Increased content of natural ATPase inhibitor in tumor mitochondria

The ATPase activity of Zajdela hepatoma and Yoshida sarcoma submitochondrial particles was several times lower than the enzyme activity in rat heart and rat liver submitochondrial particles. The content of F1-ATPase in the tumor mitochondria was found not to be very different from that in mitochondria of rat liver. Immunochemical determination of the amount of the natural ATPase inhibitor revealed that the tumor mitochondria contain 2-3-times more ATPase inhibitor than control mitochondria. It is concluded that the low ATPase activity of the tumor mitochondria results from the inhibition of the enzyme activity by the natural ATPase inhibitor.     

Succinate dehydrogenase activity of isolated myocardial mitochondria during chronic insufficiency]

Succinic dehydrogenase (SDH) activity of the isolated mitochondria myocardial cells in chronic cardiac insufficiency was studied experimentally. The highest SDH values were found in the mitochondria of condensed type. The activity of the enzyme was low in the ordinary (orthodox) mitochondria. The medium SDH activity was registered in the intermediary type of mitochondria. The ordinary and the intermediary types of mitochondria prevailed in the fraction under study reflecting a fall of the SDH activity in the myocardial tissue. Biochemical study of the mitochondrial fraction revealed a slight dissociation between the tissue respiration and the oxidative phosphorilation. The low SDH activity values seen to indicatate the state of overstrain of the energy-producing structures.     

Anisakis simplex: uncoupling of oxidative phosphorylation in the muscle mitochondria of infected fish

Adenosine triphosphatase activity stimulated by Mg2+ was greater in muscle mitochondria of fish infected with larval Anisakis simplex nematodes than in uninfected fish. When muscle mitochondria were isolated in a sucrose ethylene-glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid medium from fresh uninfected fish, they were loosely coupled, and their adenosine triphosphatase activity was comparable to that of mitochondria from rat tissue. Activity in infected fish was dose dependent, increasing with the number of worms per fish. Excretory secretory products or a cytoplasmic fraction of anisakines, when incubated with coupled rat mitochondria, also caused adenosine triphosphatase activity to increase. Storage of fish flesh caused an increase in adenosine triphosphatase activity, but such aging was not significant until 5 and 10 days after death in refrigerated and frozen samples, respectively. The Mg2+ stimulated adenosine triphosphatase activity of muscle mitochondria can be used to estimate the number of nematodes per market fish. The type of medium used to isolate the mitochondria is crucial in such studies; an ionic medium with Nagarse proteinase was optimal for fish muscle mitochondria.     

Inhibition by secalonic acid D of oxidative phosphorylation and Ca2+-induced swelling in mitochondria isolated from rat livers

The in vitro biological activity of secalonic acid D, a mycotoxin from Aspergillus ochraceus, was studied to assess its cytotoxicity for isolated rat liver mitochondria. Secalonic acid D uncoupled the oxidative phosphorylation of mitochondria and caused a mild inhibition of state 3 respiration. Secalonic acid D weakly enhanced latent ATPase activity in mitochondria but suppressed 2,4-dinitrophenol-stimulated ATPase activity. Secalonic acid D did not induce pseudoenergized swelling of mitochondria and markedly inhibited the Ca2+-induced swelling of mitochondria in KCl isotonic solution.     

Inhibition by secalonic acid D of oxidative phosphorylation and Ca2+-induced swelling in mitochondria isolated from rat livers.

The in vitro biological activity of secalonic acid D, a mycotoxin from Aspergillus ochraceus, was studied to assess its cytotoxicity for isolated rat liver mitochondria. Secalonic acid D uncoupled the oxidative phosphorylation of mitochondria and caused a mild inhibition of state 3 respiration. Secalonic acid D weakly enhanced latent ATPase activity in mitochondria but suppressed 2,4-dinitrophenol-stimulated ATPase activity. Secalonic acid D did not induce pseudoenergized swelling of mitochondria and markedly inhibited the Ca2+-induced swelling of mitochondria in KCl isotonic solution.     

Further evidence for a separate enzymic entity for the synthesis of homocitrulline, distinct from the regular ornithine transcarbamylase

Differential digitonin extraction of rat liver mitochondria and of mitochondria of livers of affected and unaffected male sparse fur mice released a lysine transcarbamylase activity from the mitochondria at a digitonin to protein ratio in between that for myokinase and glutamate dehydrogenase, but at a slightly lower ratio than the ornithine transcarbamylase activity. Homocitrulline formation by isolated rat liver mitochondria is independent of the uptake of lysine by mitochondria as evidenced by the insensitivity of homocitrulline formation to changes in the matrix pH, in contrast to citrulline formation from ornithine. High-performance liquid chromatography separates the lysine transcarbamylase activity from the ornithine transcarbamylase activity. It is concluded that the lysine transcarbamylase activity is localized outside the inner mitochondrial membrane.     

Cytoprotective activity of minocycline includes improvement of mitochondrial coupling: the importance of minocycline concentration and the presence of VDAC

Available data indicate that minocycline, an antibiotic of the tetracycline family, has cytoprotective properties due to a direct interaction with mitochondria. Yet, the data in the case of isolated mitochondria suggest discrepant or even detrimental effect(s) of the interaction. We have studied the cytoprotective activity displayed by minocycline in the case of the yeast Saccharomyces cerevisiae cells pretreated with H?O?. We demonstrated that the activity of minocycline required the presence of VDAC (voltage-dependent anion-selective channel) and provided distinct improvement of mitochondrial coupling. In the case of isolated mitochondria, we verified that minocycline exhibited uncoupler activity when applied in micromolar concentrations. However, when added in nanomolar concentrations, minocycline was able to improve the level of coupling for isolated mitochondria. The coupling improvement effect was observed in mitochondria containing VDAC but not in Δpor1 mitochondria (depleted of VDAC1, termed here VDAC) and in both types of mitoplasts. Thus, properly low concentrations of minocycline within the cell in the vicinity of VDAC-containing mitochondria enable the improvement of energy coupling of mitochondria that contributes to cytoprotective activity of minocycline.     

Some aspects of fatty acid oxidation in isolated fat-cell mitochondria from rat.

Mitochondrial were prepared from fat-cells isolated from rat epididymal adipose tissues of fed and 48 h-starved rats to study some aspects of fatty acid oxidation in this tissue. The data were compared with values obtained in parallel experiments with liver mitochondria that were prepared and incubated under identical conditions. 2. In the presence of malonate, fluorocitrate and arsenite, malate, but not pyruvate-bicarbonate, facilitated palmitoyl-group oxidation in both types of mitochondria. In the presence of malate, fat-cell mitochondria exhibited slightly higher rates of palmitoylcarnitine oxidation than liver. Rates of octanoylcarnitine oxidation were similar in liver and fat-cell mitochondria. Uncoupling stimulated acylcarnitine oxidation in liver, but not in fat-cell mitochondria. Oxidation of palmitoyl- and octanoyl-carnitine was partially additive in fat-cell but not in liver mitochondria. Starvation for 48 h significantly decreased both palmitoylcarnitine oxidation and latent carnitine palmitoyltransferase activity in fat-cell mitochondria. Starvation increased latent carnitine palmitoyltransferase activity in liver mitochondria but did not alter palmitoylcarnitine oxidation. These results suggested that palmitoylcarnitine oxidation in fat-cell but not in liver mitochondria may be limited by carnitine palmitoyltransferase 2 activity. 3. Fat-cell mitochondria also differed from liver mitochondria in exhibiting considerably lower rates of carnitine-dependent oxidation of palmitoyl-CoA or palmitate, suggesting that carnitine palmitoyltransferase 1 activity may severely rate-limit palmitoyl-CoA oxidation in adipose tissue.     

Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.     

Increased ATP-hydrolase activity of the brain mitochondria under the effect of aminazine (electron cytochemical study)]

The localization of Mg-stimulated ATPase activity was determined in the sensomotor cortex mitochondria of Wag rats after aminazin administration (15 mg/kg). Apart from normal mitochondria containing no reaction product, a number of altered mitochondria with different localization in them of the ATPase reaction product were recorded. The intact animals showed no sediment in the mitochondria. It is suggested that aminazin-induced increase in ATP-hydrolase activity is caused by two factors: neuroleptic-induced decrease in glycolytic and oxidative cell activity and increased permeability of mitochondrial membranes.     

3-Hydroxybutyrate dehydrogenase in the liver mitochondria of rats and chickens

The activity of mitochondrial 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in rat and chicken liver was found to be comparable with the activity of electron transport chain of rat liver mitochondria. This activity is absent in chicken liver mitochondria, which are devoid of the 3-hydroxybutyrate oxidase activity. Both types of mitochondria have nearly identical respiration parameters but respond differently to Mg2+. It was assumed that chicken liver mitochondria are characterized by a low rate of fatty acids oxidation due to the absence of 3-hydroxybutyrate dehydrogenase in these organelles.     

Effects of Sclerin on Energy-linked Functions and Phospholipase Activity in Mitochondria Isolated from Rat Liver and Some Plants

Sclerin (SCL) not only elevated the respiratory control ratio and ADP/O ratio in mitochondria isolated from rat liver and some plants, but was effective in maintaining the energy-linked functions in these mitochondria during aging. There was a close relationship in the effect of SCL between the liberation of fatty acid and maintenance of the energy-linked functions in mitochondria during aging. The liberation of fatty acid was mainly due to the digestion of mitochondrial phospholipids by endogenous phospholipase. SCL had no effect on the activity of phospholipase and rather raised the level of endogenous phospholipase in mitochondria during aging at 30°C. The activity of phospholipase in mitochondria was inhibited by ATP, but stimulated by DNP. It was supposed that SCL inhibits the activity of phospholipase through ATP or high-energy intermediates which is maintained in mitochondria during aging. SCL had a protective effect on the activity of DNP-activated ATPase in mitochondria stored in the cold, and, at a very low concentration, stimulated the ATP-driven NAD reduction by mitochondria.     

A method of determining 2-oxoglutarate dehydrogenase activity in intact mitochondria

A rather simple method is suggested for measuring the activity of 2-oxoglutarate dehydrogenase of intact mitochondria. The method is based on the determination of the rate of exogenic 2-oxoglutarate decrease in the mitochondrial suspension. Experiments with sodium arsenite and comparison of kinetic parameters of the 2-oxoglutarate, dehydrogenase reaction and transport of 2-oxoglutarate to mitochondria have shown that the measurable exogenic 2-oxoglutarate oxidation rate corresponds to the 2-oxoglutarate dehydrogenase activity in intact mitochondria. The method made it possible to establish the stimulating effect of ADP on the 2-oxoglutarate dehydrogenase activity of intact mitochondria and the absence of such an effect in destructed mitochondria.     

Cytochrome c oxidase activity in mitochondria of cardiomyocytes from isolated cardiac tissue incubated under long-term hypoxic conditions

Electron microscopic histochemistry based upon the oxidative polymerization of 3,3′-diaminobenzidine (DAB) was applied to identify cytochrome c oxidase activity. We found that the incubation of isolated small pieces of cardiac tissue over 72 h under hypoxic conditions caused changes in the mitochondrial ultrastructure and disorders in the functional activity of mitochondria, particularly in the IV complex of respirator chain. Small, electron-dense mitochondria appeared inside electron-light mitochondria (“mitochondria inside mitochondria”) stained positively for cytochrome c oxidase activity along the full length of crista. The results are discussed in connection with the concept of intracellular regeneration and mitochondria structural transformations during mitoptosis.     

Regulation of palmitoyl-CoA inhibition of mitochondrial adenine nucleotide transport by cytosolic fatty acid binding protein.

Defatted liver fatty acid binding protein (FABP) reverses the inhibitory effect of palmitoyl-CoA on adenine nucleotide transport in rat liver mitochondria; addition of titrating amounts of FABP to mitochondria pretreated with palmitoyl-CoA stimulates nucleotide transport and that activation parallels the removal of the inhibitor from mitochondria. This effect is specific only for FABP; all other cytosolic proteins which do not bind fatty acids do not influence nucleotide transport activity. Addition of free fatty acids (which can compete for ligand binding sites on FABP) to mitochondria pretreated with palmitoyl-CoA interferes with the reversal activity of FABP. Adding FABP alone to freshly isolated mitochondria also activates nucleotide transport activity suggesting that the originally submaximal activity is probably due to the presence of endogenous long-chain acyl-CoA esters in the mitochondrial preparation. Because FABP is present in relatively high concentration in most mammalian cells, these observations offer a likely explanation of why the potent inhibitory effects of long-chain acyl-CoA esters on adenine nucleotide transport in isolated mitochondria are not seen in the intact cell.     

The ultrahistochemical picture of the so-called reversed ATPase in the gastrocnemius muscle of the rat.

The ultrahistochemical localization of the "reversed" ATPase activity was investigated. Red muscle fibres showed permanent sarcomere contraction, enzymatic activity in the inner membrane and matrix of mitochondria, and large, osmiophilic, probably calcium-containing structures within mitochondria and on their outside. White muscle fibre sarcomeres were relaxed, and activity within their sarcoplasmic reticulum was marked, but slight in the mitochondria. The relaxed state of the sarcomere in the white muscle fibres is supposed to be connected with inactivation of myofibrillar ATPase by acid preincubation, whereas red muscle contraction indicates that acid preincubation does not inactivate their myofibrillar ATPase. That the product of its activity failed to become visible in the sarcomeres is probably due to imperfection of the method. Two sub-types of red muscle fibres were distinguished: those showing only enzymatic activity in mitochondria, and those containing large intra- and extramitochondrial osmiophilic structures. The origin and composition of these structures is difficult to explain. A relation seems to exist between their presence within mitochondria and outside.     

nHAP对大鼠肝线粒体的生物活性作用

为了探讨羟基磷灰石纳米粒子(nHAP)对大鼠肝线粒体生物活性的影响,将nHAP直接作用于线粒体,在不同浓度和时间下测定线粒体标志酶琥珀酸脱氢酶(SDH)比活性,并与对照组进行比较。结果显示,当nHAP中水含量在10%以下时,线粒体生物活性未发现改变;当nHAP浓度递增时,在等时间段内,对线粒体SDH比活性呈逐步抑制作用;在不等时间段内,nHAP对线粒体SDH比活性的抑制作用与对照组相比较差异有显著性(p<0.05)。因此,nHAP对线粒体SDH比活性的抑制有浓度和时间的依赖性。     

A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver.

The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.     

Regulation of Alternative Oxidase Activity by Pyruvate in Soybean Mitochondria

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.     

Formation and utilization of 3-hydroxy-3-methylglutarate in liver mitochondria of starved and streptozotocin-diabetic rats.

Kidney and liver mitochondria of rat, rabbit and guinea pig are able to transform 3-hydroxy-3-methylglutarate into acetoacetate, whereas ox liver mitochondria and rat mitochondria of heart, diaphragm and brain do not exhibit such an activity. Starvation and streptozotocin treatment decreases the formation of acetoacetate from 3-hydroxy-3-methylglutarate. Addition of acetoacetate and succinate to the incubation media of mitochondria results in a decrease in the transformation of 3-hydroxy-3-methylglutarate into acetoacetate. A 3-hydroxy-3-methylglutaryl-CoA hydrolase is present in rat liver mitochondria; the activity does not show appreciable changes after starvation or streptozotocin treatment.