Delta Mu Research Articles

Microsatellite variation and the differentiation of modern humans.

This study presents an analysis of 20 tetranucleotide microsatellites in 16 worldwide human populations representing the major geographic groups. Global Fst values for the 20 microsatellites are indicators of their relative validity as tools in human population genetics. Four different measures of genetic distance (Fst, DSW, delta mu 2 and Rst) have been tested and compared with each other. Neighbor-joining trees have been constructed for all the measures of genetic distance and populations. Measures of genetic distance such as Fst, which does not consider different mutational relationships among alleles and has a known relationship to differentiation by drift, and to some extent DSW, reflect what is known of human evolution, while mutation-based distances such as Rst and delta mu 2 give very different results from those recognized from other sources (genetic or archaeological). When the genetic relationship between human populations is analyzed through allelic frequencies for microsatellites, the choice of distance may be a key issue in the picture obtained of genetic relationships between human populations. The results of the present study suggest that genetic drift played the main role in generating the present distributions of microsatellite alleles and their variation among human populations; the role of mutation must have been less important owing to the time constraint imposed by the small timescale in which most human differentiation has occurred. Moreover, the results support the theory of a recent origin of modern humans, although the existence of strong bottlenecks in the origin of the various human groups seems unlikely.

Cytosine accumulation as a measure of the proton electrochemical gradient acting on the overexpressed cytosine permease of Saccharomyces cerevisiae.

The magnitude of the proton gradient (delta mu H+) driving solute accumulation in Saccharomyces cerevisiae has long been in doubt, principally because of the lack of an agreed method for assaying its electrical component, the membrane potential (delta psi). In the present work, the size of the cytosine gradient (delta mu cyt) that the yeast generated was used as a measure of the driving gradient (delta mu H+). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 H+/cytosine system. delta mu cyt, assayed in washed cell suspensions fermenting glucose and containing 0.5 or 50 mM KCl, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a first estimate, -delta mu H+ was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered delta mu cyt to a value roughly equal to the magnitude of the pH gradient (delta pH). A mathematical model was used to correct the first estimates of delta mu H+ for the effect of cytosine leakage outside the symport. In such a system, delta mu cyt cannot exceed the equivalent ratio Vmax/KmL, where Vmax and Km are kinetic parameters of the symport and L is the rate coefficient for leakage. The feasibility of assaying delta mu H+ depends on it not being much larger than that ratio. The model was tested successfully against observations made with yeast preparations depleted of ATP. After correction, -delta mu H+ during fermentation was estimated to be up to 25 mV larger than delta mu cyt and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of delta pH, delta psi was in turn deduced and compared with the maximum methylamine gradient (delta mu M) the yeast formed. The results supported the claim in the literature that, at acid pH, delta mu M is a measure of delta psi.

Cytosine accumulation as a measure of the proton electrochemical gradient acting on the overexpressed cytosine permease of Saccharomyces cerevisiae.

The magnitude of the proton gradient (delta mu H+) driving solute accumulation in Saccharomyces cerevisiae has long been in doubt, principally because of the lack of an agreed method for assaying its electrical component, the membrane potential (delta psi). In the present work, the size of the cytosine gradient (delta mu cyt) that the yeast generated was used as a measure of the driving gradient (delta mu H+). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 H+/cytosine system. delta mu cyt, assayed in washed cell suspensions fermenting glucose and containing 0.5 or 50 mM KCl, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a first estimate, -delta mu H+ was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered delta mu cyt to a value roughly equal to the magnitude of the pH gradient (delta pH). A mathematical model was used to correct the first estimates of delta mu H+ for the effect of cytosine leakage outside the symport. In such a system, delta mu cyt cannot exceed the equivalent ratio Vmax/KmL, where Vmax and Km are kinetic parameters of the symport and L is the rate coefficient for leakage. The feasibility of assaying delta mu H+ depends on it not being much larger than that ratio. The model was tested successfully against observations made with yeast preparations depleted of ATP. After correction, -delta mu H+ during fermentation was estimated to be up to 25 mV larger than delta mu cyt and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of delta pH, delta psi was in turn deduced and compared with the maximum methylamine gradient (delta mu M) the yeast formed. The results supported the claim in the literature that, at acid pH, delta mu M is a measure of delta psi.

ΔμH+ dependency of in vitro protein translocation into Escherichia coli inner‐membrane vesicles varies with the signal‐sequence core‐region composition

Signal sequences frequently contain alpha-helix-destabilizing amino acids in the hydrophobic core. Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the alpha-helix. In the precursor of the Escherichia coli outer membrane protein PhoE (pre-PhoE), a glycine residue at position -10 (Gly -10) is thought to be responsible for the break in the alpha-helix. We investigated the role of this glycine residue in the translocation process by employing site-directed mutagenesis. SDS-PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence. In vivo, no drastic differences in the translocation kinetics were observed as compared with wild-type PhoE, except when a charged residue (Arg) was substituted for Gly -10. However, the in vitro translocation of all mutant proteins into inverted inner-membrane vesicles was affected. Two classes of precursors could be distinguished. Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly -10) was almost independent of the presence of a delta mu H+, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the delta mu H+. Apparently, the delta mu H+ dependency of in vitro protein translocation varies with the signal-sequence core-region composition. Furthermore, a proline residue at position -10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.

Energizing effects of illumination on the reactivities of lysine residues of the gamma subunit of chloroplast ATP synthase.

Incubation of chloroplast thylakoids with pyridoxal 5'-phosphate for a short time (5 s) modified the lysine residues of the gamma subunit of ATP synthase. Energization of thylakoids by illumination increased the reactivity of Lys24 by a factor of three and decreased the reactivity of Lys30 to 60%. The reactivities of these residues reached their maximum and minimum values, respectively, within 1 s after the onset of illumination. Illumination of thylakoids increased the reactivities of Lys222 and Lys231 in two steps by a factor of three. The first step was completed within 1 s and the second step was completed 20-30 s after the onset of illumination. In the presence of 10 mM NH4Cl, illumination of thylakoids did not change the reactivities of these lysine residues. These results suggest that the Lys24- and Lys30-containing region of the gamma subunit changes its conformation rapidly in response to delta mu H+ and that the Lys222- and Lys231-containing region of the gamma subunit changes its conformation in two steps in response to delta mu H+ formation.

Is the Mg(2+)-ATP-dependent proton pumping activity of the synaptic vesicles a factor involved in the cerebral hypoxia?

The changes in the Mg(2+)-dependent V-type ATPase activity and the Mg(2+)-ATP-dependent H+ pumping activity of the synaptic vesicles from the cerebral cortex of rats submitted to intermittent chronic (4 weeks) mild or severe hypoxia were evaluated. The adaptation to the chronic severe hypoxia increases both the ATPase and the H+ pumping activities which are inhibited by NEM with an exponential relationship between the IC(50) values and the in vivo O2 concentration. The Mg(2+)-dependent increase in H+ pumping activity of synaptic vesicles from the rats subjected to in vivo chronic hypoxia may be antagonized by nigericin (dissipating delta pH) and by FCCP (dissipating delta pH and delta psi SV). In contrast, valinomycin (dissipating the delta psi SV) and facilitating an enhancement in delta pH) increases in vitro the H+ pumping activity that is inhibited by the addition of high concentration of K gluconate (reducing the rate of K+ efflux). The preincubation of vesicles from hypoxic rats with FCCP, but not with nigericin, inhibits the valinomycin-increased H+ pumping activity. L-glutamate increases the H+ pumping activity in synaptic vesicles from the cerebral cortex of chronic hypoxic rats, whereas other amino acids (i.e., L-aspartate and L-homocysteate) and glutamate analogs (i.e., quisqualate and ibotenate) are ineffective. The adaptation to both chronic intermittent severe hypoxia and in vivo treatment with posatireline causes a decrease in the Mg(2+)-ATPase activity consistent with the decrease in the H+ pumping one of the synaptic vesicles. The addition of nigericin into incubation medium magnifies the decrease in the H+ pumping activity, while the addition of FCCP is ineffective, suggesting that the treatment with posatireline interferes with the delta psi SV component in the delta mu H+ of the synaptic vesicles from rats submitted to chronic hypoxia. The results of the in vivo and in vitro experiments suggest that in the synaptic vesicles from hypoxic rats the delta psi SV component in delta mu H+ may be most effective in increasing the Mg(2+)-ATP-dependent H+ pumping activity.

Contribution of Na+/Ca2+ exchanger in maintaining [Ca2+]c at a stable state in rat pancreatic islets.

The effects of lowering extracellular Na+ concentration [Na+]o, on cytosolic Ca2+ concentration, [Ca2+]c were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. The total replacement of extracellular Na+ (Na+o) by equimolar N-methyl-D-(--)-glucamine caused a rapid rise in [Ca2+]c, and partial replacement of Na+o resulted in correlative rises in [Ca2+]c in accordance with the magnitude of reduced [Na+]o. The rise in [Ca2+]c induced by Na+o removal was strongly inhibited in the Ca2+o-deficient environment or by Ni2+. The [Ca2+]c rise, however, remained almost unchanged in the presence of nifedipine or SK&F 96365, and was enhanced by the addition of ouabain. The electrochemical gradients for Ca2+ (delta mu Ca2+) and Na+ (delta mu Na+) were calculated to be 39.08 and 12.8 kJ/mol, respectively, in this study, indicating a stoichiometry of 3Na+: 1 Ca2+. These results indicate that, in rat pancreatic islets, the rise in [Ca2+]c induced by lowering [Na+]o is mainly due to Ca2+ entry medicated by the Na+/Ca2+ exchanger operating with the stoichiometry of 3Na+:1 Ca2+, and that the Na+/Ca2+ exchanger plays an important role in maintaining stable-state [Ca2+]c.

Involvement of stress protein PspA (phage shock protein A) of Escherichia coli in maintenance of the protonmotive force under stress conditions.

The expression of specific PhoE mutant proteins leads to induction of the expression of the psp operon of Escherichia coli and the export of various plasmid-encoded precursors is retarded in a pspA mutant strain. Here, we have investigated the specific role of various Psp proteins in the export process. PspB and PspC are both inner membrane proteins that are involved in the regulation of the transcription of the psp operon. Precursor PhoE translocation was retarded in a pspB mutant strain to a similar extent as in a pspA mutant strain. The reduced translocation efficiencies in the various psp mutants could be complemented by expression of PspA from a plasmid, indicating that only PspA is required for efficient translocation. Mutant prePhoE proteins that can be translocated independently of the deltamu H+ appeared to translocate equally efficiently in a wild-type and in a pspA mutant strain. Furthermore, quantitative in vivo determination of the deltamu H+ showed that it specifically decreased in a pspA mutant strain upon expression of plasmid-encoded (mutant) prePhoE protein. Apparently, the translocation defects observed in a psp mutant strain are caused by a decrease of the delta mu H+ and PspA functions by maintaining the delta mu H+ under these conditions.

Effects of growth environment on recombinant plasmid stability in Saccharomyces cerevisiae grown in continuous culture.

A recombinant strain of Saccharomyces cerevisiae, containing a 2-microns-fragment-based plasmid (pYE alpha a4) was grown under non-selective conditions in continuous culture. The decrease in the population carrying the plasmid-encoded auxotrophic marker, LEU2, was examined under different physiological conditions. The difference in growth rate (delta mu) between plasmid-free and plasmid-containing cells and the rate of plasmid segregation (R) were determined using a non-linear regression technique. Loss rates were greater in defined glucose-limited cultures than in complex glucose-limited cultures. Plasmid loss was delta mu-dominated in cultures grown on defined media whereas delta mu and R were co-dominant in cultures grown on complex medium. Loss rates increased with increasing dilution rate in complex glucose-limited cultures. The reverse was found in defined glucose-limited cultures. Plasmid retention and loss kinetic determined from defined magnesium-limited cultures were not significantly different from those observed in defined glucose-limited cultures. Although plasmid retention in defined phosphate-limited culture was not significantly different from that in defined glucose-limited culture, reduced R and increased delta mu indicated an alternative physiological effect of phosphate limitation on plasmid stability.

Induction of the Escherichia coli cytochrome d by low delta mu H+ and by sodium ions.

Regulation of synthesis of cytochrome d in Escherichia coli has been studied using mutants with cytochrome-d--beta-galactosidase gene fusions. It was shown that various protonophorous uncouplers, when added to the growth medium, cause induction of the cytochrome d synthesis. The cytochrome-d-inducing activity of uncouplers correlates with their ability to inhibit such a delta mu (H+)-driven function as motility of the E. coli cells. An increase in the Na+ concentration in the growth medium from 1.5 mM to 25 mM results in induction of the cytochrome d synthesis. The cytochrome-d-inducing effect of uncouplers is much more pronounced when the Na+ concentration is high than when it is low. These data are in agreement with the assumption that cytochrome d is involved in the Na+ energetics substituting for the H+ energetics when the latter appears to be inefficient. Mutations in arcA or arcB genes (but not in fnr gene) completely prevent the increase in the cytochrome d level induced by uncouplers but are without effect on that induced by Na+. It is assumed that in the control of the cytochrome d synthesis, the Arc system is involved in the delta mu H+ sensing whereas sensing of delta mu Na+ (or of the Na+ concentration) is mediated by some other receptor system.

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd2+ influx via the Mn2+ porter down membrane potential (delta psi) was fast due to involvement of energy generated by two proton pumps--the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd2+ influx is suggested to be due to a series of toxic events elicited by Cd2+ accumulation down delta psi generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient (delta mu H+) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of delta mu (H+)-dependent ATP synthesis, (iii) acceleration of the initial Cd2+ influx down delta psi generated by the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd2+ of delta mu (H+)-dependent ATP synthesis and of pH gradient (delta pH)-linked [14C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of delta mu H+ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd2+ accumulation was prevented due to activity of the plasmid-coded Cd2+ efflux system. Consequently, delta mu (H+)-producing and -requiring processes were not affected by Cd2+.

Green function analysis of energy spectra scaling properties

The relation between the singularities of multifractal energy spectral measures and the behaviours of the Green function is studied in the framework of a tight-binding Hamiltonian. If the measure mu (E) has a seating behaviour at energy E of the form Delta mu (E)= mu (E+ delta )- mu (E- delta ) varies as delta alpha (E), it is proved that the imaginary part of the Green function P(E, epsilon ) scales as P(E, epsilon ) varies as epsilon beta (E) with beta (E)= alpha (E)+1, the reverse being also true. This is exemplified in the case of the density of states and the local density of states of the one-dimensional Fibonacci quasicrystalline chain.

Finite-size effects on the approach of complete wetting

We perform an exact model calculation of the unbinding transition of a one-dimensional interface from a substrate on the approach of two-phase coexistence. This transition is modelled in a solid-on-solid (SOS) model with discrete columns which have a partition function calculated for a finite substrate area A. In the limits of large A and a small distance mod Delta mu mod to coexistence, the partition function becomes a function of the the scaling variable x= mod Delta mu mod 23/A. The interface-to-substrate distance l diverges as l alpha mod Delta mu mod - beta , with beta =-1/3 if the two limits are taken at constant or diverging x. If x vanishes, a finite-size regime appears in which beta =-1. A related interface model in continuous space was studied earlier by Kroll and Gompper (1989) and our scaling functions are identical to theirs.

Stark effect spectroscopy of tryptophan

The change in permanent dipole moment (magnitude of delta mu) for the transition from the 1La state to the ground state of tryptophan is the key photophysical parameter for the interpretation of tryptophan fluorescence spectra in terms of static and dynamic dielectric properties of the surrounding medium. We report measurement of this parameter by means of electric field effect (Stark) spectroscopy for N-acetyl-L-tryptophanamide (NATA) in two solvents, the single tryptophan containing peptide melittin, and 5-methoxytryptophan. The values ranged from 5.9 to 6.2 +/- 0.4 Debye/f for NATA and melittin, where f represents the local field correction. The 1Lb magnitude of delta mu was much smaller. Application of Stark spectroscopy to these chromophores required decomposition of the near-UV absorption into the 1La and 1Lb bands by measurement of the fluorescence excitation anisotropy spectrum and represents an extension of the method to systems where band overlap would normally preclude quantitative analysis of the Stark spectrum. The results obtained for 5-methoxytryptophan point out limitations of this method of spectral decomposition. The relevance of these results to the interpretation of steady-state and time-resolved spectroscopy of tryptophan is discussed.

Effects of Energization and Substrates on the Reactivities of Lysine Residues of the Chloroplast ATP Synthase beta Subunit

The incubation of chloroplast thylakoids with pyridoxal 5'-phosphate for a short time (5 s) modified the lysine residues of the ATP synthase beta subunit. Except for lysine residues in the N-terminal and C-terminal regions, the glycine-rich P-loop (GGAGVGKT)-, Lys154-, and Lys167-containing peptide (P-peptide) exhibited high reactivity with pyridoxal 5'-phosphate. The energization of thylakoids or addition of substrates (ADP, Pi, ATP) affected the modifications of the P-peptide, Lys447, and Lys399. For the P-peptide, substrates inhibited the modification with 0.5 mM ADP inhibiting by 80%. Energization enhanced the inhibitory effects of substrates. For Lys447, substrates also inhibited the modification; 0.5 mM ADP inhibited by 60%. For Lys399, the reactivity depended on the transmembrane delta mu H+. With increasing delta mu H+, the reactivity decreased. These results suggest the existence of energy-dependent conformational changes at the catalytic nucleotide-binding site and around Lys399. The former increases the affinity of the site for substrates. Substrate binding at the catalytic site changes the conformation around Lys447.

The Type IA supernova 1989B in NGC 3627 (M66)

We report extensive optical photometry and spectroscopy of the Type Ia supernova 1989B. Maximum light in B occurred approximately seven days after discovery on JD 2447565.3 +/- 1.0 (1989 February 7.8 +/- 1.0) at a magnitude of 12.34 +/- 0.05. The UBV light curves of this supernova were very similar to those of other well observed Type Ia events such as SN 1981B and SN 1980N. From a comparison of the UBVRIJHK photometry, we derive an extinction for SN 1989B of E(B-V) = 0.37 +/- 0.03 mags relative to the unobscured Type Ia SN 1980N. The properties of the dust responsible for the reddening of SN 1989B appear to have been similar to those of normal dust in the Milky Way. In particular, we find no evidence for an unusually low value of the ratio of the total to selective absorption. We derive a distance modulus of delta mu(sub 0) = -1.62 +/- 0.03 mag relative to the Type Ia SN 1980N. We present optical spectra which provide essentially continuous coverage of the spectral evolution of SN 1989B over the first month following B maximum. These data show the transition from the maximum-light spectrum, in which lines of elements such as Ca, Si, S, Mg, and O are most prominent, to the Fe-dominated spectrum observed a few weeks after maximum. This transition occurred quite smoothly over a two-week period following B maximum. Comparison of the spectra of SN 1989B with data for two other well observed Type Ia supernovae -- 1981B and 1986G -- reveals subtle differences in the relative strengths of the S II and Si II absorption lines at maximum light. However, these differences disappeared within a week or so after maximum with the onset of the Fe-dominated phase.

Bacterial multidrug resistance is due to a single membrane protein which functions as a drug pump.

Multidrug transport system in proteoliposomes was reconstituted using the highly purified membrane transport protein responsible for bacterial multidrug resistance. This protein (named Smr, for staphylococcal multidrug resistance) consists of 107 amino acid residues and displays four putative transmembrane domains. The Smr protein was tagged with a FLAG epitope, and the modified protein was expressed, purified, characterized, and reconstituted into proteoliposomes. With this in vitro experimental system, it has been demonstrated that a highly purified multidrug resistance protein functions as a drug pump, which transports methyltriphenylphosphonium actively against a 10(3)-fold concentration gradient. Delta mu H+ was shown to be a driving force, and an electrogenic drug/proton antiport was suggested as the molecular mechanism of the drug transport. Of the 2 Glu residues in putative extramembrane loops of the Smr polypeptide chain, Glu-24 was shown to be involved in determining the specificity of drug resistance. Replacement of both of these Glu residues with Asp produced active Smr. In contrast, Smr was unable to protect cells from multiple drugs when a Glu-13-->Asp-13 replacement was made. We suggest that Glu-13, a unique acidic residue located in the hydrophobic domain of Smr, is directly involved in the drug/proton antiport.

A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.

Deactivation of F0F1 ATPase in intact plant mitochondria

By using a method especially adapted to intact (pea leaf) mitochondria, we studied the regulation of the F0F1 ATPase by the electrochemical proton gradient (delta mu H+) and by the matricial pH. The kinetics of decay of the ATP hydrolase activity was studied immediately after the collapse of the electrochemical proton gradient by an uncoupler. At pH 7.5, three inhibitors of the ATPase (venturicidin, tri-n-butyl tin and aurovertin), used at non-saturating concentrations, inhibited ATP hydrolysis to the same extent throughout the decay. This showed that the activity was totally controlled by the ATPase during all the decay and rules out any involvement of the phosphate or nucleotide carriers. This interpretation was confirmed by the fact that carboxyatractyloside, an inhibitor of the ATP/ADP antiporter, had a strong effect only on the initial rate of ATP hydrolysis, but not on the rate measured after some tens of seconds of decay. Oligomycin, at variance with the other ATPase inhibitors, interfered with the deactivation process, suggesting that its effect depends on the conformational state of the enzyme. Between pH 6.5 and 7.5, the hydrolase activity rose continuously and was still kinetically controlled by the ATPase. At higher pH value, the activity slightly decreased and appeared limited by at least one of the carriers. The activity of the ATPase itself, free of any transport process, seemed to increase monotonously with pH from 6.5 to 8. The electrochemical proton gradient is required to maintain the ATPase active, whereas no effect can be observed on transport processes. Matricial pH, while modulating the apparent catalytic turnover, has no marked effect on the rate of deactivation. These results, obtained with intact mitochondria, extend previous observations on the isolated enzyme and question the binding of IF1 as a rate-limiting step for ATPase deactivation.