm ATP and was abolished by N',N'-dicyclohexylcarbodiimide treatment. Thus, the rotation of the central stalk in V1 accompanies rotation of a proteolipid ring of V0 in the functioning V0V1-ATPase.

DCCD inhibits the reactions of the iron-sulfur protein in Rhodobacter sphaeroides chromatophores.

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. To understand the possible role of DCCD in inhibiting the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores. DCCD has two distinct effects on phase III of the electrochromic bandshift of carotenoids reflecting the electrogenic reactions of the bc(1) complex. At low concentrations, DCCD increases the magnitude of the electrogenic process because of a decrease in the permeability of the membrane, probably through inhibition of F(o)F(1). At higher concentrations (>150 microM), DCCD slows the development of phase III of the electrochromic shift from about 3 ms in control preparations to about 23 ms at 1.2 mM DCCD, without significantly changing the amplitude. DCCD treatment of chromatophores also slows down the kinetics of flash-induced reduction of both cytochromes b and c, from 1.5-2 ms in control preparations to 8-10 ms at 0.8 mM DCCD. Parallel slowing of the reduction of both cytochromes indicates that DCCD treatment modifies the reaction of QH(2) oxidation at the Q(o) site. Despite the similarity in the kinetics of both cytochromes, the onset of cytochrome c re-reduction is delayed 1-2 ms in comparison to cytochrome b reduction, indicating that DCCD inhibits the delivery of electrons from quinol to heme c(1). We conclude that DCCD treatment of chromatophores leads to modification of the rate of Q(o)H(2) oxidation by the iron-sulfur protein (ISP) as well as the donation of electrons from ISP to c(1), and we discuss the results in the context of the movement of ISP between the Q(o) site and cytochrome c(1).

Analysis of the role of the Listeria monocytogenes F 0F 1-ATPase operon in the acid tolerance response

As little is known about the genes involved in the induction of an acid tolerance response in Listeria monocytogenes, the role of the F 0F 1-ATPase was analyzed as a consequence of its role in the acid tolerance of a number of other bacteria and its conserved nature. It was found that acid adapted cells treated with N,N′-dicyclohexylcarbodiimide (DCCD) exhibited greatly enhanced sensitivity to low pH stress. Degenerate primers were designed to amplify and sequence a portion of the atpD gene. Subsequently, a PCR product from atpA to atpD was identified. While we were unable to create a deletion in the atpA gene, the plasmid pORI19 was inserted in a region between atpA and atpG to reduce, rather than eliminate, expression of the downstream genes. As expected this mutant displayed enhanced resistance to neomycin and exhibited slower growth than the wild type strain. This mutant could still induce an acid tolerance response and remained susceptible to DCCD treatment, but its relative acid sensitivity was difficult to assess as a consequence of its slow growth.

Calcium binding to the chloroplast andE. coli (CF0) F0 subunit III (c) of the ATP-synthase

Subunit III and c, the 8 kDa components of the chloroplast CF0, andE. coli H+ channel complexes respectively, were isolated and purified for the purpose of studying their Ca++-binding properties. Purified subunit III or c as well as the unfractionated organic-solvent soluble preparation from chloroplasts were used in a45Ca++-ligand blot assay known to detect high affinity Ca++-binding sites in proteins. Both subunit III and c showed strong45Ca++-binding. None of the other CF0 subunits bound Ca++ and of the CF1 only a weak binding was detected in the region of the α,β subunits. The Ca++-binding was inhibited after treating the proteins in solution by derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La3+. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivatizes the hydrophobically located Glu 61 (Asp 61 inE. coli), did not inhibit the Ca++-binding in either protein. The data indicate that for both proteins the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III (or c) C-terminal provide some of seven essential oxygen ligands normally required for defining a Ca++-binding site in proteins. Based on the accepted models for the hairpin conformation of the subunit III (c), it seems clear that the Ca++-binding site can form on the lumenal side of the membrane in the functional CF0 structures or on the periplasmic side of theE. coli membrane. A working hypothesis we are testing is that Ca++-binding to the CF0 (or F0) can form an easily reversible gating site such as to enhance the probability for membrane-localized H+ gradients being coupled to ATP formation under moderate energization loads, but under excess energization the local H+ ion concentration may build up high enough to displace the bound Ca++, resulting in delocalization of the H+ gradient. The latter situation seems, in chloroplasts at least, to function as a signal for over-energization; i.e., excess light absorption, a potential stress situation for plants. Lumenal acidification appears to be a trigger for initiating stress alleviation responses.

N,N'-dicyclohexylcarbodiimide cross-linking suggests a central core of helices II in oligomers of URF13, the pore-forming T-toxin receptor of cms-T maize mitochondria.

URF13 is a mitochondrially encoded, integral membrane protein found only in maize carrying the cms-T cytoplasm. URF13 is associated with cytoplasmic male sterility, Texas type, and causes susceptibility to the fungal pathogens Bipolaris maydis race T and Phyllosticta maydis. URF13 is predicted to contain three transmembrane alpha-helices and is a receptor for the pathotoxins (T-toxins) produced by B. maydis race T and P. maydis. Binding of T-toxin to URF13 leads to membrane permeability. Cross-linking of URF13 oligomers with N,N'-dicyclohexylcarbodiimide (DCCD) protects Escherichia coli cells expressing URF13 and cms-T mitochondria from the permeability caused by T-toxin or methomyl. Using mutated forms of URF13 expressed in E. coli cells, we determined the molecular mechanism of DCCD protection. We separately changed Lys-37 in helix II to isoleucine (K37I-URF13) and Lys-32 in the helix I/helix II loop region to alanine (K32A-URF13). DCCD treatment of K37I-URF13-expressing cells did not protect the cells from permeability caused by T-toxin or methomyl. DCCD cross-linking was greatly reduced in K37I-URF13 and in D39V-URF13-expressing cells, but it was unaffected in K32A-URF13-expressing cells. Binding of methomyl or T-toxin decreases DCCD cross-linking of URF13 oligomers expressed in either E. coli or cms-T mitochondria. We conclude that Asp-39 in helix II is cross-linked by DCCD to Lys-37 in helix II of an adjacent URF13 molecule and that this cross-linking protects against toxin-mediated permeabilization. Our results also indicate that helices II form a central core in URF13 oligomers.

Subunit III of the chloroplast ATP-synthase can form a Ca 2+-binding site on the lumenal side of the thylakoid membrane

Subunit III, the 8 kDa component of the chloroplast CF 0 H + channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca 2+-binding properties. After n-butanol extraction and ether precipitation, HPLC purification was accomplished using a poly(styrenedivinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic proteins of near 4 kDa molecular mass, psaI and psbK gene products associated with PSI and PSII reaction centers, respectively. Purified subunit III as well as the unfractionated organic-solvent soluble preparation were used in a 45Ca 2+-ligand blot assay known to detect high affinity Ca 2+-binding sites in proteins. Polypeptides were separated with SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with 45CaCl 2 in the presence of 10-fold excess of MgCl 2 and 200-fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca 2+ binding was inhibited after derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La 3+. Ca 2+ binding was maximum at pH 7.5–8.5 and was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivativizes the hydrophobically located Glu-61, decreased the electrophoretical mobility of isolated subunit III but did not inhibit the Ca 2+ binding. The data indicate that the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III C-terminal Val-81 provide some of seven essential oxygen ligands normally required for defining a Ca 2+-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca 2+-binding site. Based on the accepted models for the hairpin conformation of the subunit III, it does seem clear that the Ca 2+-binding site can form on the lumenal side of the membrane in the functional CF 0 structure.

Effects of dicyclohexylcarbodiimide (DCCD) treatment on coupled activities of vanadate‐sensitive ATPase from plasma membrane of maize roots

The presence of dicyclohexylcarbodiimide (DCCD) inhibited the activities of vanadate‐sensitive H+ ‐ATPase in both native and reconstituted plasma membrane of maize (Zea mays L. cv. WF9 × Mo 17) roots. Concentration dependence of DCCD inhibition on adenosine triphosphate (ATP) hydrolysis of native plasma membrane vesicles suggested that the molar ratio of effective DCCD binding to ATPase was close to 1. The DCCD inhibition of ATP hydrolysis could be slightly reduced by the addition of ATP, Mg:ATP, adenosine monophosphate (AMP), Mg:AMP and adenosine diphosphate (ADP). More hydrophilic derivatives of DCCD such as l‐ethyl‐N?‐3‐trimethyl ammonium carbodiimide (EDAC) or 1‐ethyl‐3‐3‐dimethyl‐aminopropyl carbodiimide (EDC) gave no inhibition, indicating that the effective DCCD binding site was located in a hydrophobic region of the protein. The proton transport activity of reconstituted plasma membrane at a temperature below 20°C or above 25°C was much sensitive to DCCD treatment. Build‐up of the proton gradient was analyzed according to a kinetic model, which showed that proton leakage across de‐energized reconstituted plasma membranes was not affected by DCCD, but was sensitive to the method employed to quench ATP hydrolysis. Reconstituted plasma membrane vesicles treated with DCCD exhibited a differential inhibition of the coupled H+‐transport and ATP hydrolysis. The presence of 50 μM DCCD nearly abolished transport but inhibited less than 50% of ATP hydrolysis. The above results suggest that the link between proton transport and vanadate‐sensitive ATP hydrolysis is indirect in nature.

Role of energy metabolism in conversion of nonmucoid Pseudomonas aeruginosa to the mucoid phenotype.

Phosphatidylcholine, the major component of lung surfactant, when supplied as the sole source of phosphate for Pseudomonas aeruginosa PAO1, resulted in conversion of as much as 2% of the population to the mucoid phenotype under continuous culture conditions over a 24-day culture period. In addition, growth in phosphatidylcholine resulted in the highest yields of extracellular alginate compared with other environmental conditions. Iron limitation, another environmental condition relevant to the lungs of patients with cystic fibrosis, also resulted in conversion to mucoid. Since both conditions suggested the likelihood of an energy-deprived growth environment as a common variable, the effect of direct inhibition of energy generation by N,N'-dicyclohexylcarbodiimide or gramicidin on the conversion of nonmucoid P. aeruginosa to the mucoid phenotype was examined. Both inhibitors resulted in mucoid subpopulations (0.5 and 0.8%, respectively). Severe energy stress imposed by the combination of phosphate limitation and N,N'-dicyclohexylcarbodiimide treatment resulted in conversion of 55% of the population to mucoidy during a 7-day growth period. A growth advantage of the mucoid over the nonmucoid phenotype was observed under severe nutrient deprivation by growth on unsupplemented Noble agar or in a 1/2,500 dilution of a chemically defined medium. These results clearly demonstrate a significant role for the energy state of the cell in conversion to mucoid and in selection for the mucoid phenotype.

Δψ and ΔpH generation by the H + pumps of the respiratory chain and ATPase in subcellular vesicles from marine bacterium Vibrio alginolyticus

Succinate and glycerol 3-phosphate oxidation and coupled Δψ generation by the respiratory chain of inside-out subcellular vesicles of Vibrio alginolyticus do not depend on Na + concentration and are not suppressed by submicromolar concentrations of 2-heptyl-4-hydroxyquinoline N-oxide, arresting Na +-motive NADH:quinone reductase. When the H +-permeability of membranes is diminished by adding Mg 2+ and by N, N′-dicyclohexylcarbodiimide treatment, Na +-independent NADH oxidation appears to be competent in the Δψ generation. NADH and succinate oxidations and ATP-hydrolysis result in ΔpH formation (inside acidic) in media with K + and valinomycin. Na +-dependent NADH oxidation also participates in Δ \\ ̄ gmH + generation because the inhibition of Na +-motive NADH:quinone reductase (a medium with 2-heptyl-4-hydroxyquinoline N-oxide and without added Na +) slows down ΔpH formation. It is assumed that the NADH-quinone and quinole-O 2 steps of the respiratory chain are Na +- and H +-motive, respectively. Since ATP hydrolysis by the V. alginolyticus vesicles supports uphill transport of Na + (Dibrov, P.A., Skulachev, V.P., Sokolov, M.V. and Verkhovskaya, M.L. (1988) FEBS Lett. 233, 355–358) as well as of H + (this paper), we may conclude that Na +-cycle and H +-cycle co-exist in the membrane of this bacterium.

Conserved Polar Loop Region of Escherichia coli Subunit c of the F1F0 H+-ATPase: Glutamine 42 is not Absolutely Essential, But Substitutions Alter Binding and Coupling of F1 to F0

Abstract The uncE114 mutation (Gln42----Glu) in subunit c of the Escherichia coli H+ ATP synthetase causes uncoupling of proton translocation from ATP hydrolysis (Mosher, M. E., White, L. K., Hermolin, J., and Fillingame, R. H. (1985) J. Biol. Chem. 260, 4807-4814). In the background of strain ER, the mutation led to dissociation of F1 from the membrane. Ten revertants to the uncE114 mutation were isolated, and the uncE gene was cloned and sequenced. Six of the revertants were intragenic and had substitutions of glycine, alanine, or valine for the mutant glutamate residue at position 42. The intragenic, revertant uncE genes were incorporated into an otherwise wild type chromosome of strain ER. Membrane vesicles prepared from each of the revertants showed a restoration of F1 binding to F0. The Val42 revertant differed from the other two revertants in that the ATPase activity of F1 was inhibited when membrane bound. This was shown by the stimulation of ATPase activity when F1 was released from the membrane. The Gly42 and Ala42 revertants demonstrated membrane ATPase activity that was resistant to dicyclohexylcarbodiimide treatment. Resistance was shown to be due to the increased dissociation of F1 from the membrane under ATPase assay conditions. The Ala42 revertant showed a significant reduction in ATP-dependent quenching of quinacrine fluorescence that was attributed to less efficient coupling of ATP hydrolysis to H+ translocation, whereas the other revertants showed responses very near to that of wild type. Minor changes in the F1-F0 interaction in all three revertants were indicated by an increase in H+ leakiness, as judged by reduced NADH-dependent quenching of quinacrine fluorescence. The minor defects in the revertants support the idea that residue 42 is involved in the binding and coupling of F1 to F0 but also show that the conserved glutamine (or asparagine) is not absolutely necessary in this function.

Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups.

This paper demonstrates and characterizes inactivation by N,N'-dicyclohexylcarbodiimide (DCCD) of Rb+ and Na+ occlusion in pig kidney (Na+,K+)-ATPase. Rb+ and Na+ occlusion dependent on oligomycin are measured with a manual assay. Parallel measurement of phosphorylation (by Pi plus ouabain) and Na+ or Rb+ occlusion lead to stoichiometries of 3 Na+ or 2 Rb+ per pump molecule. Inactivation of cation occlusion by DCCD shows the following features: (a) Rb+ and Na+ occlusion are inactivated with identical rates and (b) DCCD concentration dependence shows first-order kinetics and also proportionality to the ratio of DCCD to protein, (c) Rb+ and Na+ occlusion are equally protected from DCCD, by Rb+ ions with high affinity (or Na+ ions with lower affinity), (d) inactivation is only slightly pH-dependent between 6 and 8.5 but (e) is significantly accelerated by several hydrophobic amines while a water-soluble nucleophile, glycine ethyl ester has no effect, and (f) inactivation is exactly correlated with inactivation of (Na+,K+)-ATPase activity of ATP-dependent Na+/K+ exchange in reconstituted vesicles and with the magnitude of E1Na+----E2(Rb+) conformational transitions measured with fluorescence probes. The simplest hypothesis to explain the results is that DCCD modifies one (or a small number of) critical carboxyl residues in a non-aqueous cation binding domain and so blocks occlusion of 2 Rb+ or 3 Na+ ions. The results suggest further that Na+ and K+(Rb+) bind to the same sites and are transported sequentially on the same trans-membrane segments. A second effect of the DCCD treatment is a 4-8-fold shift of the conformational equilibrium E2(Rb+)----E1Rb+ toward E1Rb+. This is detected by (a) reduction in apparent Rb+ affinity for Rb+ occlusion or Rb+/Rb+ exchange in vesicles and (b) direct demonstration of an increased rate of E2(K+)----E1Na+ and decreased rate of E1Na+----E2(K+). This effect is not protected against by Rb+ ions and probably reflects modification of a second group of residues. Modification of (Na+,K+)-ATPase by carbodiimides is complex. Depending on the nature of the carbodiimide (water- or lipid-soluble), ratio of carbodiimide to protein, and perhaps source of the enzyme, inactivation might result either from modification of critical carboxyls, as suggested by this work, or from internal cross-linking as proposed by Pedemonte, C. H. and Kaplan, J. H. ((1986) J. Biol. Chem. 261, 3632-3639).

Inhibition of Na+-H+ exchange by N,N'-dicyclohexylcarbodiimide in isolated rat renal brush border membrane vesicles.

The inactivation of rat renal brush border membrane Na+-H+ exchange by the covalent carboxylate reagent N,N'-dicyclohexylcarbodiimide (DCCD) was studied by measuring 1 mM Na+ influx in the presence of a pH gradient (pHi = 5.5; pHo = 7.5) and H+ influx in the presence of a Na+ or Li+ gradient ([Na+]i = 150 mM; [Na+]o = 1.5 mM). In the presence of DCCD, the rate of Na+ uptake decreased exponentially with time and transport inhibition was irreversible. At all DCCD concentrations the loss of activity was described by a single exponential, consistent with one critical DCCD-reactive residue within the Na+-H+ exchanger. Among several carbodiimides the most hydrophobic carbodiimide, DCCD, was also the most effective inhibitor of Na+-H+ exchange. With 40 nmol of DCCD/mg of protein, at 20 degrees C for 30 min, 75% of the amiloride-sensitive 1 mM Na+ uptake was inhibited. Neither the equilibrium Na+ content nor the amiloride-insensitive Na+ uptake was significantly altered by the treatment. The Na+-dependent H+ flux, measured by the change in acridine orange absorbance, was also decreased 80% by the same DCCD treatment. If 150 mM NaCl, 150 mM LiCl, or 1 mM amiloride was present during incubation of the brush border membranes with 40 nmol of DCCD/mg of protein, then Li+-dependent H+ flux was protected 50, 100, or 100%, respectively, compared to membranes treated with DCCD in the absence of Na+-H+ exchanger substrates. The combination of DCCD and an exogenous nucleophile, e.g. ethylenediamine and glycine methyl ester, increased Na+-dependent H+ flux in the presence of 80 nmol of DCCD/mg of protein, compared to the transport after DCCD treatment alone. These findings suggest that the Na+-H+ exchanger contains a single carboxylate residue in a hydrophobic region of the protein, and the carboxylate and/or a nearby endogenous nucleophilic group is critical for exchange activity.

Binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate opens the pathway for protons through the chloroplast ATPase complex.

The effect of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate (TNP-ADP) on photophosphorylation and on the proton conductivity of the thylakoid membrane has been investigated. The results show that TNP-ADP is a potent competitive inhibitor of photophosphorylation (Ki = 1-2 microM). Moreover, in the absence of ADP and Pi, TNP-ADP accelerates basal electron transport of chloroplasts. Addition of ADP, which promotes release of the analogue from CF1, completely reverses this effect of TNP-ADP; likewise Pi alone reverses stimulation of electron transport by TNP-ADP. Dicyclohexylcarbodiimide treatment, which is known to close CF0 to H+, completely abolishes the effect of TNP-ADP. The measurements of the alkalization of the medium and the acidification of the thylakoid lumen following single turnover flashes showed that binding of TNP-ADP to CF1 increased membrane permeability for H+. Further results suggest that binding of TNP-ADP to the catalytic site of CF1 opens the CF0-CF1 complex for H+. Since ADP, as well as Pi alone, reverses the effect, it is concluded that TNP-ADP induces a conformation of the CF0-CF1 complex similar to the one triggered by simultaneous binding of ADP plus Pi. This may be achieved by interaction of the TNP residue with the Pi binding site. Thus it seems that the status of the catalytic site(s) in CF1 can be transmitted to the CF0 part to control proton flux through the ATPase complex in an economically reasonable way.

Energy transduction by the reconstituted b-c1 complex from yeast mitochondria. Inhibitory effects of dicyclohexylcarbodiimide.

A purified cytochrome b-c1 complex isolated from yeast mitochondria has been reconstituted into proteoliposomes. The reconstituted comp]lex catalyzed antimycin A-sensitive electron transfer from different analogues of coenzyme Q to cytochrome c. The reconstituted complex was also capable of energy conservation as indicated by uncoupler-stimulated rates of electron transfer, electrogenic proton ejection, and reversed electron flow from cytochrome b to coenzyme Q2 in the presence of antimycin A driven by a valinomycin-induced K+-diffusion potential (negative inside). Close to four protons were ejected per two electrons transported through the reconstituted b-c1 complex with ferricyanide as an artificial and impermeable electron acceptor.l The H+/2e- ratio decreased to two in the presence of the proton-conducting agent, carbonyl cyanide m-chlorophenylhydrazone. The same processes were studied in parallel in energy-conserving site 2 of rat liver mitochondria with similar results. In the reconstituted b-c1 complex, dicyclohexylcarbodiimide (DCCD) blocked the function of the electrogenic proton translocating device in the forward direction of proton ejection as well as in the backwards direction, measured as reversed electron flow from cytochrome b to coenzyme Q2 driven by a K+-diffusion potential. The primary effect of DCCD is localized on the proton ejection process, as the low proton conductance of the proteoliposome membrane was totally preserved after DCCD treatment.

Energy transduction in Escherichia coli. The role of the Mg2+ATPase.

Inverted membrane vesicles from strain 7, a wild type Escherichia coli K12 strain, actively transport calcium with energy supplied either by respiration or by ATP. These vesicles also have energy-linked quenching of quinacrine fluorescence. Membranes of strain 7, depleted of Mg2+ATPase by EDTA treatment, lack both activities. Membrane vesicles from strain NR70, a mutant lacking the Mg2+ATPase, show neither calcium transport nor energy-linked fluorescence quenching. Neither EDTA treatment nor genetic loss of the Mg2+atpase causes a reduction in respiration. Purified Mg2+ATPase from strain 7 can bind to EDTA-treated membrane vesicles from either strain 7 or NR70. This binding restored both calcium transport and fluorescence quenching, driven either by respiration or by ATP. Dicyclohexylcarbodiimide treatment mimics the effect of the Mg2+ATPase in the case of respiration-driven reactions. Treatment with EDTA, while not essential for the binding of the Mg2+ATPase to membrane vesicles of NR70, produced better restoration of both activities. The rate of restoration of fluorescence quenching showed a time lag which may indicate that binding of the Mg2+ATPase is a relatively slow process. Antiserum prepared against the Mg2+ATPase inhibited the quenching of quinacrine fluorescence when driven by ATP but not when driven by respiration. Addition of antiserum prior to addition of Mg2+ATPase prevented the restoration of fluorescence quenching, whether driven by respiration or ATP. These results clearly show that MG2+ATPase has an important role not only in catalyzing ATP synthesis and hydrolysis but also in maintaining the energized membrane state.