Proton electrochemical gradient and phosphate potential in mitochondria

The paper reports an analysis of the relationship between deltamuH the proton electrochemical potential difference, and deltaGp, the phosphate potential. Depression of deltamuH and deltaGp has been obtained by titration with: (a) carbonylcyanide trifluoromethoxyphenylhydrazone; (b) nigericin (+ valinomycin); (c) KCl (+ valinomycin); and (d) rotenone. The uncoupler depresses deltamuH more than nigericin (+ valinomycin), KCl (+ valinomycin) and rotenone at equivalent deltaGp. The deltaGp/deltamuH ratio is about 3 at high values of deltamuH. When deltaGp and deltamuH are depressed by nigericin (4 valinomycin) the deltaGp/deltamuH ratio remains constant. When deltaGp and deltamuH are depressed by uncouplers, the deltaGp/deltamuH ratio increases hyperbolically tending to infinity while deltamuH tends to zero. The absence of constant proportionality between deltaGp and deltamuH indicates that the proton gradients driving ATP synthesis presumably operate within microscopic environments.     

Modification of flow-force relationships by external ATP in yeast mitochondria

The purpose of this work is to measure protonmotive force and cytochrome reduction level under different respiratory steady states in isolated yeast mitochondria. The rate of respiration was varied by using three sets of conditions: (a) different external phosphate concentrations with a fixed concentration of ADP (ATP synthesis) and (b) different concentrations of carbonylcyanide m-chlorophenylhydrazone in the presence of oligomycin and carboxyatractylate (uncoupling) either in the absence or (c) in the presence of external ATP. ADP plus phosphate stimulates respiration more than uncoupler at the same protonmotive force value. However, the relationships between respiratory rate and protonmotive force were similar when stimulation was induced either by ADP + Pi or by carbonylcyanide m-chlorophenylhydrazone in the presence of ATP. At the same respiratory rate, cytochrome a + a3 is more reduced by uncoupler than by ADP + Pi additions. However, the relationships between respiratory rate and reduction level of cytochrome-c oxidase are similar both under ATP synthesis and with uncoupling conditions in the presence of external ATP. Control of respiration exerted by cytochrome-c oxidase, and support the view the condition mentioned above. This control was low when the respiratory rate was varied by the ATP synthesis rate; it increased as a function of the respiratory rate with uncoupler in the absence of ATP. ATP decreased this control under uncoupling conditions. These results suggest a regulatory effect of external ATP on cytochrome-c oxidase, and support the view that the relationships between respiratory rate and protonmotive force, on the one hand, and respiratory rate and the reduction level of cytochrome-c oxidase, on the other, depend respectively on the kinetic regulations of the system.     

A correlation between respiration and synthesis of ATP in mitochondria at different degree of uncoupling of oxidative phosphorylation

It is known that mitochondrial respiration in state 3 is due to three simultaneous and independent processes: synthesis of ATP (1), endogenous passive proton leakage (2), and proton leakage by protonophoric uncoupler (3). The total rate of processes (2) and (3) is equal to the product of respiration rate in state 4 and coefficient KR, which is defined as the ratio of the deltamuH+ value in state 3 to that in state 4. It is shown that it is possible to calculate both the rates of processes (1), (2) and (3) separately and the protonophoric activity of uncoupler using the coefficient KR and other coefficients, which are determined as the ratio of deltamuH+ values in state 3 or in state 4 to its maximal value. Simple methods of determination of these coefficients were developed, which are based on the study of the dependence of respiration rate in states 3 and 4 on the concentration of protonophoric uncoupler. It was found that the uncoupling action of palmitate, a natural uncoupler of oxidative phosphorylation, unlike classic uncoupler-protonophores DNP and FCCP, depends not only on its protonophoric activity but also on the inhibition of the process (1).     

Mitochondrial bioenergetics during exposure of rats to perfluidone,a fluorinated arylalkylsulphonamide

Apart from the symptoms of poisoning which the fluorinated arylalkylsulphonamides share with the classical protonphore and uncoupler of oxidative phosphorylation, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), the direct correlation between the lipophilic weak acid properties of these chemicals and their biological activity suggests that permeation of the inner mitochondrial membrane could be the initial step in the molecular mechanism of their biological activity. Mitochondria isolated from the livers of rats intraperitoneally exposed to varying doses (0–80 mg/kg body wt.) of perfluidone (1,1,1-trifluoro-N-(2 methyl-4-(phenylsulphonyl)phenyl methanesulphonamide), a fluorinated arylalkylsulphonamide pesticide, exhibit the following dose-dependent features: (i) increased state-4 respiration: stimulation being maximal (≥400%) at 80 mg perfluidone per kg body wt.), (ii) release of respiratory control by ADP: least respiratory control ratios (RCRs) (≤1.2) were obtained at 80 mg perfluidone per kg body wt., (iii) reduced ADP/O ratios, (iv) increased mitochondrial passive swelling, (vi) reduced rates of mitochondrial proton ejection during succinate oxidation, (vi) reduced rates of respiration-dependent Ca²⁺ accumulation and (vii) an enhanced oligomycin-sensitive ATPase action. These features which are qualitatively identical to those of the classical protonophore FCCP, suggest that permeation of the inner mitochondrial membrane by perfluidone is accompanied by a movement of protons into the matrix such that the proton motive force required for ATP synthesis and ion transport becomes small or not formed at all.     

Formations of electrochemical proton gradient and adenosine triphosphate in proteoliposomes containing purified adenosine triphosphatase and bacteriorhodopsin.

Proteoliposome vesicles containing both bacteriorhodopsin of Halobacterium halobium and H+-translocating ATPase [EC 3.6,1.3] of a thermophilic bacterium, PS3, (TF0-F1) were reconstituted by either the dialysis method or the sonication method. Generation of the electrochemical proton gradient (deltamuH+) in these vesicles was measured using 9-aminoacridine for estimation of the chemical (deltapH) component and 8-anilinonaphthalene sulfonate for the electrical (deltaphi) component). In illuminated bacteriorhodopsin-vesicles the deltamuH+ reached 180-190 mV when reconstituted by the dialysis method and 210-220 mV when reconstituted by the sonication method. Vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method generated a deltapH+ of about 200 mV on addition of ATP, while vesicles prepared by the sonication method generated very little deltamuH+, if any. These vesicles generated similar deltamuH+ on illumination to that found in bacteriorhodopsin-vesicles. Using vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method, light dependent ATP synthesis was measured in relation to deltamuH+ formation. It was necessary to generate a deltamuH+ of above 170 mV for demonstration of appreciable formation of ATP and the greater the deltamuH+, the faster the rate of ATP synthesis.     

The action of heavy metals on the gametes of the marine mussel, Mytilus edulis (L.)--I. Copper-induced uncoupling of respiration in the unfertilized egg

The direct addition of Cu2+ to unfertilized eggs of Mytilus edulis results in a stimulation of respiration with maximal stimulation occurring at a Cu2+ concentration of ca 0.5 mM. By contrast, the addition of Zn2+ has no effect on egg respiration. The uncoupler CCCP produces a 5/6 fold stimulation of egg respiration but the addition of ADP leads to only a small release of respiration. In contrast, sperm respiration is unaffected by Cu2+, inhibited by Zn2+ and CCCP produces only a small respiratory stimulation. The addition of Cu2+ to respiring Mytilus mantle tissue mitochondria produces an initial stimulation of State 4 oxidation which is then followed by a progressive inhibition. It is suggested that respiration in the unfertilized egg may be inhibited by a high ATP/ADP ratio in the cytosol. Respiration can, therefore, be released by either the addition of a H+-translocating uncoupler or by Cu2+ which may act by stimulating mitochondrial K+ influx.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (delta mu H+) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and delta mu H+ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of delta mu H+, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on delta mu H+; for example, the excess of oligomycin produced a 90% inhibition of the respiration while delta mu H+ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing delta mu H+ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of delta mu H+, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and delta mu H+.     

Early metabolic effects and mechanism of ammonium transport in yeast

Studies were performed to define the effects and mechanism of NH+4 transport in yeast. The following results were obtained. Glucose was a better facilitator than ethanol-H2O2 for ammonium transport; low concentrations of uncouplers or respiratory inhibitors could inhibit the transport with ethanol as the substrate. With glucose, respiratory inhibitors showed only small inhibitory effects, and only high concentrations of azide or trifluoromethoxy carbonylcyanide phenylhydrazone could inhibit ammonium transport. Ammonium in the free state could be concentrated approximately 200-fold by the cells. Also, the addition of ammonium produced stimulation of both respiration and fermentation; an increased rate of H+ extrusion and an alkalinization of the interior of the cell; a decrease of the membrane potential, as monitored by fluorescent cyanine; an immediate decrease of the levels of ATP and an increase of ADP, which may account for the stimulation of both fermentation and respiration; and an increase of the levels of inorganic phosphate. Ammonium was found to inhibit 86Rb+ transport much less than K+. Also, while K+ produced a competitive type of inhibition, that produced by NH4+ was of the noncompetitive type. From the distribution ratio of ammonium and the pH gradient, an electrochemical potential gradient of around -180 mV was calculated. The results indicate that ammonium is transported in yeast by a mechanism similar to that of monovalent alkaline cations, driven by a membrane potential. The immediate metabolic effects of this cation seem to be due to an increased [H+]ATPase, to which its transport is coupled. However, the carriers seem to be different. The transport system studied in this work was that of low affinity.     

Calcium transport in membrane vesicles of Streptococcus cremoris

Rightside-out membrane vesicles of Streptococcus cremoris were fused with proteoliposomes containing the light-driven proton pump bacteriorhodopsin by a low-pH fusion procedure reported earlier [Driessen, A.J.M., Hellingwerf, K.J. & Konings, W.N. (1985) Biochim. Biophys. Acta 808, 1-12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing delta psi with a concomitant increase in delta pH stimulates Ca2+ uptake while dissipation of the delta pH results in a reduced rate of Ca2+ uptake. Also an artificially generated delta pH, interior acid, can drive Ca2+ uptake in S. cremoris membrane vesicles. Ca2+ uptake depends strongly on the presence of external phosphate while Ca2+-efflux-induced proton flux is independent of the presence of external phosphate. Ca2+ accumulation is abolished by the divalent cation ionophore A23187. Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone or inhibition of the membrane-bound ATPase by N,N'-dicyclohexylcarbodiimide strongly inhibits Ca2+ release. Further studies on Ca2+ efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+ exit from intact cells is an electrogenic process. It is concluded that Ca2+ efflux in S. cremoris is mediated by a secondary transport system catalyzing exchange of calcium ions and protons.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium.

The light-dependent uptake of triphenylmethylphosphonium (TPMP+) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (deltapH) across the membrane, and TPMP+ was used as an index of the electrical potential difference, deltapsi. Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, deltapH amounts to 0.6-0.8 pH unit. Its value can be increased to 1.5-2.0 by either incubating the cells with TPMP+ (10(-3) M) or at low external pH (5.5). --deltapH can be lowered by uncoupler or by nigericin. The TPMP+ uptake by the cells indicates a large deltapsi across the membrane, negative inside. It was estimated that in the light, at pH 6.6, deltapsi might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, deltamuH+/F, amounts under these conditions to about 140 mV. The effects of different ionophores on the light-drive proton extrusion by the cells were in agreement with the effects of these compounds on --deltapH.     

Activation of latent K+ uniport in mitochondria treated with the ionophore A23187

Addition of A23187 plus EDTA to energized mitochondria in KCl medium determines a rapid osmotic swelling due to K+ uptake. The swelling is fully reversed by uncoupler, is stimulated by quinine, and is accompanied by membrane depolarization and increased rate of respiration. A23187-treated mitochondria passively swell in K+ thiocyanate at neutral pH, under conditions where the H+-K+ antiporter appears to be silent. These data indicate that A23187 activates electrophoretic K+ flux, supporting the notion that Mg2+ depletion unmasks several ionic conductance pathways whose concerted interplay could provide a sensitive regulation of mitochondrial volume homeostasis.     

Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation.

The effects of three tetrachlorobiphenylols [2',3',4',5'-tetrachloro-2-biphenylol (1); 2',3',4',5'-tetrachloro-4- biphenylol (2); and 2',3',4',5'-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3',5,5'-tetrachloro-4,4'-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase) activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1-3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5-7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (greater than or equal to 0.4 microM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1-7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

Transport of cyclitols by a proton symport in Klebsiella aerogenes.

The respiration and the ATP content of Klebsiella aerogenes in the presence of various inhibitors were compared to the transport of scyllo-inositol. The ATPase was found to be inhibited by dicyclohexyl carbodiimide. The transport has been tested in anaerobiosis and aerobiosis. From the results obtained it is concluded that either ATP or respiration can sustain the transport activity in independent manner. 2. The energy derived from the respiratory chain reactions or the ATP hydrolysis results in electrogenic extrusion of protons. The electrochemical potential created drives the accumulation of scyllo-inositol, as shown by an increase of pH of the medium on addition of the substrate to cells in anaerobiosis. With non-induced cells no change in pH occurs, which demonstrates that proton flow is really linked to the transport. No H+/Na+ or K+ exchange is observed and the proton conductor carbonylcyanide m-chlorophenylhydrazone abolishes the pH shift caused by substrate addition. The stoichiometry of the symport H+/cyclitol is 1 and the half-maximum value of the pH variation as a function of the amount of scyllo-inositol added corresponds to a concentration of scyllo-inositol very close to the KT of influx.     

On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

Washed chloroplast thylakoid membranes upon exposure to [3H]ADP retain a tightly bound [3H]ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg2+ results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound [3H]ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme [Larson, E. M., Umbach, A., & Jagendorf, A. T. (1989) Biochim. Biophys. Acta 973, 75-85]. We present evidence that this is not the case. The Mg2(+)- and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22 degrees C and of about 15 s at 37 degrees C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg2+ and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound [3H]ADP parallels the onset of ATPase activity, although some [3H]ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound [3H]ADP being at a catalytic site and being replaced as this Mg2(+)- and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperglycemia Preserves Brain Mitochondrial Respiration During Anoxia

We examined brain mitochondrial function in normo- (5 mM) and hyperglycemic (50 mM) cats after 8 min of anoxia. In anoxic normoglycemic cats, mitochondrial state 3 respiration with NAD-linked substrates glutamate or pyruvate (both plus malate) was inhibited 30-50%. The uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) maximally stimulated respiration, indicating that inhibition of phosphorylation, not impairment of electron transport, substrate transport, or oxidation was present. State 3 respiration with succinate (plus rotenone) was unaffected. Mitochondrial respiratory control ratios trended toward reductions whereas ADP/O ratios remained unchanged. In contrast, brain mitochondria from anoxic hyperglycemic cats showed no such inhibition of state 3 respiration and no differences in function from normo- and hyperglycemic control animals except for trends toward loose coupling. Significantly higher brain tissue glucose concentrations were present in hyperglycemic controls as the only metabolite difference compared to normoglycemic controls. At the end of anoxia, hyperglycemic cats exhibited significantly higher cortical lactate and glucose levels but similarly reduced high-energy phosphate concentrations compared to normoglycemic cats. These results demonstrate that increased availability of glucose to gray matter as a consequence of hyperglycemia maintains normal mitochondrial state 3 respiration during exposure to anoxia. Previous survival studies have shown that lower serum glucose concentrations during anoxia are relatively brain protective. This result indicates that the presently described alterations in mitochondrial respiration must be fully reversible.     

Intracellular calcium and adenosine 3'',5''-cyclic monophosphate as mediators of potassium-induced aldosterone secretion.

We compared the action of K+ on aldosterone secretion from isolated bovine adrenal glomerulosa cells with that of ionophore A23187. Addition of either 50 nM-A23187 or 8 mM-K+ to perifused cells induces a similar initial aldosterone-secretory responses, and a similar sustained increases in Ca2+ entry. However, K+-induced secretion is more sustained than is A23187-induced secretion, even though each agonist appears to act by increasing Ca2+ entry into the cells. When [3H]inositol-labelled cells are stimulated by 8 mM-K+, a small decrease in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is observed. This decrease is not accompanied by an increase in inositol trisphosphate (InsP3) concentration. Also, if [3H]arachidonic acid-labelled cells are exposed to 8 mM-K+, there is no increase in [3H]diacylglycerol production. When [3H]inositol-labelled cells are stimulated by 50 nM-A23187, a small decrease in PtdIns(4,5)P2 is observed. This decrease is not accompanied by an increase in InsP3. The cyclic AMP content of K+-treated cells was approximately twice that in A23187-treated cells. If cells are perifused simultaneously with 50 nM-forskolin and 50 nM-A23187, the initial aldosterone-secretory response is similar to that induced by A23187 alone, and the response is sustained rather than transient, and is similar to that seen during perifusion of cells with 8 mM-K+. This dose of forskolin (50 nM) causes an elevation of cyclic AMP concentration in A23187-treated cells, to a value similar to that in K+-treated cells. These results indicate that, in K+-treated cells, a rise in cyclic AMP content serves as a positive sensitivity modulator of the Ca2+ message, and plays a key role in mediating the sustained aldosterone-secretory response.     

Effect of micromolar concentrations of free Ca2+ ions on pyruvate dehydrogenase interconversion in intact rat heart mitochondria.

1. The mitochondrial content of active (dephospho) pyruvate dehydrogenase (PDHA) was found to be severalfold higher at an extramitochondrial Ca2+ concentration of 2 microM (pCa6) than at pCa7. The nature of the respiratory substrate did not affect this finding. 2. This Ca2+-dependence was shown in state-4 and 50%-state-3 conditions [see Chance & Williams (1956) Adv. Enzymol. 17, 65-134], but was absent in the presence of excess ADP (state 3). 3. Na+ and Mg2+ ions shifted the pCa value required for a maximal PDHA content to lower values. This was attributed to a stimulation of mitochondrial Ca2+ egress and an inhibition of uptake, respectively. Na+ ions diminished pyruvate dehydrogenase phosphate phosphatase activity in mitochondria which had been extensively depleted of Ca2+ ions by incubation with EGTA, raising the possibility of a direct inhibitory effect of Na+ ions, unrelated to Ca2+ movements. 4. Mg2+ ions lowered the mitochondrial PDHA content at pCa 6.24 and 6.48, but had only minimal effects in the presence of EGTA. 5. The effects of P1 and bicarbonate ions on PDHA content were also studied, as possible effectors of mitochondrial Ca2+ transport. Bicarbonate ions abolished the response to Ca2+ ions, by generating maximal values of PDHA content, but such a response was still observed when physiological concentrations of both P1 and bicarbonate were used. 6. The pCa of the medium in the range 6.33 to over 7 affected PDHA content, with only very minor changes in state-4 rates of O2 uptake and no change in [ATP]/[ADP] ratio or in mitochondrial [NADH]/[NAD+] ratio, provided that Mg2+ ions were present. Thus the effect of Ca2+ ions on PDHA content is unlikely to be mediated by changes in [ATP]/[ADP] and [NADH]/[NAD+] ratio and is more likely to be direct. Equally, changes in the [acetyl-CoA]/[CoA] ratio in response to Ca2+ ions when the substrate was pyruvate were the converse of those required to mediate changes in interconversion, and are probably secondary to changes in PDHA content.     

Ca2+ ionophores affect phosphoinositide metabolism differently than thyrotropin-releasing hormone in GH3 pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) by a phospholipase C (or phosphodiesterase) and elevates cytoplasmic-free Ca2+ concentration ([Ca2+]i) in GH3 pituitary cells. To explore whether hydrolysis of PtdIns-4,5-P2 is secondary to the elevation of [Ca2+]i, we studied the effects of Ca2+ ionophores, A23187 and ionomycin. In cells prelabeled with [3H]myoinositol, A23187 caused a rapid decrease in the levels of [3H]PtdIns-4,5-P2, [3H]PtdIns-4-P, and [3H]PtdIns to 88 +/- 2%, 88 +/- 4%, and 86 +/- 1% of control, respectively, and increased [3H]inositol bisphosphate to 200 +/- 20% at 0.5 min. There was no increase in [3H] Ins-P3; the lack of a measurable increase in [3H]Ins-P3 was not due to its rapid dephosphorylation. In cells prelabeled with [14C]stearic acid, A23187 increased [14C]diacylglycerol and [14C]phosphatidic acid to 166 +/- 20% and 174 +/- 17% of control, respectively. In cells prelabeled with [3H]arachidonic acid, A23187, but not TRH, increased unesterified [3H]arachidonic acid to 166 +/- 8% of control. Similar effects were observed with ionomycin. Hence, Ca2+ ionophores stimulate phosphodiesteratic hydrolysis of PtdIns-4-P but not of PtdIns-4,5-P2 and elevate the level of unesterified arachidonic acid in GH3 cells. These data demonstrate that Ca2+ ionophores affect phosphoinositide metabolism differently than TRH and suggest that TRH stimulation of PtdIns-4,5-P2 hydrolysis is not secondary to the elevation of [Ca2+]i.     

Location of an oligomycin-insensitive and magnesium ion-stimulated adenosine triphosphatase in rat spleen mitochondria.

1. When rat spleen mitochondria are incubated with oxidizable substrates, added MgCl2 (greater than 150 muM free concentration) markedly stimulates state-4 respiration and lowers both the respiratory control and ADP/O ratios; this effect is reversible on addition of excess of EDTA. 2. With [gamma-32P]ATP as substrate, an Mg2+-stimulated ATPase (adenosine triphosphate) was identified in the atractyloside-insensitive and EDTA-accessible space of intact rat spleen mitochondria. 3. Oligomycin has no effect on the activity of the Mg2+-stimulated ATPase at a concentration (2.0mug/mg of protein) that completely inhibits the atractyloside-sensitive reaction. Of the two ATPase activities, only the atracytoloside sensitive reaction is stimulated (approx. 40%) by dinitrophenol. 4. On digitonin fractionation the atractyloside-insensitive Mg2+-stimulated ATPase co-purifies with the outer membrane-fraction of rat spleen mitochondria, whereas (as expected) the atractylosidesensitive activity co-purifies with the inner-membrane plus matrix fraction. 5. Stoicheiometric amounts of ADP and Pi are produced as the end products of ATP hydrolysis by purified outer-membrane fragments; no significant AMP production is detected during the time-course of the reaction. 6. The outer-membrane ATPase is present in rat kidney cortex and heart mitochondria as well as in spleen, but is absent from rat liver, thymus, brain, lung, diaphragm and skeletal muscle.     

The substrate proton of the pyruvate kinase reaction

The pyruvate kinase reaction occurs in separate phosphate- and proton-transfer stages: (formula; see text) K+, Mg2+, and Mg.ADP are known to be required for the phosphoryl transfer step, and K+ and Mg2+ with allosteric stimulation by MgATP are important for proton transfer. This paper uses the isotope trapping method with 3H-labeled water to identify the proton donor and determine when in the sequence of the catalytic cycle it is generated. When the enzyme was allowed to exchange briefly with 3H2O (pulse phase) and then diluted into a mixture containing PEP, ADP, and the cofactor K+, Mg2+, or Co2+ in D2O (chase phase), an amount of [3H]pyruvate was formed in great excess of the amount expected from steady-state catalysis in the diluted 3H-labeled water. With K+, Mg2+, and ADP at pH 6-9.5 in the pulse phase, a limit of 1.25 enzyme equiv of 3H were trapped. The concentration of PEP required for half-maximum trapping was 14-fold greater than its steady-state Km. Therefore, the rate constant for dissociation of the donor proton is estimated to be 14 times the steady-state rate of [3H]pyruvate formation, approximately 109 s-1, or 1500 s-1. At pD 6.4, Mg2+ and ADP were required in the chase, indicating that the ADP in the pulse was not bound tightly enough to be used in the chase. At pD 9.4, ADP was not required in the chase, only Mg2+ or Co2+, making it possible to limit the chase to one turnover from hybrid labeled complexes such as E.K.Mg.CoADP or E.K.Co.MgADP and PEP.(ABSTRACT TRUNCATED AT 250 WORDS)