Failure of atractyloside to inhibit synaptosomal mitochondrial energy transduction

Studies on synaptosome mitochondrial respiration are complicated by “free” mitochondria. Veratridine stimulation of synaptosomal respiration was due to increased Na⁺ cycling at the synaptosome membrane associated with increased oxidative phosphorylation of intraterminal ADP and was inhibited by oligomycin, ouabain or Na⁺ free medium. Atractylate or carboxyatractyloside failed to block veratridine-stimulated respiration but inhibited exogenous-ADP-stimulated respiration. Protein synthesis in the synaptosome fraction was inhibited by oligomycin, valinomycin or 2,4-dinitrophenol but was unaffected by excess atractylate. No change in synaptosomal adenine nucleotide content was found in the presence of atractylate, although a significant decrease in the [ATP]/[ADP] was found with oligomycin, veratridine or valinomycin. These findings show that atractylate does not modify intraterminal mitochondrial energy transduction and indirectly suggest an impermeability of the synaptosome membrane to atractylate.     

Thyroid hormone inhibition of synaptosome amino acid uptake and protein synthesis.

Abstract— 3,3′,5-Triiodothyronine (T₃) inhibited L-[¹⁴C]leucine uptake into synaptosomes. Inhibition was competitive with a K_i of 3.1 × 10^?5m . Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l -Thyroxine and analogues inhibited the incorporation of l -[¹⁴C] leucine into cerebral synaptosome protein. At 50 μm , the triiodo-compounds were more inhibitory than tetraiodo->3,5-triiodo-l -thyronine >3,3′,5-triiodothyropro-pionic> l -thyroxine >3,5-diiodo-l -tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5-Triiodothyronine had no effect on respiratory control or 2,4-DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [¹⁴C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm -ouabain. 3,3′,5-Triiodothyronine had no effect on synaptosome fraction ATPase or Na-K ATPase. Addition of T₃ induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm ) or cycloheximide (50μg/ml). [¹⁴C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5-triiodothyronine. There was no inhibition of [¹⁴C]proline uptake or incorporation. The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.     

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.     

Relationships between the neuronal sodium/potassium pump and energy metabolism. Effects of K+, Na+, and adenosine triphosphate in isolated brain synaptosomes

The relationships between Na/K pump activity and adenosine triphosphate (ATP) production were determined in isolated rat brain synaptosomes. The activity of the enzyme was modulated by altering [K+]e, [Na+]i, and [ATP]i while synaptosomal oxygen uptake and lactate production were measured simultaneously. KCl increased respiration and glycolysis with an apparent Km of about 1 mM which suggests that, at the [K+]e normally present in brain, 3.3-4 mM, the pump is near saturation with this cation. Depolarization with 6-40 mM KCl had negligible effect on ouabain-sensitive O2 uptake indicating that at the voltages involved the activity of the Na/K ATPase is largely independent of membrane potential. Increases in [Na+]i by addition of veratridine markedly enhanced glycoside-inhibitable respiration and lactate production. Calculations of the rates of ATP synthesis necessary to support the operation of the pump showed that greater than 90% of the energy was derived from oxidative phosphorylation. Consistent with this: (a) the ouabain-sensitive Rb/O2 ratio was close to 12 (i.e., Rb/ATP ratio of 2); (b) inhibition of mitochondrial ATP synthesis by Amytal resulted in a decrease in the glycoside-dependent rate of 86Rb uptake. Analyses of the mechanisms responsible for activation of the energy-producing pathways during enhanced Na and K movements indicate that glycolysis is predominantly stimulated by increase in activity of phosphofructokinase mediated via a rise in the concentrations of adenosine monophosphate [AMP] and inorganic phosphate [Pi] and a fall in the concentration of phosphocreatine [PCr]; the main moving force for the elevation in mitochondrial ATP generation is the decline in [ATP]/[ADP] [Pi] (or equivalent) and consequent readjustments in the ratio of the intramitochondrial pyridine nucleotides [( NAD]m/[NADH]m). Direct stimulation of pyruvate dehydrogenase by calcium appears to be of secondary importance. It is concluded that synaptosomal Na/K pump is fueled primarily by oxidative phosphorylation and that a fall in [ATP]/[ADP][Pi] is the chief factor responsible for increased energy production.     

Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials. An explanation for the apparent uncoupler insensitivity of ATP synthesis

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (delta mu H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 mumol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 mumol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the delta mu H+ was 200 mV (inside negative). When K+ was added to the suspensions the measured delta mu H+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of delta mu H+ adjustment, it was found that lowering delta mu H+ from 200 mV ([K+]in/[K+]out = 1000) to 100 mV ([K+]in/[K+]out = 40) had no effect on the rate of methane formation and on the intracellular ATP content. At delta mu H+ values below 100 mV ([K+]in/[K+]out less than 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when delta mu H+ was adjusted to values lower 50 mV ([K+]in/[K+]out less than 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri. Protonophoric uncouplers like 3,5,3',4'-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF 6847) were found not to dissipate delta mu H+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

METHYL MERCURY INHIBITION OF SYNAPTOSOME AND BRAIN SLICE PROTEIN SYNTHESIS: IN VIVO AND IN VITRO STUDIES

Subacute methyl mercury (MeHg) intoxication was induced in adult rats following the daily intragastric administration of 1 mg MeHg/100 g body weight. Decreased [¹⁴C]leucine incorporation into cerebral and cerebellar slice protein was found. Weight loss occurred during the latent and neurotoxic phases but pair feeding did not reveal a significant defect in amino acid incorporation into slice protein. There was no decline in synaptosome protein synthesis in vitro during the latent phase but a significant decline in cerebellar and cerebral synaptosome synthesis was found during the neurotoxic phase. MeHg in vitro inhibited cerebral slice and synaptosome protein synthesis at half maximal concentrations of 7.5 and 12.5 μM respectively. Inhibition of synthesis in synaptosomes was non-competitive with K₁ of 4 × 10^?6M. MeHg had no effect on [¹⁴C]leucine or [¹⁴C]proline uptake into synaptosomes. There was no significant inhibition of synaptosome basal ATPase or Na + K ATPase at concentrations of MeHg (12 μM) giving half maximal inhibition of protein synthesis. No preferential inhibition of the chloramphenicol (55S) or cycloheximide sensitive components of synaptosome fraction protein synthesis was found, suggesting that the inhibition is common to both mitochondrial and extramitochondrial protein synthesizing systems. Addition of nucleotides and/or atractylate failed to influence protein synthesis and did not reverse the MeHg inhibition. Mannitol, as a replacement for the predominant cation species of the incubation medium, gave 40% inhibition of protein synthesis in the control but protected against further inhibition by MeHg.     

ATP synthesis in Staphylococcus aureus cells during induction of membrane potentials and proton gradient

The dynamics of ATP synthesis in Staphylococcus aureus cells was studied during membrane potential induction and K+ gradient generation in the presence of valinomycin. The starting level of intracellular ATP was 0.05 mM. Valinomycin (30 micrograms/ml) caused an increment of the intracellular ATP level up to 0.25 mM. The protonophore uncoupler, m-chlorinecarbonylcyanidephenylhydrazonium, and the H+-ATPase inhibitor, N,N'-dicyclohexylcarbodiimide, effectively suppress ATP synthesis induced by valinomycin. No ATP synthesis occurs at K+ concentration of 200 mM. The transmembrane gradient formation results in the synthesis of a smaller amount of ATP (0.10 mM).     

K+-independent effects of valinomycin in photosynthetic systems

With chromatophores ofRhodospirillum rubrum, valinomycin inhibited electron transport in the presence or absence of K⁺. NH₄Cl had no effect on photophosphorylation but uncoupled with valinomycin present. ATPase activity was stimulated by NH₄Cl plus valinomycin but not by either alone. K⁺ partially reversed the inhibition of phosphorylation and the stimulation of ATPase by valinomycin plus NH₄Cl.With chloroplasts, valinomycin inhibited coupled but not basal electron transport. The inhibition was only partially reversed by uncouplers. Valinomycin stimulated the light-activated Mg²⁺-dependent ATPase similar to several uncouplers such as quinacrine, methylamine, and S-13. In addition, valinomycin inhibited delayed light emission and stimulated the H⁺/e^– ratio. These contrasting activities in chloroplasts are not easily explained.Contribution number 389 of the Charles F. Kettering Research Laboratory.     

Changes in energy charge and block of protein synthesis in rabbit reticulocytes under the action of valinomycin

Valinomycin blocks protein synthesis at the elongation stage in intact reticulocyte while being without action on a cell free system. We demonstrate here that this membrane mediated inhibition is accompanied by a change of the energy charge (ATP/ADP/AMP ratio) of the cells. There seems to be an accumulation of AMP without a proportional gain in ADP. The change in energy charge in itself can be the cause of the inhibition of protein synthesis. We show also that valinomycin can cause a decrease of the energy charge at concentrations where its action as an ionophore is not seen and independent of a possible activation of the Na-K dependent ATPase.Present address: Meir Hospital, Kfar Saba, Israel     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

The sodium cycle. II. Na+-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na+ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na+-motive respiratory chain of V. alginolyticus. Na+ loaded cells incubated in a K+ or Li+ medium fail to synthesize ATP in response to lactate addition. The addition of Na+ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na+ level. Neither lactate oxidation nor delta psi generation coupled with this oxidation is increased by external Na+ in the Na+-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse delta pNa, i.e., [Na+]in greater than [Na+]out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K+-loaded cells incubated in a high Na+ medium, i.e., when delta pNa of the proper direction [( Na+]in less than [Na+]out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K+ medium (delta pNa is low) is decreased by CCCP even without monensin. Artificial formation of delta pNa by adding 0.25 M NaCl to the K+-loaded cells (Na+ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na+ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na+ instead of H+ as the coupling ion.     

Inhibition by lead ion of Electrophorus electroplax (Na+ + K+)-adenosine triphosphatase and K+-p-nitrophenylphosphatase.

Inorganic lead ion in micromolar concentrations inhibits Electrophorus electroplax microsomal (Na+ + K+)-adenosine triphosphatase ((Na+ + K+)-ATPase) and K+-p-nitrophenylphosphatase (NPPase). Under the same conditions, the same concentrations of PbCl2 that inhibit ATPase activity also stimulate the phosphorylation of electroplax microsomes in the absence of added Na+. Enzyme activity is protected from inhibition by increasing concentrations of microsomes, ATP, and other metal ion chelators. The kinetics follow the pattern of a reversible noncompetitive inhibitor. No kinetic evidence is elicited for interactions of Pb2+ with Na+, K+, Mg2+, ATP, or p-nitrophenylphosphate. Na+- ATPase, in the absence of K+, and (Na+ + K+)-NPPase activity at low [K+] are also inhibited. ATP inhibition of NPPase is not reversed by Pb2+. The calculated concentrations of free [Pb2+] that produce 50% inhibition are similar for ATPase and NPPase activities. Pb2+ may act at a single independent binding site to produce both stimulation of the kinase and inhibition of the phosphatase activities.     

Oxidative phosphorylation in nuclei isolated from rat thymus

1. Nuclei were isolated from rat thymus in 0.25 M sucrose-3 mM CaCl₂. Fractionation of thymus tissue showed that the nuclear fraction could be contaminated with 10% intact cells at the most.

2. The nuclear fraction contained about 50% of the cellular ATP. Under anaerobic conditions nuclear ATP is degraded. When, after an anaerobic incubation, the nuclei are incubated under aerobic conditions endogenous nuclear ATP is resynthesized. The amount of ATP found cannot be accounted for by mitochondria or intact cells. It could also be shown that the ATP synthesis is not due to such contamination.

3. Nuclear ATP synthesis and respiration were inhibited by amytal, rotenone, and carbon monoxide. The latter inhibition was reversed by light. 2,4-Dinitrophenol abolished ATP synthesis and accelerated respiration. Oligomycin inhibited respiration and phosphorylation. The inhibition of respiration by oligomycin could be released by dinitrophenol. ATP synthesis was also inhibited by lewisite. This inhibition could be reversed by BAL. No inhibition, however, was found with arsenite or arsenite + BAL.

4. A P:O ratio between 0.6 and 1.0 was obtained for nuclear phosphorylation. This must be considered as a minimal value.

5. It is concluded that thymus nuclei are capable of respiratory-chain-linked oxidative phosphorylation.     

Plasma membrane potential modulates chemotactic peptide-stimulated cytosolic free Ca2+ changes in human neutrophils

The relationship between fMet-Leu-Phe-induced changes in the cytosolic free Ca2+ concentration [( Ca2+]i), plasma membrane potential depolarization, and metabolic responses was studied in human neutrophils. Receptor-activated depolarization occurred both at high and resting [Ca2+]i, but was inhibited at very low [Ca2+]i. Phorbol 12-myristate 13-acetate-induced plasma membrane depolarization, on the contrary, was independent of [Ca2+]i. The threshold fMet-Leu-Phe concentration for plasma membrane depolarization (10(-8) M) was at least 1 log unit higher than that for [Ca2+]i increases (5 X 10(-10) M) and coincident with that for NADPH oxidase activation. Nearly maximal [Ca2+]i increases were elicited by 3 X 10(-9) fMet-Leu-Phe in the absence of any significant plasma membrane potential change. This observation allowed us to investigate the effects of artificially induced plasma membrane depolarization and hyperpolarization at low fMet-Leu-Phe concentrations (10(-9) to 3 X 10(-9) M) which did not perturb plasma membrane potential. Depolarizing (gramicidin D at 10(-7) to 10(-6) M or KCl at 50 mM) and hyperpolarizing (valinomycin at 4 microM) treatments had little influence on unstimulated [Ca2+]i levels, whereas fMet-Leu-Phe-induced transients were significantly altered. Gramicidin D and KCl decreased the fMet-Leu-Phe-induced [Ca2+]i increases in Ca2+-containing or in Ca2+-free media. Valinomycin, on the contrary, increased receptor-stimulated [Ca2+]i increases, and the effect was larger in the presence of extracellular Ca2+. Valinomycin also strongly potentiated secretion. It is suggested that plasma membrane depolarization in human neutrophils is a physiological feedback mechanism inhibiting receptor-dependent [Ca2+]i changes.     

Inhibition by valinomycin of atractyloside binding to the membrane-bound ADP/ATP carrier: Counteracting effect of cations

The atractyloside binding capacity of rat heart mitochondria, but not the binding affinity, was markedly decreased by preincubation of the mitochondria with valinomycin in isotonic KCl medium. Maximum inhibition was attained with 5 ng of valinomycin per mg of mitochondrial protein; it corresponded to a 40% decrease of the atractyloside binding capacity. The inhibitory effect of valinomycin was maximal between pH 7.0 and 7.5. It was more marked for heart mitochondria than for liver mitochondria. Valinomycin inhibition of atractyloside binding to heart mitochondria was counteracted by nigericin and FCCP, by sublytic concentrations of cationic surfactants such as cetyltrimethylammonium bromide, and by low concentrations of trivalent and divalent metal ions at acidic pH's still compatible with atractyloside binding, i.e., down to pH 5.5; trivalent metal ions were more effective than divalent metal ions. The effect of valinomycin was also counteracted by exceedingly high concentrations of K+ (more than 300 mM), resulting in a substantial increase in the ionic strength. These results were discussed in terms of the relation between the atractyloside binding capacity of the inner mitochondrial membrane and the surface potential of this membrane.     

Control of proteoliposomal cytochrome c oxidase: the partial reactions

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith-Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by delta pH and by delta psi can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.     

Covalent modification of the catalytic sites of the H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ADP: modification of the catalytic site 2 (loose) and the catalytic site 3 (open) impairs multi-site, but not uni-site catalysis of both ATP synthesis and ATP hydrolysis

The H(+)-ATPase from chloroplasts, CF(0)F(1), was isolated and purified. The enzyme contained one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]AD(T)P leads to a tight binding of the azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and after UV-irradiation, the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-(32)P]ADP, the covalently bound label was found exclusively at beta-Tyr-362, i.e. binding occurs only to catalytic sites. Incubation conditions with 2-azido-[alpha-(32)P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, either to catalytic site 2 or to catalytic site 3. For measurements of the degree of inhibition by covalent modification, CF(0)F(1) was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential. The rate of ATP synthesis was 120 s(-1), and the rate of ATP hydrolysis was 20 s(-1), both measured under multi-site conditions. Covalent modification of either catalytic site 2 or catalytic site 3 inhibited both ATP synthesis and ATP hydrolysis, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that modification of one catalytic site, either site 2 or site 3, is sufficient to completely block multi-site ATP synthesis and ATP hydrolysis. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with covalently modified CF(0)F(1) and with non-modified CF(0)F(1). The result was that uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent modification of either catalytic site 2 or site 3. The results indicate cooperative interactions between catalytic nucleotide binding sites during multi-site catalysis, whereas neither uni-site ATP synthesis nor uni-site ATP hydrolysis require interaction with other sites.     

ORGANIC MERCURIAL ENCEPHALOPATHY: IN VIVO AND IN VITRO EFFECTS OF METHYL MERCURY ON SYNAPTOSOMAL RESPIRATION

—A reproducible model of subacute methyl mercury (MeHg) intoxication was developed in the adult rat following the daily intragastric administration of 10 mg methyl mercury/kg body wt. Synaptosomes isolated from animals during the latent phase of mercury neurotoxicity (6-10 days) demonstrated no significant change in respiratory control, State 3, State 4, or 2,4-dinitrophenol stimulated respiration with succinate, glutamate or pyruvate plus malate. During the neurotoxic phase, a significant decline in respiratory control was evident with all substrates. Cerebellar synaptosomes revealed qualitatively similar but quantitatively greater inhibition of 2,4-dinitrophenol stimulated respiration during the latent and neurotoxic phases with glutamate. In vitro studies of synaptosome respiration, oxidative phosphorylation and respiratory control with 5-15 μm -methyl mercury revealed a stimulation of initial State 4 respiration, loss of RCI, inhibition of State 3 but no change in the gramicidin or 2,4-dinitrophenol uncoupled rate supported by pyruvate-malate. Phosphate did not relieve the State 3 inhibition. At 25 μm -methyl mercury and above, considerable inhibition of electron transfer occurred. At this concentration, cytochrome c oxidase was inhibited 50%. Isosmotic replacement of medium KC1 by mannitol reduced the MeHg stimulation of State 4 respiration but had no effect on MeHg inhibition of ADP stimulated respiration. Half-maximal stimulation of State 4 respiration by MeHg occurred at [K]⁺⋍ 6 mm . These findings are compatible with an energy-linked methyl mercury induced cation translocation across the synaptosome (mitochondrial) membrane.     

Effect of heliomycin on the respiration and oxidative phosphorylation of the liver mitochondria of the rat

The effect of heliomycin and known uncouplers of oxidative phosphorylation on respiration and oxidative phosphorylation was studied comparatively. Heliomycin, as well as 2,4-dinitrophenol, valinomycin and gramicidin S inhibited the mitochondrial synthesis of ATP. This process was inhibited completely by heliomycin at a concentration of 1.5 x 10(-5) M. The synthesis of inorganic pyrophosphate, the other macroergic compound, was also inhibited by heliomycin, ATPase and pyrophosphatase of uncoupled mitochondria being not inhibited by the antibiotic. Like 2,4-dinitrophenol, heliomycin stimulated the synthesis of ATPase and respiration in intact mitochondria. Probably, heliomycin inhibited the synthesis of ATP and pyrophosphate by uncoupling the processes of respiration and oxidative phosphorylation. It was shown earlier that heliomycin, a specific inhibitor of bacterial RNA synthesis, also affected energy metabolism of bacterial cells by inhibiting the synthesis of ATP and active transport.