Pyruvate metabolism in castor-bean mitochondria.

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.     

The effect of fluorocitrate on oxygen consumption and Ca2+ transport in the mitochondria of liver cells]

The effect of fluorocitrate on oxidative reactions and energy production systems of rat liver mitochondria has been studied. It was shown that oxidation of endogenous substrates and malate with pyruvate as well as the phosphorylation of the added ADP were inhibited by fluorocitrate. Inhibition of oxygen consumption by fluorocitrate induced the efflux of Ca2+ ions from mitochondria and a decrease in the Ca(2+)-accumulating capacity. The effect of fluorocitrate on Ca2+ transport in mitochondria is due to activation of the Ca-efflux pathway in those sensitive to ruthenium red.     

Cytosolic Ca2+ regulates the energization of isolated brain mitochondria by formation of pyruvate through the malate-aspartate shuttle

The glutamate-dependent respiration of isolated BM (brain mitochondria) is regulated by Ca2+(cyt) (cytosolic Ca2+) (S0.5=225±22 nM) through its effects on aralar. We now also demonstrate that the α-glycerophosphate-dependent respiration is controlled by Ca2+(cyt) (S0.5=60±10 nM). At higher Ca2+(cyt) (>600 nM), BM accumulate Ca2+ which enhances the rate of intramitochondrial dehydrogenases. The Ca2+-induced increments of state 3 respiration decrease with substrate in the order glutamate>α-oxoglutarate>isocitrate>α-glycerophosphate>pyruvate. Whereas the oxidation of pyruvate is only slightly influenced by Ca2+(cyt), we show that the formation of pyruvate is tightly controlled by Ca2+(cyt). Through its common substrate couple NADH/NAD+, the formation of pyruvate by LDH (lactate dehydrogenase) is linked to the MAS (malate-aspartate shuttle) with aralar as a central component. A rise in Ca2+(cyt) in a reconstituted system consisting of BM, cytosolic enzymes of MAS and LDH causes an up to 5-fold enhancement of OXPHOS (oxidative phosphorylation) rates that is due to an increased substrate supply, acting in a manner similar to a 'gas pedal'. In contrast, Ca2+(mit) (intramitochondrial Ca2+) regulates the oxidation rates of substrates which are present within the mitochondrial matrix. We postulate that Ca2+(cyt) is a key factor in adjusting the mitochondrial energization to the requirements of intact neurons.     

The role of calcium ions in the regulation of rat thymocyte pyruvate oxidation by mitogens.

1. Calcium concentrations in the nanomolar range cause a specific stimulation of the oxidation of pyruvate by isolated mitochondria from rat thymus that is sufficient to account precisely for the stimulation of pyruvate oxidation observed when rat thymocytes are incubated with the mitogens concanavalin A or ionophore A23187. 2. Higher concentrations of Ca2+ (more than 50 nM) inhibit the oxidation of NAD+-linked substrates by rat thymus mitochondria without affecting the oxidation of succinate or ascorbate+ NNN'N'-tetramethyl-p-phenylendiamine. 3. The addition of Ni2+ or Co2+ (2mM) to rat thymocytes prevents the response to concanavalin A at the level of pyruvate oxidation without affecting the stimulation of glycolysis induced by this mitogen. In contrast, the complete metabolic response to the ionophore A23187 is abolished by these ions. Ni2+ and Co2+ interfere with the ability of the ionophore to transport Ca2+ across the plasma membrane. 4. Concanavalin A, but not ionophore A23187, increases the respiratory inhibition induced by Ni2+ and Co2+. 5. These results support the view that mitogens stimulate lymphocyte pyruvate oxidation through an increase in cellular Ca2+ uptake.     

Role of calcium ions in the regulation of intramitochondrial metabolism. Properties of the Ca2+-sensitive dehydrogenases within intact uncoupled mitochondria from the white and brown adipose tissue of the rat.

1. Increasing concentrations of both Ca2+ and Sr2+ (generated by using EGTA buffers) resulted in 4-fold increases in the initial activity of pyruvate dehydrogenase within intact uncoupled mitochondria from rat epididymal adipose tissue incubated in the presence of the ionophore A23187, ATP, Mg2+ and oligomycin. The k0.5 values (concentrations required for half-maximal effects) for Ca2+ and Sr2+ were 0.54 and 7.1 microM respectively. In extracts of the mitochondria, pyruvate dehydrogenase phosphate phosphatase activity was stimulated about 4-fold by Ca2+ and Sr2+, with k0.5 values of 1.08 and 6.4 microM respectively. 2. NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase appeared to be rate-limiting in the oxidation of threo-Ds-isocitrate and oxoglutarate by uncoupled mitochondria from brown adipose tissue of cold-adapted rats. Ca2+ (and Sr2+) diminished the Km for the oxidation of both threo-Ds-isocitrate and oxoglutarate. The kinetic constants for these oxidations were very similar to those obtained for the activities of NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of the mitochondria. In particular, the k0.5 values for Ca2+ were all in the range 0.2--1.6 microM and Sr2+ was found to mimic Ca2+, but with k0.5 values about 10 times greater. 3. Overall, the results of this study demonstrate that the activities of pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase may all be increased by Ca2+ and Sr2+ within intact mitochondria. In all cases the k0.5 values are close to 1 and 10 microM respectively, as found for the separated enzymes. Experiments on brown-adipose-tissue mitochondria incubated in the presence of albumin suggest that it may be possible to use the sensitivity of the dehydrogenases to Ca2+ as a means of assessing the distribution of Ca2+ across the mitochondrial inner membrane.     

Regulation of citric acid cycle by calcium

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.     

Acetoacetate and malate effects on succinate and energy production by O2-deprived liver mitochondria supplied with 2-oxoglutarate

Acetoacetate provision to Ca(2+)-loaded liver mitochondria (less than 40 micrograms-ion Ca2+ x g protein-1), supplied with 2 mM Pi and 2-oxoglutarate as substrate, was found to prevent the mitochondrial deenergization and Ca2+ release induced by either rotenone during aerobic incubations or by O2 deprivation. Under the latter condition, the acetoacetate-promoted Ca2+ retention was entirely supported by ATP produced anaerobically at the succinylthiokinase step of the tricarboxylic acid cycle and was therefore abolished by addition of oligomycin. Surprisingly, oligomycin was also found to trigger Ca2+ release in rotenone-inhibited mitochondria in the presence of acetoacetate under aerobic conditions, unless a Pi acceptor was supplied. ADP deprivation at the succinylthiokinase step is likely to be involved. As estimated from rates of succinate production in O2-deprived mitochondria or from respiration rates in rotenone-inhibited mitochondria at supramaximal acetoacetate concentrations (above 1.2 mM) in the presence of a Pi acceptor, ATP production by substrate-level phosphorylation was close to 10 mumol.g protein-1.min-1 and appeared to be limited by rates of ketone body transport across the inner membrane. The rates of anaerobic energy production obtained by coupling 2-oxoglutarate oxidation to acetoacetate reduction were markedly higher than those obtained by reactions involved in the anaerobic metabolism of amino acids, simulated by providing 2-oxoglutarate and malate to mitochondria. Energy production was limited by rates of oxidant equivalent generation under the latter condition. Our data suggest that acetoacetate could effectively contribute to sustaining anaerobic energy production from endogenous substrates in liver tissue.     

Regulation of gluconeogenesis by norepinephrine, vasopressin, and angiotensin II: a comparative study in the absence and presence of extracellular Ca2+1

In hepatocytes isolated from fasted rats, vasopressin and angiotensin II stimulate the rate of gluconeogenesis from lactate or pyruvate in a Ca2+-dependent manner similar to that previously reported for norepinephrine. Actions of the peptide hormones on gluconeogenesis from glycerol or sorbitol, reduced substrates that require oxidation before they enter the gluconeogenic pathway at triosephosphate, also resemble those of norepinephrine. Stimulation of glucose production from these substrates is observed only in the presence of extracellular Ca2+. Actions of the peptide hormones on gluconeogenesis from dihydroxyacetone or fructose, the oxidized counterparts of glycerol and sorbitol, respectively, do not resemble those of norepinephrine. While norepinephrine enhances rates of glucose production from dihydroxyacetone or fructose in the absence of extracellular Ca2+, vasopressin and angiotensin II are ineffective either in the absence or presence of extracellular Ca2+. When the oxidation-reduction state in hepatocytes metabolizing dihydroxyacetone is altered by adding an equimolar concentration of ethanol (to provide cytosolic reducing equivalents), the results are similar to those obtained when cells are incubated with the reduced counterpart of dihydroxyacetone, glycerol, i.e., the peptide hormones cause an apparent increase in the rate of glucose production in a Ca2+-dependent manner. If, on the other hand, hepatocytes are incubated with glycerol or sorbitol and an equimolar concentration of pyruvate (to provide a cytosolic hydrogen acceptor), the peptide hormones, unlike norepinephrine, are ineffective in stimulating gluconeogenesis in the absence of extracellular Ca2+. These results indicate that whereas many of the actions of vasopressin and angiotensin II are similar to those of alpha 1-adrenergic agents, there are major differences in the manner in which the hormones act at various sites to regulate gluconeogenesis.     

The use of calcium uptake by small amounts of mitochondria from pancreatic islets to study mitochondrial respiration: the effects of diazoxide and sodium

The net uptake of 45Ca into mitochondria from pancreatic islets is stimulated by substrates that transfer reducing equivalents to various sites of the respiratory chain, such as succinate or glycerol 3-phosphate (site II), malate plus pyruvate (site I) or ascorbate plus TMPD (site III). Diazoxide, a known inhibitor of insulin release in vivo and in vitro, strongly inhibited net 45Ca uptake supported by glycerol phosphate and succinate and weakly inhibited 45Ca uptake supported by the other substrates. These results suggest that diazoxide, although not completely specific, is predominately an inhibitor at site II of the respiratory chain. This result is consistent with previous work that showed diazoxide inhibits the enzyme activity of the mitochondrial glycerol phosphate dehydrogenase in islets. Sodium ion inhibited the net accumulation of 45Ca by islet mitochondria suggesting a similarity between islet mitochondria and those of heart and some other endocrine tissues.     

On the Function of Mitochondrial Metabolism during Photosynthesis in Spinach (Spinacia oleracea L.) Leaves (Partitioning between Respiration and Export of Redox Equivalents and Precursors for Nitrate Assimilation Products)

The functioning of isolated spinach (Spinacia oleracea L.) leaf mitochondria has been studied in the presence of metabolite concentrations similar to those that occur in the cytosol in vivo. From measurements of the concentration dependence of the oxidation of the main substrates, glycine and malate, we have concluded that the state 3 oxidation rate of these substrates in vivo is less than half of the maximal rates due to substrate limitation. Analogously, we conclude that under steady-state conditions of photosynthesis, the oxidation of cytosolic NADH by the mitochondria does not contribute to mitochondrial respiration. Measurements of mitochondrial respiration with glycine and malate as substrates and in the presence of a defined malate:oxaloacetate ratio indicated that about 25% of the NADH formed in vivo during the oxidation of these metabolites inside the mitochondria is oxidized by a malate-oxaloacetate shuttle to serve extramitochondrial processes, e.g. reduction of nitrate in the cytosol or of hydroxypyruvate in the peroxisomes. The analysis of the products of the oxidation of malate indicates that in the steady state of photosynthesis the activity of the tricarboxylic acid cycle is very low. Therefore, we have concluded that the mitochondrial oxidation of malate in illuminated leaves produces mainly citrate, which is converted via cytosolic aconitase and NADP-isocitrate dehydrogenase to yield 2-oxoglutarate as the precursor for the formation of glutamate and glutamine, which are the main products of photosynthetic nitrate assimilation.     

The nature and control of the tricarboxylate cycle in beetle flight muscle.

The only exogenous substrates oxidized by mitochondria isolated from the flight muscle of the Japanese beetle (Popillia japonica) are proline, pyruvate and glycerol 3-phosphate. The highest rate of oxygen consumption is obtained with proline. The oxidation of proline leads to the production of more NH3 than alanine, indicating a functioning glutamate dehydrogenase (EC 1.4.1.2). Studies of mitochondrial extracts confirm the presence of a very active glutamate dehydrogenase, and this enzyme is found to be activated by ADP and inhibited by ATP. These extracts also show high alanine aminotransferase activity (EC 2.6.1.2) and a uniquely active "malic'' enzyme (EC 1.1.1.39). The "malic'' enzyme is activated by succinate and inhibited by ATP and by pyruvate. It is suggested that the input of tricarboxylate-cycle intermediate from proline oxidation is balanced by the formation of pyruvate from malate, and the complete oxidation of the majority of the pyruvate. Studies of the steady-state concentrations of mitochondrial CoASH and CoA thioesters during proline oxidation show a high succinyl (3-carboxypropionyl)-CoA content which falls on activating respiration with ADP. There is a concomitant rise in CoASH. However, the reverse transition, from state-3 to state-4 respiration, causes only very slight changes in acylation. The reasons for this are discussed. Studies of the mitochondrial content of glutamate, 2-oxoglutarate, malate, pyruvate, citrate and isocitrate during the same phases of proline oxidation give results consistent with control at the level of glutamate dehydrogenase and isocitrate dehydrogenase during proline oxidation, with the possibility of further control at "malic'' enzyme. During the oxidation of pyruvate all of the tricarboxylate-cycle intermediates and NAD(P)H follow the pattern of changes described in the blowfly (Johnson & Hansford, 1975; Hansford, 1974) and isocitrate dehydrogenase is identified as the primary site of control.?2OAuthor     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria.

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.     

Identification of a Ca2+-ATPase in brown adipose tissue mitochondria: regulation of thermogenesis by ATP and Ca2+

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca(2+) concentration from 0.05 up to 0.70 mum. It is not known how the rise of Ca(2+) concentration activates BAT thermogenesis. In this report we compared the effects of Ca(2+) in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca(2+)-ATPase bound to the inner membrane of BAT mitochondria. A Ca(2+)-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca(2+) enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca(2+) concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 mum. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca(2+) did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca(2+)-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca(2+) concentrations similar to those noted in the cell during adrenergic stimulation.     

The role of mitochondria in modifying calcium-sensitive cytoplasmic metabolic activities. Modification of pyruvate kinase activity

1. The modification of pyruvate kinase activity in vitro was examined by altering the environmental [Mg(2+)]/[Ca(2+)] ratio with EDTA on the one hand and isolated rat liver mitochondria on the other. 2. Controlled additions of Ca(2+) and EDTA caused pyruvate kinase activity to be alternately and rapidly switched on and off. 3. By being able to accumulate Ca(2+) in preference to Mg(2+) rat liver mitochondria were able to alter the [Mg(2+)]/[Ca(2+)] ratio in the vicinity of pyruvate kinase and thereby modify the activity of this enzyme. 4. The possible role of mitochondria in modifying pyruvate kinase and other ion-sensitive cytoplasmic enzyme activities is discussed.     

Oxidations of various substrates and effects of the inhibitors on purified mitochondria isolated from <Emphasis Type="Italic">Kalanchoë pinnata</Emphasis>

Kalanchoë pinnata mitochondria readily oxidized succinate, malate, NADH, and NADPH at high rates and coupling. The highest respiration rates usually were observed in the presence of succinate. The high rate of malate oxidation was observed at pH 6.8 with thiamine pyrophosphate where both malic enzyme (ME) and pyruvate dehydrogenase were activated. In CAM phase III of K. pinnata mitochondria, both ME and malate dehydrogenase (MDH) simultaneously contributed to metabolism of malate. However, ME played a main function: malate was oxidized via ME to produce pyruvate and CO₂ rather than via MDH to produce oxalacetate (OAA). Cooperative oxidation of two or three substrates was accompanied with the dramatic increase in the total respiration rates. Our results showed that the alternative (Alt) pathway was more active in malate oxidation at pH 6.8 with CoA and NAD⁺ where ME operated and was stimulated, indicating that both ME and Alt pathway were related to malate decarboxylation during the light. In K. pinnata mitochondria, NADH and NADPH oxidations were more sensitive with KCN than that with succinate and malate oxidations, suggesting that these oxidations were engaged to cytochrome pathway rather than to Alt pathway and these capacities would be desirable to supply enough energy for cytosol pyruvate orthophosphate dikinase activity.     

ELECTRON MICROSCOPE HISTOCHEMICAL EVIDENCE FOR A PARTIAL OR TOTAL BLOCK OF THE TRICARBOXYLIC ACID CYCLE IN THE MITOCHONDRIA OF PRESYNAPTIC AXON TERMINALS

Respiration-linked, massive accumulation of Sr²⁺ is used to reveal the coupled oxidation of pyruvate, α-oxoglutarate, succinate, and malate by in situ mitochondria. All of these substrates were actively oxidized in the dendritic and perikaryal mitochondria, but no α-oxoglutarate or succinate utilization could be demonstrated in the mitochondria of the presynaptic axon terminals. A block at an early step of α-oxoglutarate and succinate oxidation is proposed to account for the negative histochemical results, since the positive reaction with pyruvate and malate proves that these mitochondria possess an intact respiratory chain and energy-coupling mechanism essential for Sr²⁺ accumulation. This indicates that the mitochondria in the axon terminals would be able to generate energy for synaptic function with at least some of the respiratory substrates. With regard to the block in the tricarboxylic acid cycle, the oxaloacetate necessary for citrate formation is suggested to be provided by fixation of CO₂ into some of the pyruvate.     

Effect of fatty acids and ketones on the activity of pyruvate dehydrogenase in skeletal-muscle mitochondria.

The presence of palmitoyl-L-carnitine and acetoacetate (separately) decreased flux through pyruvate dehydrogenase in isolated mitochondria from rat hind-limb muscle. The effect of acetoacetate was dependent on the presence of 2-oxoglutarate and Ca2+. Palmitoylcarnitine, but not acetoacetate, also decreased the mitochondrial content of active dephospho-pyruvate dehydrogenase (PDHA). This effect was large only in the presence of EGTA. Addition of Ca2+-EGTA buffers stabilizing pCa values of 6.48 or lower gave near-maximal values of PDHA content, irrespective of the presence of fatty acids or ketones when mitochondria were incubated under the same conditions used for the flux studies, i.e. at low concentrations of pyruvate. There was, however, a minor decrement in PDHA content in response to palmitoylcarnitine oxidation when the substrate was L-glutamate plus L-malate. Measurement of NAD+, NADH, CoA and acetyl-CoA in mitochondrial extracts in general showed decreases in [NAD+]/[NADH] and [CoA]/[acetyl-CoA] ratios in response to the oxidation of palmitoylcarnitine and acetoacetate, providing a mechanism for both decreased PDHA content and feedback inhibition of the enzyme in the PDHA form. However, only changes in [CoA]/[acetyl-CoA] ratio appear to underlie the decreased PDHA content on addition of palmitoylcarnitine when mitochondria are incubated with L-glutamate plus L-malate (and no pyruvate) as substrate. The effect of palmitoylcarnitine oxidation on flux through pyruvate dehydrogenase and on PDHA content is less marked in skeletal-muscle mitochondria than in cardiac-muscle mitochondria. This may reflect the less active oxidation of palmitoylcarnitine by skeletal-muscle mitochondria, as judged by State-3 rates of O2 uptake. In addition, Ca2+ concentration is of even greater significance in pyruvate dehydrogenase interconversion in skeletal-muscle mitochondria than in cardiac-muscle mitochondria.     

The role of mitochondria in modifying the cellular ionic environment. Calcium-induced respiratory activities in mitochondria isolated from various tumour cells

Cyclic stimulation by Ca(2+) of respiration in mitochondria isolated from Ehrlich ascites-tumour cells occurs only when low phosphate concentrations (approx. 0.5mm) are also included in the incubation system. Under these circumstances the extra oxygen consumed is related stoicheiometrically to the amount of Ca(2+) taken up by the mitochondria; the values are similar to those obtained with mitochondria from rat liver in the absence of added phosphate. In contrast with liver mitochondria, up to 280nmol of Ca(2+)/mg of protein can be added to ascites mitochondria without causing any deleterious effect. Respiration in mitochondria isolated from the Yoshida ascites hepatoma (HA 130) and from the Morris hepatomas 5123C and 9618A is also stimulated by Ca(2+) in a cyclic manner. However, that in mitochondria from regenerating rat liver responds to Ca(2+) in the same way as those from normal rat liver. ADP-stimulated respiration in mitochondria from Ehrlich ascites tumour cells, but not from rat liver, is inhibited by low amounts of Ca(2+).     

The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.     

Isolation and properties of flight muscle mitochondria of the bumblebee Bombus terrestris (L.)

This report describes the isolation procedure and properties of tightly coupled flight muscle mitochondria of the bumblebee Bombus terrestris (L.). The highest respiratory control index was observed upon oxidation of pyruvate, whereas the highest respiration rates were registered upon oxidation of a combination of the following substrates: pyruvate + malate, pyruvate + proline, or pyruvate + glutamate. The respiration rates upon oxidation of malate, glutamate, glutamate + malate, or succinate were very low. At variance with flight muscle mitochondria of a number of other insects reported earlier, B. terrestris mitochondria did not show high rates of respiration supported by oxidation of proline. The maximal respiration rates were observed upon oxidation of α-glycerophosphate. Bumblebee mitochondria are capable of maintaining high membrane potential in the absence of added respiratory substrates, which was completely dissipated by the addition of rotenone, suggesting high amount of intramitochondrial NAD-linked oxidative substrates. Pyruvate and α-glycerophosphate appear to be the optimal oxidative substrates for maintaining the high rates of oxidative metabolism of the bumblebee mitochondria.