Effect of Sclerin on Amino Acid Incorporation into Mitochondria Isolated from Rat Liver

Though sclerin (SCL) stimulated amino acid incorporation into the protein fraction of post mitochondrial supernatant of rat liver homogenate, it had no effect on the incorporation into the isolated mitochondria at pH 7.2, despite of its stimulating effect on mitochondrial oxidative phosphorylation. SCL stimulated amino acid incorporation into the mitochondria at pH 6.1, and to some extent maintained the activity on that in mitochondria during aging in hypotonic Tris-HCl buffer (pH 7.2). Since SCL prevented leakage of amino acids from the mitochondria into these buffers, it was suggested that SCL may protect a structure of mitochondrial membrane which appeared to have a significance on transport of amino acids. In liver slices, SCL stimulated amino acid incorporation only into the extra-mitochondrial fraction for the first 3 min, but gradually turned to stimulate incorporation into mitochondria within 30 min.     

Effect of Sclerin on Incorporation of Oleic Acid into Phospholipids and Activity of Phospholipase in Mitochondria

Sclerin (SCL) stimulated the oxidation and the incorporation into the phospholipids of Na-[1-¹⁴C]-oleate in mitochondria isolated from rat liver, preventing the depression of the phosphorylating functions and protecting 2,4-dinitrophenol (DNP)-activated ATPase in mitochondria during incubation with oleate. Also, SCL markedly enhanced the activity of phospholipase to hydrolyze endogenous substrates in mitochondria. The increase in the activity was due to reconstruction of phospholipids through esterification of oleate in mitochondrial membrane, but not to the de novo enzyme synthesis. It was concluded that the level of endogenous phospholipase in mitochondria during incubation reflects the energy- dependent reacylation of the lysophospholipids produced by the action of phospholipase in mitochondrial membrane.     

Mitochondria Isolated from Rat Brown Adipose Tissue and Liver

Mitochondrial fractions, relatively free from contamination by other cytoplasmic structures, have been isolated by differential centrifugation from homogenates of brown adipose tissue from starved rats. It was possible in such fractions to distinguish two types of mitochondria in this tissue. Type I mitochondria, when morphologically intact, are limited by a bilaminar membrane and show regular parallel cristae. In isolated fractions, a proportion of these mitochondria are swollen, vacuolation occurring within the cristae between their limiting membranes. Type II mitochondria are distinguished from the more numerous type I bodies by the opaque appearance of their matrix. They are limited by a membrane which is in part single, and in part double. They show a few, but crisply outlined internal membranes. Vacuolation of this type of mitochondrion has not been observed. Vacuolation comparable to that in brown fat mitochondria was also observed between the two laminae of the enclosing membrane and within the cristae of liver mitochondria.     

Monoamine Oxidase Activity of Mitochondria Prepared from Rat Liver and Rat Heart

Abstract: The effect of agents that change the respiratory state of the mitochondrion on tyramine oxidation was investigated. Neither uncoupler nor ADP and P_t in the presence of substrate produced any change in the rate of tyramine oxidation, as judged by direct measurement of tyramine oxidation or by H₂O₂ production. We conclude that previously reported depression of monoamine oxidase activity by stimulated respiration was due to oxygen depletion.     

Energy-linked Functions of Submitochondrial Particles Prepared from Mung Bean Mitochondria

Submitochondrial particles from mung bean mitochondria (Phaseolus aureus) are able to catalyze an energy-linked reduced nicotinamide adenine dinucleotide-nicotinamide adenine dinucleotide phosphate transhydrogenase reaction supported by ATP or by aerobically generated high energy intermediates. The energy transfer pathway appears to differ from that utilized for oxidative phosphorylation.     

Bivalent Metal Ions Modulate Cd2+ Effects on Isolated Rat Liver Mitochondria

We have studied Cd²⁺-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd²⁺] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 M, Cd²⁺ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 M, Cd²⁺ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO₃ or NH₄NO₃ medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd²⁺ effects on mitochondria treated in sequence with 100 M of Ca²⁺, Sr²⁺, Mn²⁺ or Ba²⁺(Me²⁺) and 7.5 M RR, as well as the alterations in Cd²⁺ action on the uptake of ¹³⁷Cs⁺ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca²⁺ or RR. The evidence obtained indicate that Ca²⁺ exhibits a synergestic action on all Cd²⁺ effects examined, whereas Sr²⁺ and Mn²⁺, conversely, are antagonistic. In the presence of RR, the Cd²⁺ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd²⁺, conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd²⁺-stimulated swelling and ¹³⁷Cs⁺ (analog of K⁺) uptake in the acetate media. For the first time, we have shown that Cd²⁺-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.     

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。     

Effect of Synthetic Detergents on the Swelling and the ATPase of Mitochondria Isolated from Rat Liver

A study was made of the effects of various types of detergents on the swelling of isolated mitochondria and on mitochondrial ATPases which are activated by Mg or DNP respectively. The rate of swelling was measured in the Beckman spectrophotometer by following the decrease in turbidity of dilute suspensions of these organelles. It was found that non-ionic detergents containing a nonyl phenoxy side chain or anionic detergents caused swelling of the mitochondria and activation of Mg-ATPase. On the other hand, cationic detergents promoted the clumping of mitochondria and did not activate Mg-ATPase. DNP-ATPase was inhibited by all of the detergents tested. It would appear from these observations that the inhibition of DNP-ATPase is not related to a gross change in the morphology of the organelles; in contrast, the activation of Mg-ATPase definitely is correlated with swelling of the isolated mitochondria. These data also suggest that the ionic detergents combine with charged sites on the protein moiety of the lipoprotein in the mitochondrial surface, whereas the non-ionic detergents form inclusion compounds with the lipide moiety, thereby altering the mitochondrial structure and permeability.     

Flow Cytometric Analysis of the Effects of High Protein Diet On Isolated Rat Liver Mitochondria

Abstract. We have investigated changes that occur in mitochondria obtained from the livers of rats that had been maintained on a high protein diet (80% casein instead of 20%) for 6 months. Liver homogenates were separated by centrifugation into a mitochondrial fraction, a nuclear fraction and the supernatant fluid of the nuclear fraction (nuclear wash). Rhodamine-123 was used to selectively stain mitochondria depending upon their membrane. potential. the stained organelles were processed through a flow cytometer where the fluorescent stains were excited by the 488 nm wavelength of a laser and the resultant fluorescence signals analysed. After 6 months on a high protein diet, mitochondria displayed an increase in the fluorescence associated with rhodamine-123 uptake in both mitochondrial and nuclear wash fractions, while mitochondrial fluorescence in the nuclear fraction showed a heterogeneous distribution. This was interpreted as an increase in membrane potential in most of the liver mitochondria under these nutritional conditions, with a certain degree of heterogeneity. These functional changes may be correlated with morphological alterations previously reported and show the usefulness of flow cytometry for biochemical analysis of isolated mitochondria.     

大鼠急性应激时肝线粒体质子跨膜转运活性的调控

大鼠烫伤早期(烫伤后30min),肝线粒体质子和电子传递速度均加快,线粒体能化态跨膜电位降低(均以琥珀酸为底物),线粒体膜脂流动性降低。皮下注射去甲肾上腺素后也有上述现象发生。推测急性应激通过儿茶酚胺类作用于肝细胞,导致线粒体内膜有序性增强所致。     

Microcalorimetric Investigation on Effects of LaIII on Metabolic Activity of Mitochondria Isolated from Hybrid Rice

Rare earth elements (REEs) have beneficial influence on plant growth and are widely used in agriculture practice, but little is known about the behavior of the REEs in mitochondria of plant cell. Thermogenic metabolic curves were determined by the ampoule method at 303 K using a TAM air isothermal microcalorimeter in mitochondria isolated from hybrid rice Liangyoupeijiu (Oryza sativa L.), and the effect of La^III on its mitochondrial metabolism was investigated. From the obtained heat flux curves, the crucial parameters including as activity recovery rate constant (k) and maximum heat power (P_max) were determined. Application of La³⁺ in concentrations ranging from 0 to 120 μg/ml significantly increased k and P_max values with the high point reaching 346 and 222% of the control, respectively. Concentrations from 140–150 μg/ml had the opposite effect. These results were consistent with previous reports on the effects of REEs on plant growth. It was concluded that the La‐induced change of mitochondrial metabolic activity is a possible mechanism by which La^III ions influences hybrid rice growth.     

Changes in Properties of Barley Leaf Mitochondria Isolated from NaCl-Treated Plants

Treatment of barley (Hordeum vulgare) seedlings with 400 millimolar NaCl for 3 days resulted in a reduction in plant growth and an increase in the leaf content in ions (K⁺ + Na⁺) and proline. Purified mitochondria were successfully isolated from barley leaves. Good oxidative and phosphorylative properties were observed with malate as substrate. Malate-dependent electron transport was found to be only partly inhibited by cyanide, the remaining oxygen uptake being SHAM sensitive. The properties of mitochondria from NaCl-treated barley were modified. The efficiency of phosphorylation was diminished with only a slight decrease in the oxidation rates. In both isolated mitochondria and whole leaf tissue of treated plants, the lower respiration rate was due to a lower cytochrome pathway activity. In mitochondria, the activity of the alternative pathway was not modified by salt treatment, whereas this activity was increased in whole leaf tissue. The possible participation of the alternative pathway in response to salt stress will be discussed.     

大鼠肝线粒体中的脂肪酸分析

用双 2 乙基己基酚酞酸酯 (DEHP)诱导大鼠肝过氧化物酶体增殖 ,然后用蔗糖密度梯度离心法分离大鼠肝线粒体 ,用毛细管气相色谱法测定肝线粒体中的脂肪酸含量。测定结果 :所测 1 4种脂肪酸的总量 ,青年正常组大于青年诱导组 (P <0 .0 1 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。不饱和脂肪酸与脂肪酸总量的比例 ,老年诱导组大于老年正常组 (P <0 .0 5 ) ,青年正常组大于老年正常组 (P <0 .0 5 )。长链脂肪酸与脂肪酸总量的比例 ,老年正常组小于老年诱导组 (P <0 .0 5 )。结果表明 ,用DEHP诱导大鼠肝过氧化物酶体增值 ,影响肝线粒体脂肪酸正常代谢 ,使线粒体膜结构发生变化 ,这种变化 ,青年鼠与老年鼠不同     

Effects of Insect-Growth-Regulatory Benzimidazole Derivatives on Cultured Integument of the Rice Stem Borer and Mitochondria from Rat Liver

The larvicidal activities of benzimidazole derivatives with a terpenoid side chain on the rice stem borer and the silkworm were compared with such in vitro activities as the growth inhibition of the cultured integument of the rice stem borer and the respiration inhibition of rat liver mitochondria. Each larvicidal activity is parallel with these in vitro activities. The comparisons of their activities with those of rotenone and diflubenzuron indicate that the benzimidazoles mainly acted as respiration inhibitors in their larvicidal activity as well as causing cuticular growth inhibition. The activity of 1-(3,7-dimethyl-7-ethoxy-2-octenyl)-2-methylbenzimidazole, the most potent compound tested as a respiration inhibitor, was found to be about 6-fold higher than that of rotenone. In the respiratory chain, the site between NADH and ubiquinone was blocked, indicating that the larvicidal benzimidazoles shared a mode of action with those of rotenone, piericidins, and ubicidines.     

Some Biochemical Properties of Mitochondria Isolated from Euglena gracilis*

SYNOPSIS. Mitochondria were isolated from Euglena gracilis strain Z by pressure-breakage of the cells and sucrose-cushion centrifugation. Multiple peaks (2-4) were observed in the rate of phosphorylation with Mg-ADP-phosphate concentration curves. The phosphorylative and oxidative activities were highest with NADH as the substrate, moderate with succinate, and lowest with glutamate. Inhibition of phosphorylation with 2,4-dinitrophenol and carbonyl cyanide, m-chlorophenylhydrazone gave sigmoidal concentration curves, with the extent of inhibition by DNP depending on the substrate used. Inhibition of phosphorylation by valinomycin, atractyloside, or carboxyatractyloside was only ～ 60%. Oligomycin inhibited phosphorylation in 2 phases at low and high concentrations; it inhibited Mg-ATPase in a sigmoidal fashion. Both phosphorylation and oxidation had discontinuities in Arrhenius plots at 34 C and 18 C. The relative Mg²⁺-dependent nucleoside triphosphatase activity was: 1 for ATP and GTP, 0.6 for ITP, 0.15 for CTP and and UTP; with Ca²⁺ in place of Mg²⁺ this activity was 0.35. Both DNP and CCCP stimulated the Mg-ATPase 50-200%. The optimal pH for the stimulation was ～ 7 regardless of the uncoupler used, and ～ 8 without the uncouplers. The few differences observed between mitochondria from Euglena and those from other sources are probably due to the fragmentation of the reticular mitochondrial structure during isolation and not to unique characteristics of these mitochondria.     

Photochemical Activity of Chloroplasts Isolated from Etiolated Plants

The rates of phosphorylation, ferricyanide, and dye reductionwere determined with chloroplasts isolated from Linum usitatissimumgrown in darkness and subjected to periods of light of differentduration. With an increased period of illumination, the chlorophyllcontent increased as did also the rate of the three processesmeasured, but no correlation between these two factors was observed.Neither was there any correlation between the rate of any photochemicalreaction and the plastoquinone content. It was concluded thatsome unspecified factor, possibly structural, which developsduring illumination must control the rate of the photochemicalreactions.     

Microcalorimetric Investigation on Metabolic Activity and Effects of La (III) in Mitochondria Isolated from Indica Rice 9311

Thermogenic metabolic curves were determined by the ampoule method at 303 K using a TAM air isothermal microcalorimeter in mitochondria isolated from rice 9311 (Oryza sativa L). From the thermogenic curves the activity recovery rate constant k and the maximum heat power P _m were obtained. Both were positively correlated to the protein content of rice mitochondria. The corresponding correlation coefficients were 0.9959 and 0.9950, respectively, indicating that the in vitro metabolic activity of mitochondria can be reliably expressed by these parameters. Addition of La (III) ions in concentrations ranging from 0 to 130 μg/mL resulted in significantly higher k and P _m values. Concentrations from 140 to 180 μg/mL had the opposite effect. These results are consistent with previous reports on the effects of rare earth elements on plant growth. We propose that the lanthanum-induced change of mitochondrial metabolic activity is a possible mechanism by which La (III) ions influence indica rice 9311 growth.     

Repression of Pyruvate Kinase in Candida utilis and Rat Liver by Sclerin through Energy Charge

Though sclerin (SCL) only slightly inhibited the activity of pyruvate kinase (PK) in crude extract of Candida utilis, markedly repressed the level of that in the growing cells in a glucose medium. The repression of PK was largely recovered by 2,4-dinitrophenol (DNP), and SCL rather raised the level in the cells growing on gluconate.

SCL also slightly inhibited the activity of a partially purified PK from rat liver, and, when orally administered, or incubated with the liver slices, obviously lowered the level of PK in the liver and liver slices. The effect of SCL in the liver slices was reversed by DNP. SCL stimulated the oxidative phosphorylation in mitochondria prepared from the fresh liver, and served to maintain the activity in the liver slices during incubation.

Both activity of PK from Candida utilis and rat liver was remarkably inhibited by adenylate energy charge in vitro.

It is concluded that SCL represses the level of PK in these cells and tissues through a high energy charge by stimulating the oxidative phosphorylation.     

Circadian Rhythms of Oxidative Phosphorylation: Effects of Rotenone and Melatonin on Isolated Rat Brain Mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.