Intact Mitochondrial Preparations Research Articles

A common coupling mechanism for A-type heme-copper oxidases from bacteria to mitochondria

Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome c oxidase (CcO). CcO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H+/e- stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CcO's hydrophobic core and on whether bacterial and mitochondrial CcOs operate via the same coupling mechanism. To resolve this, we exploited the unique amenability to mitochondrial DNA mutagenesis of the yeast Saccharomyces cerevisiae to introduce single point mutations in the hydrophilic pathways of CcO to test function. From adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria, we definitely established that the D-channel, and not the H-channel, is the proton pump of the yeast mitochondrial enzyme, supporting an identical coupling mechanism in all forms of the enzyme.

Muscling in on mitochondrial sexual dimorphism; role of mitochondrial dimorphism in skeletal muscle health and disease.

Mitochondria are no longer solely regarded as the cellular powerhouse; instead, they are now implicated in mediating a wide-range of cellular processes, in the context of health and disease. A recent article in Clinical Science, Ventura-Clapier et al. highlights the role of sexual dimorphism in mitochondrial function in health and disease. However, we feel the authors have overlooked arguably one of the most mitochondria-rich organs in skeletal muscle. Many studies have demonstrated that mitochondria have a central role in mediating the pathogenesis of myopathologies. However, the impact of sexual dimorphism in this context is less clear, with several studies reporting conflicting observations. For instance in ageing studies, a rodent model reported female muscles have higher antioxidant capacity compared with males; in contrast, human studies demonstrate no sex difference in mitochondrial bioenergetics and oxidative damage. These divergent observations highlight the importance of considering models and methods used to examine mitochondrial function, when interpreting these data. The use of either isolated or intact mitochondrial preparations in many studies appears likely to be a source of discord, when comparing many studies. Overall, it is now clear that more research is needed to determine if sexual dimorphism is a contributing factor in the development of myopathologies.

Preparation of intact mitochondria using free-flow isoelectric focusing with post-pH gradient sample injection for morphological, functional and proteomics studies

Mitochondria play essential roles in both energy metabolism and cell signaling, which are critical for cell survival. Although significant efforts have been invested in understanding mitochondrial biology, methods for intact mitochondria preparation are technically challenging and remain to be improved. New methods for heterogeneous mitochondria purification will therefore boost our understanding on their physiological and biophysical properties. Herein, we developed a novel recycling free-flow isoelectric focusing (RFFIEF) with post-pH gradient sample injection (post-PGSI) for preparative separation of mitochondria. Crude mitochondria of rabbit liver obtained from differential centrifugation were purified by the developed method according to their pI values as six fractions. Transmission electron microscope images revealed that intact mitochondria existed in two fractions of pH 6.24 and 6.61, degenerative mitochondria were in two fractions of pH 5.46 and 5.72, and inner membrane vesicles (IMVs) appeared in the fractions of pH 4.70 and 5.04. Membrane potential measurement proved a dramatic difference between intact mitochondria and IMVs, which reflected the bioactivity of obtained populations. Particularly, proteomics analyses revealed that more number of proteins were identified in the intact fractions than that of IMVs or crude mitochondria, which demonstrated that RFFIEF could be powerful tool for the preparation of intact organelle as well as their proteomic and in-depth biological analysis.

Determination of sorbitol in the presence of high amount of mannitol from biological samples

Preparation of intact mitochondria requires the use of an osmoticum in high concentration that preserves the mitochondrial structure and prevents physical swelling and rupture of membranes. For this purpose mannitol is widely accepted as a component of the homogenization medium. The formation of sorbitol in plant mitochondria can be an important element of plant response to salinity and drought. The large difference in the concentration of possibly formed sorbitol and mannitol of the homogenisation medium makes the determination of sorbitol difficult. Gas-chromatography with flame ionization detection (GC-FID), gas-chromatography coupled to quadrupole mass spectrometry (GC-QPMS) and liquid chromatography/tandem mass spectrometry (LC/MS-MS) using hydrophilic interaction chromatography (HILIC) were tested to quantify this highly polar sugar alcohol: Although all three methods offered satisfactory results, the HPLC/MS-MS technique proved to be the best.

Topological Probes of Monoamine Oxidases A and B in Rat Liver Mitochondria: Inhibition by TEMPO-Substituted Pargyline Analogues and Inactivation by Proteolysis

TEMPO-substituted pargyline analogues differentially inhibit recombinant human monoamine oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria, suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane [Upadhyay, A., and Edmondson, D. E. (2009) Biochemistry 48, 3928]. This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the m- and p-amido TEMPO pargylines are not as absolute with the rat enzymes. Similar patterns of reactivity are observed for rat MAO A and B in mitochondrial outer membrane preparations expressed in Pichia pastoris or isolated from rat liver. In intact yeast mitochondria, recombinant rat MAO B is inhibited by the pargyline analogue whereas MAO A activity shows no inhibition. Intact rat liver mitochondria exhibit an inhibition pattern opposite to that observed in yeast where MAO A is inhibited and MAO B activity is unaffected. Protease inactivation studies show specificity in that MAO A is sensitive to trypsin whereas MAO B is sensitive to β-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast upon trypsin treatment and MAO B is readily inactivated by β-chymotrypsin in yeast but not in rat liver. These data show MAO A is oriented on the cytosolic face and MAO B is situated on the surface facing the intermembrane space of the mitochondrial outer membrane in rat liver. The differential mitochondrial outer membrane topology of MAO A and MAO B is relevant to their inhibition by drugs designed to be cardioprotectants or neuroprotectants.

Survey of Fusarium species for plasmid-like DNA and some evidence for its occurrence in a strain of F. merismoides

Twenty-three strains representing thirteen species of Fusarium have been screened for the presence of plasmid-like DNA. Only one strain of F. merismoides showed evidence for the presence of this material, which could be extracted both from whole mycelium and from a preparation of intact mitochondria. It sedimented with mitochondrial DNA in caesium chloride gradients, and neither intercalating agents nor sodium dodecylsulphate were effective in eliminating it from growing mycelium.

Characterization of an RNase P activity from HeLa cell mitochondria. Comparison with the cytosol RNase P activity.

A ribonuclease P-like activity was partially purified from HeLa cell mitochondria by DEAE-cellulose and octyl-Sepharose chromatography. RNase P-like activity can be quantitatively recovered from intact mitochondrial preparations treated with micrococcal nuclease, strongly suggesting that the enzyme is localized within the organelles. Mitochondrial RNase P (mtRNase P) cleaves the precursor to Escherichia coli suppressor tRNATyr at the same site as E. coli RNase P, producing the mature 5'-end of tRNATyr. The sensitivity of mtRNase P to pretreatment with nucleases or Pronase indicates that the enzyme has essential RNA and protein components. Although the ionic requirements of mtRNase P are similar to those of the RNase P activity isolated from the post-mitochondrial cytosol fraction, the chromatographic properties of mtRNase P are distinct. Mitochondrial RNase P is probably a part of the mitochondrial RNA processing machinery of mammalian mitochondria, being responsible for the endonucleolytic cleavage of the RNA transcripts at the 5'-side of the tRNA sequences.

Reye's syndrome: Salicylates and mitochondrial functions

The effects of aspirin (acetylsalicylate, ASA) and related compounds in the presence of Ca 2+ on the oxidative metabolism of isolated rat liver mitochondria were studied. Intact mitochondrial preparations preincubated with ASA + Ca 2+ exhibited a transient stimulation of the state 4 respiratory rate with NAD +-linked substrates, followed by an inhibition which could not be released by the addition of ADP or uncoupler. Maximum respiratory rates were achieved by subsequent addition of NAD + or succinate. The Ca 2+-transport inhibitors ruthenium red and ethylene glycol-bis-(β-aminoethyl ether) N, N′-tetraacetic acid (EGTA) prevented these effects. Five brands of commercial aspirin were tested and were as effective as purified ASA. Tylenol (acetaminophen) could reproduce these effects only at much higher (≥ 10-fold) concentrations. Other salicyl derivatives showed results qualitatively similar to ASA, with potencies in the order: acid ⋙ASA > alcohol ≥ catechol > amide, salicylate being approximately 10-fold more potent than ASA. The magnitude of the effect seen depended on the Ca 2+ (endogenous + exogenous) and salicylate concentrations/mg mitochondrial protein, and on the length of the preincubation. Added inorganic phosphate was also required. That salicylate + Ca 2+ induces an increase in the permeability of the mitochondrial inner membrane was demonstrated by the observation that 90% of the intramitochondrial NAD(P) + was released into the surrounding medium upon preincubation of intact mitochondria with these agents. Salicylate + Ca 2+ had virtually no effect on respiration with succinate (+ rotenone) as substrate at salicylate concentrations which markedly affected NAD +-linked substrate oxidation. The presence of rotenone in the preincubation mixture prevented the damaging effects of salicylate + Ca 2+ on the mitochondrial membrane, suggesting that the redox state of intramitochondrial pyridine nucleotides can modulate these effects. The results reported here are similar to those reported previously by our laboratory for the effects of Reye's plasma and allantoin + Ca 2+, and indicate that, like these agents, salicylate and salicyl compounds can potentiate the Ca 2+-induced damage to the mitochondrial inner membrane and may be another factor responsible for Reye's syndrome.

Inhibition of mitochondrial aldehyde dehydrogenase by malondialdehyde

Rat liver mitochondria contain an aldehyde dehydrogenase (ALDH, EC 1.2.1.3) with a low K m for acetaldehyde (2 μm) which is susceptible to inhibition by a variety of agents including p-chloromercuribenzoate and arsenite. This low K m ALDH isozyme has been shown to be decreased in activity following CCl 4 treatment in vivo and by lipid peroxidation in vitro. Malondialdehyde, the most studied product of lipid peroxidation, was investigated for possible inhibitory effects on the low K m ALDH for rat liver mitochondria. Malondialdehyde was found to be a potent inhibitor of the enzyme. The enzyme was equally sensitive to inhibition when intact mitochondrial preparations were compared with disrupted mitochondria. The extent of enzyme inhibition was dependent on malondialdehyde concentration and time of incubation. High concentrations of malondialdehyde completely inhibited low K m mitochondrial ALDH activity. Malondialdehyde was found to inhibit the low K m ALDH irreversibly. The rate of ALDH inactivation exhibited two phases, one a rapid rate phase (5–15 s) and a second slower rate phase, both of which showed rates which were dependent on malondialdehyde concentration. These data show that malondialdehyde is a potent, high affinity, irreversible inhibitor of the low K m ALDH of rat liver mitochondria. This ALDH isozyme is considered to be the most important enzyme in acetaldehyde metabolism. Malondialdehyde inhibition of the low K m ALDH may be important since both ethanol and acetaldehyde are thought to stimulate hepatic lipid peroxidation.

Labeling of complex III, with [35S]diazobenzenesulfonate: orientation of this electron transfer segment in the mitochondrial inner membrane.

[34S]Diazobenzenesulfonate has been used to tag the surface-exposed polypeptides of isolated complex III. All nine different component polypeptides were labeled, indicating that each is at least partially exposed on the surface of the isolated, detergent-dispersed complex. Labeling studies were also conducted on the membrane-bound complex. Preparations of intact mitochondria and submitochondrial particles were separately labeled with [35S]diazobenzenesulfonate in order to determine the distribution of the polypeptides of complex III between the outer (cytoplasmic) and inner (matrix) surfaces of the mitochondrial inner membrane, respectively. Polypeptides II and III were the only components labeled in a significant amount in submitochondrial particles (i.e., from the matrix side). Polypeptides III, IV, and VI were heavily labeled in mitochondria (i.e., from the cytoplasmic side). Polypeptides I,II, V, and VII were also labeled in mitochondria but to a much lesser extent. Polypeptides VIII and IX were not significantly labeled from either side of the membrane. The labeling data and information obtained from previous crosslinking studies [Smith, R.J. & Capaldi, R.A. (1977) Biochemistry 16, 2629-2633] are used to derive a picture of the arrangement of complex III in the mitochondrial inner membrane.

Glutamate Dehydrogenase Activity in Roots: Distribution in a Seedling and Storage Root, and the Effects of Red and Far‐red Illuminations

Abstract Pisum seedling and Pastinaca storage roots contained high glutanrate dehydrogenase (GDH) activity in areas of reported rapid growth and high phytoctrome content. A similar distribution was observed for malate dehydrogenase. Freeze‐thawings of mitochondrial preparations from Pisum roots always resulted in increases of GDH specific activity; however, the observed increases were much larger with basal than apical sections. Both intact and freeze‐thawed mitochondrial preparations from seedling roots exhibited increases in GDH activity with time after isolation. In intact mitochondrial preparations from roots of etiolated seedlings, an increase in malate dehydrogenase activity was observed similar to that of GDH activity; however, no increased malate dehydrogenase activity was noted in preparations from light‐grown seedlings.Illuminating Pisum seedlings with far‐red light slowly increased GDH activity in roots over a period of two weeks. Since these observed increases were not due to direct exposure of roots to light, other factors were likely involved.

Carnitine palymityltransferase in neonatal and adult heart and liver mitochondria. Effect of phospholipase C treatment.

Carnitine palmityltransferase was measured in neonatal and adult rat heart and liver mitochondria. When compared to the adult, no developmental differences in liver mitochondrial carnitine palmityltransferase were seen 24 hours postpartum. In neonatal heart, enzyme activity was initially low, reaching adult levels by 20 days. Polarographic measurements of oxygen consumption revealed normal levels of palmityl coenzyme A oxidation in the presence of carnitine and of palmitylcarnitine oxidation in both neonatal heart and liver. Phospholipase C treatment produced higher specific activities of carnitine palmityltransferase in neonatal and activities of carnitine palmityltransferase in neonatal and adult liver, probably due to decreases in total mitochondrial protein. Phospholipase C treatment had no effect on adult rat heart carnitine palmityltransferase but increased neonatal heart enzyme activity to adult levels. Low levels of measured enzyme activity were proposed to be due to "masking" of enzyme expression in intact mitochondrial preparations.

Action of halothane upon mitochondrial respiration

The inhibitory action of halothane upon respiration was studied with rat liver mitochondria (RLM 3), beef heart mitochondria (HBHM), and electron-transport particles (ETP). With intact mitochondrial preparations the oxidation of NADH-linked substrates but not of succinate was markedly suppressed by low concentrations of halothane (<2 m m as determined by gas-liquid chromatography). This inhibitory action of halothane was completely reversible. In contrast, a number of other mitochondrial processes were found to be sensitive in an irreversible manner at higher concentrations of the anesthetic. Likewise, the oxidation of added NADH by HBHM, ETP, and detergent-disrupted RLM was found to be sensitive in a reversible manner to low concentrations of halothane. The energy-dependent transfer of electrons from succinate to NAD by ETP H was also sensitive to halothane. On the other hand, the NADH-ferricyanide reductase and the succinic oxidase activities of ETP and the NADH-cytochrome c reductase activity of microsomes were all insensitive to halothane. The site of inhibition by halothane appears to be in the vicinity of the rotenone-sensitive site of complex I (NADH-CoQ reductase). A number of other general anesthetics inhibited respiration at or near the same site as halothane.

The mitochondrial oxidation of quinol monophosphates.

1. Mitochondria from ox heart and rat liver catalysed a slow cyanide-sensitive oxidation of 2,3-dimethylnaphthaquinol monophosphate, duroquinol monophosphate, menadiol 1-phosphate and menadiol 4-phosphate. 2. The release of P(i) was concomitant with oxygen uptake. 3. The oxidation was somewhat stimulated by Ca(2+) and P(i), and weakly inhibited by 2,4-dinitrophenol. 4. The quinol monophosphates effected a rapid reduction of free cytochrome c, and consequently addition of cytochrome c greatly increased the rate of the mitochondrial oxidation of 2,3-dimethylnaphthaquinol monophosphate. 5. This quinol phosphate interacts with the electron-transport chain at the level of cytochrome c. 6. Polylysine promoted an interaction between 2,3-dimethylnaphthaquinol monophosphate and cytochrome oxidase. Thus, although polylysine blocks mitochondrial oxidations via reduced cytochrome c, the oxidation of the quinol phosphate was strongly stimulated. 7. This stimulation was most effective in the most intact mitochondrial preparations and was inhibited by ADP and by P(i). 8. The implications of these results for factors limiting the rate of quinol phosphate oxidation, the mode of action of stimulators and the mechanism of P(i) formation are discussed.