Measurement by a flow dialysis technique of the steady-state proton-motive force in chromatophores from Rhodospirillum rubrum. Comparison with phosphorylation potential.

1. In the light a transmembrane electrical potential of 100 mV has been estimated to occur in chromatophores from Rhodospirillum rubrum. The potential was determined by measuring the steady-state distribution of the permeant SCN- across the chromatophore membrane using a flow dialysis technique. The potential was not observed in the dark, nor in the presence of antimycin. It was dissipated on the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The potential was reduced by between 15 and 20 mV when ADP and Pi were added. Hydrolysis of ATP by the chromatophores generated a membrane potential of about 80 mV. 2. Using a flow dialysis technique light-dependent uptake of methylamine was observed only in the presence of concentrations of SCN- that were 500-fold higher than were used to measure the membrane potential. It is concluded that the pH gradient across the illuminated chromatophore membrane is insignificant except in the presence of relatively high concentrations of a permeant anion like thiocyanate. Further evidence that a negligible pH gradient was generated by the chromatophores is that addition of K+ and nigericin to illuminated chromatophores did not stimulate uptake of SCN-. 3. In the light of chromatophores established and maintained a phosphorylation potential of up to 14 kcal/mol. If a phosphorylation potential of this magnitude is to be poised against a proton-motive force that comprises solely a membrane potential of approx. 100 mV, then at least five protons must be translocated for each ATP synthesised via a chemiosmotic mechanism.     

Effect of oligomycin on NADH oxidation and its coupled phosphorylation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum

Summary NADH oxidation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum is significantly stimulated by the addition of phosphate (Pi) and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. K _m values for Pi in NADH oxidation and phosphorylation are 10^–3 m and 8×10^–4 m, respectively. These K _m values are almost the same as in corresponding photophosphorylation and oxidative phosphorylation catalyzed with chromatophores. As in the case of NADH oxidation with chromatophores, NADH oxidation with the particulate fraction has an optimal pH at 7.5 without additions, which is shifted to 6.9 by the addition of Pi and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. The optimal pH for coupled phosphorylation is 6.9. 10 g per ml of oligomycin can suppress stimulation of NADH oxidation by Pi, or by the energy trapping system, and prevent the shift of optimal pH. The particulate fraction can catalyze Pi-incorporation into glucose-6-phosphate without externally added ATP, so that Pi-incorporation is inhibited by oligomycin. From these findings, it is concluded that NADH oxidation in the particulate fraction is tightly coupled to phosphorylation.     

Postillumination adenosine triphosphate synthesis in Rhodospirillum rubrum chromatophores. I. Conditions for maximal yields.

The very low level of postillumination ATP synthesis in chromatophores was markedly stimulated when permeant anions (thiocyanate or perchlorate) or permeant cations (potassium in the presence of valinomycin) were added to the light stage. Although these compounds stimulated also light-induced proton uptake in chromatophores the pH dependence of both photoreactions was different. Proton uptake peaked at pH 6.5 while the amount of postillumination ATP was maximal when the light stage was carried out around pH 7.7. The increased yield of ATP at the more alkaline pH could not be explained by a slower decay of the high energy state at this pH, since the decay rate was faster at pH 7.7 than at pH 6.5. The proton concentration gradient which is maintained across the chromatophore membrane in the light was also found to increase when the external pH was raised from 6.0 to 8.0. Only a minimal amount of postillumination ATP was formed when this gradient was below 2.1 pH units, but above this value the ATP yield rose steeply as a function of the increasing pH gradient. In light of these results it is suggested that in order to obtain a high yield of postillumination ATP synthesis in chromatophores two conditions are required: the particles have to be loaded with a sufficient number of protons and a light-induced pH gradient above a certain threshold value has to be maintained across their membrane. The low yield of postillumination ATP in chromatophores and the increase obtained by adding permeating ions, is thus explained by similar variations in the extent of the pH gradient, which exceeded the threshold value only in the presence of the permeating ions.     

Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient

A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane-bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed. In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium. In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5-7.5 mM free Mg2+ was very high and could not be measured. In the presence of 0.24-0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.     

Synthesis of pyrophosphate by chromatophores of Rhodospirillum rubrum in the light and by soluble yeast inorganic pyrophosphatase in water-organic solvent mixtures

Chromatophores of Rhodospirillum rubrum contain a membrane-bound pyrophosphatase that synthesizes pyrophosphate when an electrochemical H+ gradient is formed across the chromatophore membrane upon illumination. In this report it is shown that MgCl2 and Pi have different effects on the synthesis of pyrophosphate in the light depending on whether initial velocities or steady-state levels are examined. When the water activity of the medium is reduced by the addition of organic solvents, soluble yeast inorganic pyrophosphatase (no H+ gradient present) synthesizes pyrophosphate in amounts similar to those synthesized by the chromatophores in totally aqueous medium during illumination, (H+ gradient present). The pH, MgCl2 and Pi dependence for the synthesis of pyrophosphate by the chromatophores at steady-state is similar to that observed at equilibrium with the soluble enzyme in the presence of organic solvents. The possibility is raised that a decrease in water activity may play a role in the mechanism by which the energy derived from the electrochemical H+ gradient is used for the synthesis of pyrophosphate in chromatophores of R. rubrum.     

Inorganic phosphate in Ehrlich ascites tumor cells and its distribution across the cell membrane

A regulatory function of the cell membrane in controlling the cytoplasmic level of Pi has been proposed, and in Ehrlich ascites tumor cells an active influx of primary phosphate has been reported in the literature. In the present study, Ehrlich cells were incubated at 1.5--50 mM extracellular Pi at pH 7.4 (Pi mainly secondary phosphate) and at pH 6.0 (mainly primary phosphate), and the measured cell Pi was compared with the value expected from a passive distribution of Pi. At a low extracellular Pi concentration the cell Pi was 3--6 mumol/g or even more. It is suggested that a major part of this cell Pi can be accounted for by enzymic release of Pi during the sampling procedure. If this interpretation is correct, the present results show that both ionic species of Pi are in electrochemical equilibrium across the cell membrane at steady state. Moreover, in vivo the concentration of free Pi in the cytosol will presumably be maintained at a steady-state level of about 0.4 mM, one order of magnitude below the directly measured values. This implies that the ratio [ATP]/[ADP][Pi] which is important in the regulation of energy metabolism, is higher than reported in the literature.     

The distribution of inorganic phosphate and malate between intra- and extramitochondrial spaces. Relationship with the transmembrane pH difference

The steady-state distribution of inorganic phosphate and malate between the intra- and extramitochondrial spaces was measured in suspensions of nonrespiring and respiring rat liver mitochondria in which the transmembrane pH difference was incrementally varied. In respiration-inhibited mitochondria, the slope of log [Pi]in/[Pi]out (ordinate) versus delta pH approached 2 by either chemical or isotopic determination of [Pi], and the slope of log [malate]in/[malate]out versus delta pH was 2.0 with an extrapolated log [Pi]in/[Pi]out value of 0.3 at delta pH = 0. We conclude that the distribution of Pi and malate for nonrespiring mitochondria were quantitatively consistent with those predicted by exchange of Pi- for OH- (or cotransport with H+) and of malate 2- for Pi2-. In respiring mitochondria using glutamate + malate as substrate, there was very little pH dependence of Pi or malate accumulation (the slopes were less than 0.5) unless n-butylmalonate (inhibitor of Pi-dicarboxylate exchange) was added before the glutamate and malate, in which case the distribution patterns at delta pH less than 0.4 were similar to those in nonrespiring mitochondria. In either case, however, after reaching a maximal value of 1.1, log [Pi]in/[Pi]out did not further increase with increasing delta pH. Thus, in normally metabolizing mitochondria, the distributions of Pi and malate are not directly correlated with the difference in pH across the membrane.     

Effects of pH indicators on various activities of chromatophroes of Rhodospirillum rubrum.

1. The effects of pH indicators on activities for ATP hydrolysis in the dark and ATP-Pi exchange in the dark were examined with chromatophores from Rhodospirillum rubrum. Of thirty-one pH indicators tested, eleven (metanil yellow, 2, 4-dinitrophenol, ethyl orange, bromocresol green, resazurin, neutral red, bromthymol blue, alpha-naphtholphthalein, o-cresolphthalein, phenolphthalein, and alizarin yellow G) almost completely inhibited the activities for ATP formation and ATP-Pi exchange at concentrations of 1 mM, and were studied in detail. 2. Of the eleven pH indicators, those other than alpha-naptholphthalein, o-cresolphthalein and phenolphthalein, when assayed at appropriate concentrations, inhibited ATP-Pi exchange, but not ATP hydrolysis. In ATP-Pi exchange, these eight pH indicators at the concentrations described above were competitive against Pi, and non-competitive against ATP. The remaining three kinds of pH indicators were non-competitive against either Pi or ATP, when assayed at concentrations of the dyes that inhibited both activities. 3. The amounts of pH indicators bound with chromatophores were measured. No correlation was found between the amounts of the bound dyes and the extents of their inhibition of either ATP formation or ATP-Pi exchange. 4. Ethyl orange (pKa=4.1) and 2, 4-dinitrophenol (pKa=3.9) stimulated ATP hydrolysis to the greatest extent. The latter dye was hardly bound with chromatophores. 5. The stimulatory effects of pH indicators on ATP hydrolysis were hardly affected by extraction of quinones from chromatophores. 6. Most of the pH indicators stimulated both succinate-cytochrome c2 and NADH-cytochrome c2 reductions in the dark. 7. The mechanism of uncoupling of the electron transfer system and the phosphorylation system by pH indicators and the mechanism of the coupling are discussed.     

Role of phosphate on the ADP-induced hysteretic inhibition of mitochondrial adenosine 5'-triphosphatase. Effects of the natural protein inhibitor

Preincubation of F1-ATPase with ADP and Mg2+ leads to ADP binding at regulatory site inducing a hysteretic inhibition of ATP hydrolysis, i.e., an inhibition that slowly develops after Mg-ATP addition (Di Pietro, A., Penin, F., Godinot, C. and Gautheron, D.C. (1980) Biochemistry 19, 5671-5678). It is shown here that inorganic phosphate (Pi) together with ADP during preincubation abolishes the time-dependence of the inhibition after the addition of the substrate Mg-ATP. This preincubation in the presence of both Pi and ADP slowly leads to a conformation of the enzyme immediately inhibited after the addition of the substrate Mg-ATP. The Pi effect is half-maximal at 35 microM and pH 6.6, whereas a limited effect is induced at pH 8.0. The preincubation of F1-ATPase with Pi and ADP must last long enough (t1/2 = 5 min). The effects can be correlated to the amount of Pi bound to the enzyme, 1 mol Pi per mol (apparent KD of 33 microM) at saturation. Pi neither modifies the ADP binding nor the final level of the concomitant inhibition. When Pi is not present in the preincubation, the final stable rate of ADP-induced hysteretic inhibition is always reached when a near-constant amount of Pi has been generated during Mg-ATP hydrolysis. Kinetic experiments indicate that preincubation with ADP and Pi decreases both Vmax and Km which would favor a conformational change of the enzyme. Taking into account the Pi effects, a more precise model of hysteretic inhibition is proposed. The natural protein inhibitor IF1 efficiently prevents the binding of Pi produced by ATP hydrolysis indicating that the hysteretic inhibition and the IF1-dependent inhibition obey different mechanisms.     

Model for prebiotic pyrophosphate formation: Condensation of precipitated orthophosphate at low temperature in the absence of condensing or phosphorylating agents

Summary The formation of pyrophosphate (PPi) by condensation of orthophosphate (Pi) at low temperature (37°C) in the absence of condensing or phosphorylating agents could have been an ancient process in chemical evolution. In the present investigation the synthesis of³²PPi from³²Pi was carried out at pH 8.0 and PPi was found in larger amounts in the presence of insoluble Pi (with calcium or manganese ions) than in its absence (with magnesium ions, or with no divalent cations present). After 10 days of incubation in the presence of precipitated calcium phosphate, about 1.6 nmol/ml of PPi was formed (0.057% yield relative to insoluble Pi). The hypothesis that the reaction is dependent on precipitated Pi was reinforced by the effect of adding dimethyl sulfoxide (2.1–9.9 M) in the presence of magnesium ions: the amount of magnesium phosphate precipitated in the presence of the organic solvent was proportional to the amount of PPi formed. The large and negative activation entropies found in aqueous media with calcium ions and in a medium containing 11.3 M dimethyl sulfoxide with magnesium ions suggest that the reaction was favored by a hydrophobic phenomenon at the surface of solid Pi. This reaction could serve as a model for prebiotic formation of PPi.     

Modulation of proton pumping efficiency in bacterial ATP synthases

The ATP synthase in chromatophores of Rhodobacter caspulatus can effectively generate a transmembrane pH difference coupled to the hydrolysis of ATP. The rate of hydrolysis was rather insensitive to the depletion of ADP in the assay medium by an ATP regenerating system (phospho-enol-pyruvate (PEP) and pyruvate kinase (PK)). The steady state values of DeltapH were however drastically reduced as a consequence of ADP depletion. The clamped concentrations of ADP obtained using different PK activities in the assay medium could be calculated and an apparent Kd approximately 0.5 microM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate to the assay medium. The Kd for this effect was about 70 microM. Analogous experiments were performed in membrane fragment from Escherichia coli. In this case, however, the hydrolysis rate was strongly inhibited by Pi, added up to 3 mM. Inhibition by Pi was nearly completely suppressed following depletion of ADP. The Kd's for the ADP and Pi were in the micromolar range and submillimolar range, respectively, and were mutually dependent from the concentration of the other ligand. Contrary to hydrolysis, the pumping of protons was rather insensitive to changes in the concentrations of the two ligands. At intermediate concentrations, proton pumping was actually stimulated, while the hydrolysis was inhibited. It is concluded that, in these two bacterial organisms, ADP and phosphate induce a functional state of the ATP synthase competent for a tightly coupled proton pumping, while the depletion of either one of these two ligands favors an inefficient (slipping) functional state. The switch between these states can probably be related to a structural change in the C-terminal alpha-helical hairpin of the epsilon-subunit, from an extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to a retracted one, in which ATP hydrolysis and proton pumping are loosely coupled.     

Regulation of Spinach Leaf Sucrose Phosphate Synthase by Glucose-6-Phosphate, Inorganic Phosphate, and pH

Sucrose phosphate synthase was partially purified from spinach leaves and the effects and interactions among glucose-6-P, inorganic phosphate (Pi), and pH were investigated. Glucose-6-P activated sucrose phosphate synthase and the concentration required for 50% of maximal activation increased as the concentration of fructose-6-P was decreased. Inorganic phosphate inhibited sucrose phosphate synthase activity and antagonized the activation by glucose-6-P. Inorganic phosphate caused a progressive increase in the concentration of glucose-6-P required for 50% maximal activation from 0.85 mm (minus Pi) to 9.9 mm (20 mm Pi). In the absence of glucose-6-P, Pi caused partial inhibition of sucrose phosphate synthase activity (about 65%). The concentration of Pi required for 50% maximal inhibition decreased with a change in pH from 6.5 to 7.5. When the effect of pH on Pi ionization was taken into account, it was found that per cent inhibition increased hyperbolically with increasing dibasic phosphate concentration independent of the pH. Sucrose phosphate synthase had a relatively broad pH optimum centered at pH 7.5. Inhibition by Pi was absent at pH 5.5, but became more pronounced at alkaline pH, whereas activation by glucose-6-P was observed over the entire pH range tested. The results suggested that glucose-6-P and Pi bind to sites distinct from the catalytic site, e.g. allosteric sites, and that the interactions of these effectors with pH and concentrations of substrate may be involved in the regulation of sucrose synthesis in vivo.     

Study of pH- and temperature-induced transitions in F-protein (phosphofructokinase) by spectroscopy and microcalorimetry methods

The temperature- and pH-induced transitions in F-protein (phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11] have been studied by means of microcalorimetry and fluorescence and CD spectroscopy. An increase in pH from approx. 6.0 to approx. 8.0 causes a change in the protein state which seems to correspond to a shift of the dimer-tetramer equilibrium in favour of the tetramers. In the absence of phosphate, stability of the protein to temperature- and urea-induced denaturation at pH 6.0 is higher than that at pH 8.0. An addition of 150 mM phosphate results in a pronounced increase in the protein's stability in such a way that the protein becomes more stable at pH 8.0 than at pH 6.0. The shift of the denaturational heat capacity peak induced by the phosphate binding exceeds 25 degrees C at pH 8.0 and 9 degrees C at pH 6.0.     

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Role of phosphate in initial iron deposition in apoferritin

Ferritins from microorganisms to man are known to contain varying amounts of phosphate which has a pronounced effect on the structural and magnetic properties of their iron mineral cores. The present study was undertaken to gain insight into the role of phosphate in the early stages of iron accumulation by ferritin. The influence of phosphate on the initial deposition of iron in apoferritin (12 Fe/protein) was investigated by EPR, 57Fe M?ssbauer spectroscopy, and equilibrium dialysis. The results indicate that phosphate has a significant influence on iron deposition. The presence of 1 mM phosphate during reconstitution of ferritin from apoferritin, Fe(II), and O2 accelerates the rate of oxidation of the iron 2-fold at pH 7.5. In the presence or absence of phosphate, the rate of oxidation at 0 degrees C follows simple first-order kinetics with respect to Fe(II) with half-lives of 1.5 +/- 0.3 or 2.8 +/- 0.2 min, respectively, consistent with a single pathway for iron oxidation when low levels of iron are added to the apoprotein. This pathway may involve a protein ferroxidase site where phosphate may bind iron(II), shifting its redox potential to a more negative value and thus facilitating its oxidation. Following oxidation, an intermediate mononuclear Fe(III)-protein complex is formed which exhibits a transient EPR signal at g' = 4.3. Phosphate accelerates the rate of decay of the signal by a factor of 3-4, producing EPR-silent oligonuclear or polynuclear Fe(III) clusters. In 0.5 mM Pi, the signal decays according to a single phase first-order process with a half-life near 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monomeric alkaline phosphatase of Vibrio cholerae.

Alkaline phosphatase has been purified to homogeneity from two strains of Vibrio cholerae. The enzymes from both strains are single polypeptides of molecular weight 60,000. Both of the enzymes have pH optima around 8.0 and can act on a variety of organic phosphate esters, glucose-1-phosphate being the best substrate. The enzymes are unable to hydrolyze ATP and AMP. Although they have identical Km values, the two enzymes differ significantly in Vmax with p-nitrophenyl phosphate as substrate. The enzymes from the two strains also differ in their sensitivity to EDTA, Pi, and metal ions and activities of the apoenzymes. Ca2+ reactivated the apoenzymes most.     

The detection and characterization by electron-paramagnetic-resonance spectroscopy of iron–sulphur proteins and other electron-transport components in chromatophores from the purple bacterium Chromatium

Low-temperature e.p.r. (electron-paramagnetic-resonance) spectroscopy was used to detect electron-transport components in Chromatium chromatophores with e.p.r. signals in the g=2.00 region. High-potential iron protein (E(m8.0)=+325mV, where E(m8.0) is the midpoint potential at pH8) and a second component (g=1.90, E(m8.0)=+285mV) are oxidized in illuminated chromatophores. Two iron-sulphur proteins (g=1.94) with E(m8.0)=-290mV and E(m8.0)=-50mV are present. One (E(m8.0)=-50mV) is reduced on illumination. A component (g=1.82) with E(m8.0)=-135mV is photoreduced at 10 degrees K. The midpoint potential of this component is altered by o-phenanthroline and pH. The properties of this component suggest that it is the primary electron acceptor of a photochemical system. Another component (g=1.98) also has some of the properties of a primary electron acceptor, but its function cannot be completely defined. These results show that iron-sulphur proteins are present in the electron-transport system of Chromatium and indicate their role in electron transport.     

Deficiency of uncoupler-stimulated adenosine triphosphatase activity in yeast mitochondria

Oligomycin-sensitive ATPase activity was studied in isolated yeast mitochondria. The protonophore CCCP, at a concentration which completely inhibited ATP synthesis, induced only a low rate of hydrolysis of externally added ATP, and the extent of hydrolysis was dependent upon phosphate (Pi) concentration. CCCP promoted hydrolysis of intramitochondrial ATP. However, hydrolysis of externally added ATP was total in a medium containing potassium phosphate plus valinomycin. Without ionophores, ATPase activity was only observed at high external pH or with detergent-treated mitochondria. Under state 4 conditions, external ATP had access to the catalytic nucleotide site of ATPase as shown by 32Pi-ATP exchange experiments. These results are discussed in terms of a limitation of the translocase-mediated ATP/ADP exchange in uncoupled mitochondria.     

Physico-chemical characterization of proteins by capillary electrophoresis

The electrophoretic mobility of proteins was successfully determined by means of capillary electrophoresis (CE) with various background electrolytes (BGEs). The objective was focused on the variation in BGE physico-chemical composition and the consequential impact on the observed protein charge. Experimental and calculated mobilities, according to Henry's equation, versus ionic strength have been compared. For positively-charged lysozyme, a good agreement between observed and calculated mobilities was observed using triethanolamine chloride at pH 7.0 as the BGE. Mobility close to zero was shown using borate (pH 8.0) and phosphate (pH 7.0) at a low ionic strength of about 20 mmol l⁻¹, and as a consequence, specific adsorption of oxyanions was evidenced. Lysozyme retention in the case of reversed-phase high-performance liquid chromatography (RP-HPLC) was decreased by the presence of phosphate ions. CE and HPLC are complementary tools for characterizing the behaviour of lysozyme. On the other hand, the mobility of the negatively-charged α-lactalbumin remained constant as regards phosphate at pH 7.0 in the 20–200 mmol l⁻¹ range, contrary to the decrease that had been expected with the increasing ionic strength. β-Lactoglobulin exhibited increasingly lower mobilities than those expected of boric acid/borate at pH 7.0 and 8.0 (I=20 mmol l⁻¹).     

31P-NMR studies of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis

31P NMR was applied to an examination of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis. Resonances from sugar phosphates, inorganic phosphate, adenylates and arginine phosphate were identified. Two peaks of Pi were identified corresponding to intracellular and extracellular Pi. Anoxia produced an expected decrease in peak intensities of ATP and arginine phosphate while the peak of intracellular Pi was enhanced and shifted to indicate intracellular acidification during anoxia. Spectra of whole larvae were monitored over a temperature range from -30 degrees to +25 degrees C. No abrupt alterations in the spectra were seen at the point of extracellular freezing which occurs at about -8 degrees C but temperature had dramatic effects upon the peak intensities of ATP and arginine phosphate. A reversible increase/decrease in peak intensities, relative to Pi, was observed as temperature was raised/lowered. At 15 degrees and -20 degrees C, the beta peak of ATP was 64% and 2% of the peak intensity of Pi while that of arginine phosphate was 78% and 11%, respectively. This temperature effect was not an artifact of instrumentation (as model solutions containing Pi, ATP and arginine phosphate did not show this effect) or a result of changes in the total amounts of these compounds in the cell with temperature. Rather it is apparent that these molecules become restricted in their rotational movement as temperature is lowered perhaps via binding to subcellular components. Changes in the amounts of freely soluble ATP and arginine phosphate with temperature could have important implications for metabolism and its control. Analysis of the effect of temperature on the chemical shift of Pi was also used to determine pH in the intracellular and extracellular compartments. Temperature change had no effect on extracellular (hemolymph) pH which remained constant at 6.1-6.3. Intracellular pH varied with temperature, however, from pH 6.8 at 15 degrees C to pH 7.3 at -12 degrees C with a change, delta pH/delta 0, of -0.0185 degrees C consistent with alphastat regulation.