Isolation of mitochondria from embryogenic cultures of Picea abies (L.) Karst. and Abies cephalonica Loud.: characterization of a K ATP + channel

A valuable method to isolate and purify mitochondria from embryonal masses of two coniferous species (Picea abies [L.] Karst. and Abies cephalonica Loud.) is described. Crude mitochondria from both species were shown to be intact, oxygen consuming (with malate plus glutammate, succinate and NADH as substrates) and well coupled (respiratory control ratio ca. 4). The oxidation of the substrates was only partially KCN-insensitive (alternative oxidase) in some cases. However, these fractions were contaminated by membranes (e.g. plasmalemma, tonoplast, Golgi and endoplasmic reticulum). After purification by a discontinuous Percoll gradient (18, 23, 40%, v/v), three mitochondrial populations were separated. The 0/18 interface fraction was composed mainly of broken and uncoupled mitochondria, while the other two (18/23 and 23/40 interface fractions) contained intact and coupled mitochondria, but only 23/40 interface fraction revealed to be better purified starting from both coniferous embryonal masses. In the latter purified fraction, the presence of a cyclosporin A-sensitive K_ATP⁺ channel was demonstrated. These findings were discussed in the light of the potential use of these mitochondrial fractions in bioenergetic studies, or in the involvement of these organelles to stress response in conifers.     

Purification and characterization of the phosphate/hydroxyl ion antiport protein from rat liver mitochondria.

The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.     

Preparation of highly purified mitochondria ofNeurospora crassa on a Percoll gradient

Mitochondria isolated from Neurospora crassa were purified by centrifugation in a Percoll density gradient. Enzyme activities and cytochrome differential spectra revealed a high purity of the mitochondria. As compared with a crude mitochondrial fraction the purified mitochondria exhibited a high respiratory activity and a fine ADP/O ratio. Electrophoresis of nucleic acids demonstrated the absence of cytoplasmic rRNA.     

Galactosyltransferase activities in mitochondrial outer membrane: biosynthesis of dolichylmonophosphate-galactose

Mitochondrial outer membranes were prepared from mouse liver homogenates by swelling purified mitochondria in phosphate buffer and were purified on a discontinuous sucrose gradient. Assays for marker enzymes and controls in electron microscopy confirmed the purity and homogeneity of this subfraction. Mitochondrial outer membranes had significant galactosyltransferase activity when incubated with UDP-[14C]galactose: 14C-labelling was found in products extractable with organic solvents and in a residual precipitate. Addition of exogenous dolichylmonophosphate loaded into phosphatidylcholine liposomes strongly enhanced the incorporation of [14C]galactose into chloroform/methanol (2:1, v/v) -extractable products. Thin-layer chromatography of these 2:1 extracts showed that the increase of [14C]galactose incorporation was attributable to the synthesis of a new galactosylated lipid, 'lipid L'. This 'lipid L' has been purified on silicic acid columns by elution with chloroform/methanol (1:1, v/v). The purified 'lipid L' was labile in acid and released [14C]galactose. It had the same chromatographic behaviour as dolichylmonophosphate-mannose in neutral, acid and alkaline solvent systems. Upon incubation in presence of [3H]dolichylmonophosphate and UDP-[14C]galactose, purified 'lipid L' contained both 3H- and 14C-labelling. 'Lipid L', synthesized by mitochondrial outer membranes, was therefore characterized as dolichylmonophosphate-galactose.     

Preparation and properties of mitochondria from cowpea nodules

Mitochondria were isolated from nodules of cowpea (Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar O₂ uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD⁺-malic enzyme which was 12-fold lower in the mitochondria from nodules.     

Large-scale purification procedure of spinach leaf mitochondria —isolation and immunological studies of the F1–ATPase

A large–scale purification procedure for mitochondria from spinach ( Spinacia oteracea L, cv Medania) leaves is described. It involves differential centrifugation and density gradient centrifugation on a self–generating gradient of Percoll, From 3 kg of spinach leaves, 150 mg mitochondrial protein are obtained. The thylakoid contamination is lower than 0.2% on a chlorophyll basis. The mitochondria oxidize malate and glycine with state 3 rates of 108 and 140 nmol (mg protein)^-1 min^-1, with respiratory control ratios of 2,7 and 3,8 and ADP/O ratios of 2,0 and 2.1, respectively. The present large–scale purification procedure will facilitate further biochemical and molecular biological studies of leaf mitochondrial proteins.
A pure and active catalytic moiety of the F₁–ATPase (EC 3,6,1,3) was purified from the isolated mitochondria. The yield was 5 mg of F₁–ATPase from 150 mg mitochondria. The F₁–ATPase contained five polypeptides of apparent molecular mass 54 kDa (α), 52 kDa (β), 33 kDa (γ), 22 kDa (ω) and 11 kDa (ɛ). An additional component at 24 kDa was present in variable amounts in some preparations and was therefore not ascribed to the ATPase complex. The enzyme catalyzed ATP hydrolysis at a rate of 12.5 nmol (mg protein)^-1 min^-1. Antibodies against the spinach mitochondrial F₁–ATPase cross–reacted only with the a and β subunits of F₁–ATPases of spinach chloroplasts, photosynthetic bacteria Rhodospirillum rubrum and beef heart mitochondria.     

Preparation of leaf mitochondria from Arabidopsis thaliana

Arabidopsis thaliana is, perhaps, the most important model species in modern plant biology. However, the isolation of organelles from leaves of this plant has been difficult. Here, we present two different protocols for the isolation of mitochondria, yielding either highly functional crude mitochondria or highly purified mitochondria. The crude mitochondria were well coupled with the substrates tested (malate + glutamate, glycine and NADH), exhibiting respiratory control ratios of 2.1–3.9. Purified mitochondria with very low levels of chlorophyll contamination were obtained by Percoll gradient centrifugation, yielding 1.2 mg of mitochondrial protein from 50 g of leaves.     

Heterogeneity of Maize Root Mitochondria from Plants Grown in the Presence of Ammonium

Mitochondria isolated from root tissue of maize plants grown on a modified Knop solution containing 10.9 mM nitrate ± 7.2 mM ammonium were purified on the discontinuous Percoll density gradient with polyvinylpyrrolidone (PVP) added. The presence of PVP allowed separation of several mitochondrial fractions of a different density. Contrary to mitochondria isolated from plants grown in the presence of nitrate alone, revealing only two fractions, the mitochondria from NH₄ ⁺/NO₃ ^–-plants were distributed in four fractions. Total amount of mitochondria, as well as specific activities of some nitrogen metabolism enzymes and tricarboxylic acid (TCA) cycle enzymes of all mitochondrial fractions, and respiratory activities of two lower density fractions isolated from plants grown on mixed nitrogen were higher in comparison to mitochondria from nitrate-grown plants.     

Adrenaline activates oxidative phosphorylation of rat liver mitochondria through alpha 1-receptors

We studied the effects and mode of action of epinephrine on the oxidative phosphorylation of rat liver mitochondria. With either succinate or beta-hydroxybutyrate as substrate, i.v. injection of 1.5 microgram/100 g epinephrine increased the respiratory rates by 30-40% in state 3 (with ADP), and by 20-30% in state 4 (after ADP phosphorylation), so that the respiratory control ratio (state 3/state 4) changed little. The respiratory stimulation by epinephrine was maximal 20 minutes after its injection. The action of epinephrine on mitochondria was blocked by pretreatment of the animals with the alpha 1-antagonist prazosin but not by treatment with the beta-antagonist propranolol. I. v. injection of 10 micrograms/100 g phenylephrine evoked the same mitochondrial response as epinephrine. I. v. administration of 50 micrograms/100 g dibutyryl cyclic AMP enhanced glycaemia but did not affect mitochondrial respiration. Epinephrine therefore has an alpha 1-type of action on mitochondrial oxidative phosphorylation.     

Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate-Nitrate Reductase of Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans was purified over 200-fold by use of salt fractionation, gel filtration, and ion-exchange chromatography. The purified enzyme was specific for NADPH and catalyzed reduction of nitrate, cytochrome c from isolated mitochondria of Aspergillus, and mammalian cytochrome c. An S(0.725) (20, w) of 7.8 was derived with sucrose density gradient centrifugation, and a Stokes radius of 6.4 nm was derived by gel filtration on Sephadex G-200. From these values, a molecular weight of 197,000 was computed, assuming v = 0.725 cm(3)/g. The spectral properties of the purified enzyme suggested a flavine component was present but revealed no pattern indicative of a hemoprotein. A cytochrome c, similar to the cytochrome c from isolated mitochondria, was found unassociated with the nitrate reductase after ion-exchange chromatography. No NADPH-nitrate reductase activity was detected in isolated mitochondria. Spectrally discernable reduction of the flavine component of the enzyme at 450 nm was noted after reaction with NADPH. This reduction was inhibited by p-chloromercuribenzoate but not by KCN. The addition of nitrate to NADPH reduced enzyme caused a reoxidation of the flavine component via a reaction which was inhibited by KCN but not by p-chloromercuribenzoate. The half-life of the purified enzyme at 37 C was 20 min for NADPH-nitrate reductase and 35 min for NADPH-cytochrome c reductase.     

Rhodamine 6G, inhibitor of both H-ejections from mitochondria energized with ATP and with respiratory substrates

Rhodamine 6G inhibited ATP hydrolysis by oligomycin-sensitive ATPase, purified from rat liver mitochondria, in good accord with the dose-response curve for its inhibition of energy transduction of ATP synthesis in mitochondria, but it did not inhibit ATP hydrolysis by purified F₁. Rhodamine 6G also inhibited both H⁺-ejections from mitochondria energized with respiratory substrates and with ATP.

The present findings show that the inhibitory effect of rhodamine 6G on energy transduction is not due to a modification of the transport system for adenine nucleotides, P_i, and respiratory substrates, and that the inhibition sites of rhodamine 6G are on components related with H⁺-ejection by redox components and also on F₀.     

Mitochondria from Zea mays leaf tissues: Differentiation of carbon assimilation and photorespiratory activity between mesophyll and bundle sheath cells

A procedure was developed to obtain intact and purified mitochondria from mesophyll and bundle sheath tissues of Zea mays L. cv. I.N.R.A. 180, an NADP⁺-malic enzyme type C₄ plant. There was little cross-contamination between the two mitochondrial fractions.
Both types of mitochondria oxidized NADH, succinate and malate with respiratory control. In mesophyll mitochondria malate oxidation was highly sensitive to KCN (85–90% inhibition of first state 3) and showed good respiratory control. In bundle sheath mitochondria malate oxidation was less sensitive to cyanide (75-80% inhibition) and showed poor respiratory control. Malate and NADH appeared to be the best substrates for respiratory activity. Mesophyil mitochondria could not oxidize glycine, whereas bundle sheath mitochondria could.
The results indicate that mesophyll and bundle sheath mitochondria of Zea mays are differentiated, not only with respect to the decarboxylation of malate but also with respect to the decarboxylation phase of photorespiration.     

脱辅基细胞色素c定向于线粒体的转运特异性研究

用分离纯化的完整线粒体和部分细胞器组分,初步研究了脱辅基细胞色素c在细胞内转运的特异性。完整线粒体用差速离心和密度梯度离心的方法,从幼龄鸡心肌组织中获得,对胞内几种细胞器标志酶比活力的测量表明,纯化的线粒体单胺氧化酶活力提高25.6倍,腺苷酸激酶活力提高3.59倍,细胞色素c氧化酶活力提高5.48倍,外膜完整性达90%以上,呼吸控制率大于20。以上数据表明该纯化的线粒体受胞内其它囊泡成分污染少,外膜完整并具有较高的氧化磷酸化偶联效率;在纯化线粒体的同时,得到另两种细胞器组分-内质网和溶酶体囊泡。体外转录翻译的apo.c与上述几个组分的结合实验表明,完整线粒体与apo.c的结合能力明显高于其它组分。     

Properties of substantially chlorophyll-free pea leaf mitochondria prepared by sucrose density gradient separation

Mitochondria isolated from pea leaves (Pisum sativum L. var Massey Gem) and purified on a linear sucrose density gradient were substantially free of contamination by Chl and peroxisomes. They showed high respiratory rates and good respiratory control and ADP/O ratios. Malate, glutamate, succinate, glycine, pyruvate, α-ketoglutarate, NADH, and NADPH were oxidized but little or no oxidation of citrate, isocitrate, or proline was detected. The oxidation of NADPH by the purified mitochondria did not occur via a transhydrogenase or phosphatase converting it to NADH. NADPH oxidation had an absolute requirement for added Ca²⁺, whereas NADH oxidation proceeded in its absence. In addition, oxidation of the two substrates showed different sensitivities to chelators and sulfhydryl reagents, and faster rates of O₂ uptake were observed with both substrates than with either alone. This indicates that the NADPH dehydrogenase is distinct from the exogenous NADH dehydrogenase.     

Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates

Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P)H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be -171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F(1) complex, α-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be -291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H(2)O(2) challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.     

Liposomal and Mitochondrial Cytochrome Oxidase Display Similar Bioenergetic Properties

Abstract

Cytochrome c oxidase, the terminal electron acceptor of the respiratory chain of mitochondria, is an integral membrane protein. The bioenergetic properties of cytochrome oxidase can be studied only when the macromolecule is inserted in a phospholipid bilayer, either in situ or after reconstitution into liposomal membranes. Reintegration of purified cytochrome oxidase in liposomes allows quantitative tests of mechanistic hypothesis concerning the functional properties of the enzyme. Small unilamellar vesicles are prepared by sonication of purified soybean asolectin, and reconstitution of cytochrome oxidase in the bilayer is carried out according to the cholate/dialysis procedure. The proteoliposomes are shown to mimick the mitochondrial state of the enzyme in so far as liposomal cytochrome oxidase : a) displays the same vectorial orientation, the cytochrome c binding site being externally exposed, b) pumps protons in the physiological inside/outside direction, and c) is functionally controlled by the transmembrane electrochemical gradient, i.e. displays respiratory control.     

Expression of Classical Mitochondrial Respiratory Responses in Homogenates of Rat Forebrain

Respiratory studies of brain mitochondria have, in general, been limited to purified preparations. Conventional procedures for mitochondrial isolation yield relatively small and potentially selected subfractions of mitochondria. Examination of respiratory responses of homogenates of rat forebrain indicated that key respiratory properties of mitochondria are fully expressed in these preparations. In a high K+ buffer, comparable to those commonly used for purified mitochondria, forebrain homogenates exhibited many of the characteristics of oxygen uptake by "free" mitochondria: requirement for both pyruvate and malate for maximal respiration, stimulation (over threefold) by ADP, stimulation by uncoupling agent [carbonyl cyanide m-chlorophenylhydrazone (CCCP)], but little effect of digitonin. In a modified Krebs-Ringer phosphate buffer (a physiological buffer), respiratory responses were primarily due to mitochondria enclosed in synaptosomes: respiration with glucose was markedly stimulated by CCCP, further stimulated by pyruvate, and extensively inhibited by digitonin (which disrupts the cholesterol-rich synaptosomal membranes). Studies with purified mitochondria and synaptosomes supported the specificity of these responses. These data indicate that classical mitochondrial responses are expressed in whole brain homogenates and, under appropriate conditions, provide functional measures of the total pools of free and synaptosomal mitochondria.     

Mitochondria produce reactive nitrogen species via an arginine-independent pathway

We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS.     

Density Gradient Study of Victorin-Binding Proteins in Oat (Avena sativa) Cells

Victorin-binding proteins (VBPs) in oat (Avena sativa) cells were identified using native victorin and anti-victorin polyclonal antibodies. Homogenates of oat tissues were fractionated in continuous or discontinuous sucrose density gradients or with an aqueous two-phase method, and covalent binding sites of victorin were detected by western blotting. In a 20 to 45% (w/w) sucrose continuous density gradient, the 100-kD VBP was located in fractions of 37 to 44% sucrose, with a peak at 39% sucrose. Based on marker enzyme assays, plasma membranes peaked at 39 to 41% sucrose, mitochondria peaked at 41%, but Golgi and endoplasmic reticulum were in lower density fractions, peaking at 28 to 29% and 22 to 24% sucrose, respectively. The 100-kD VBP was not found in plasma membranes purified by the aqueous two-phase method or in mitochondria purified by discontinuous density gradient centrifugation. Victorin binding to 65- and 45-kD proteins was detected in all fractions in the continuous sucrose density gradients. The 65- and 45-kD proteins were both detected in purified plasma membranes, but only the 65-kD protein was detected in purified mitochondria. The subcellular location of VBPs was the same in sensitive and resistant oat cells.     

Mitochondrial development in liver of foetal and newborn rats

THE DEVELOPMENT OF THE INNER MITOCHONDRIAL MEMBRANE IN FOETAL AND NEONATAL RAT LIVER WAS STUDIED BY FOLLOWING THREE PARAMETERS: (1) the activity of several respiratory enzymes in homogenates and purified mitochondria, (2) the spectrophotometric determination of cytochrome content in the mitochondria and (3) the cardiolipin content in both homogenates and purified mitochondria. Respiratory-enzyme activities of homogenates of foetal liver were one-quarter to one-twentieth of those of homogenates of adult liver, and the enzyme specific activities in purified mitochondria from foetal liver were one-half to one-eighth of those in mitochondria from adult liver. The cardiolipin content of liver homogenates increased approximately twofold during the development period, but there was no significant change in the cardiolipin content of purified mitochondria. It is concluded that cell mitochondrial content approximately doubles in the immediate postnatal period. There was no evidence for an increase in the relative amount of cristae protein in mitochondria during this period to account for increases in mitochondrial enzyme specific activity, since cardiolipin and cytochrome concentrations remained unchanged and electron micrographs revealed no differences. The cause of the lower respiratory-enzyme specific activity in foetal liver mitochondria is unclear. Qualitative differences in respiratory units in foetal and mature animals are suggested.