Mitochondrial Membrane Preparations Research Articles

A receptor for protein import into potato mitochondria

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.

Inhibition of complex I by hydrophobic analogues of N-methyl-4-phenylpyridinium (MPP+) and the use of an ion-selective electrode to measure their accumulation by mitochondria and electron-transport particles

N-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, kills dopaminergic neurons after its accumulation in mitochondria where it inhibits Complex I of the respiratory chain. MPP+ inhibits respiration by binding to both a hydrophobic and a hydrophilic site on Complex I and this inhibition is increased by the lipophilic tetraphenylboron anion (TPB-) which facilitates movement of MPP+ through membranes and its penetration to the hydrophobic binding site on Complex I. To investigate the inhibition of respiration by MPP(+)-like compounds, we have measured simultaneously NADH-linked mitochondrial respiration and the uptake and accumulation of the N-benzyl-4-styrylpyridinium and N-ethyl-4-styrylpyridinium cations in mitochondria using ion-selective electrodes. The data provide direct evidence that TPB- increases the inhibition not by increasing matrix concentration but by facilitating access to the inhibitory sites on Complex I. We have also compared the rates of uptake of MPP+ analogues of varied lipophilicity by the inner membrane and the development of inhibition of NADH oxidation, using an inverted mitochondrial inner membrane preparation and appropriate ion-selective electrodes. These experiments demonstrated that the amount of MPP+ analogue bound to the inner membrane greatly exceeded the quantity required for complete inhibition of NADH oxidation. Moreover, binding to the membrane occurred much more rapidly than the development of inhibition with all MPP+ analogues tested. This suggests that the attainment of a correct orientation of these compounds within the membrane and the binding site may be a rate-limiting step in the development of inhibition.

Inhibitory Effect of NaCl on Hog Kidney Mitochondrial Membrane-Bound Monoamine Oxidase: pH and Temperature Dependences

For a further understanding of the inhibitory effect of NaCl on hog kidney mitochondrial monoamine oxidase (MAO), the activity for benzylamine as substrate was assayed spectrophotometrically in the absence and presence of NaCl for mitochondrial outer membrane preparations as well as whole mitochondria. The effect of CaCl2 was also examined for comparison. The inhibition by NaCl but not CaCl2 was strongly pH dependent. The pH dependence of the inhibitory effect of NaCl in phosphate buffer was parallel to the pH dependence of the MAO activity itself. The point at which the slope of the Arrhenius plot in the absence of NaCl decreases with increasing temperature was to be 32.3°C at pH 7.0 and 30.4°C at pH 7.5 in phosphate buffer, while the Arrhenius plot in the presence of NaCl exhibited discontinuities without change in the slope in small temperature ranges, 39.2°C-40.0°C and 33.0°C-34.2°C. It was estimated that the inhibitory effect of NaCl was due to a pH and temperature sensitive cooperative state change involving MAO protein and boundary lipids, while the effect of CaCl2 could be induced by specific Ca2+ binding to acidic phospholipids.

Application of fourier transform Raman spectroscopy to biomembranes: 1064 NM excited Raman spectra of hog kidney mitochondrial outer membranes

AbstractFourier transform Raman spectra with 1064 nm excitation were measured at ambient temperature on hog kidney mitochondrial outer membrane preparations of ultracentrifuged pellet and almost fluorescence‐free Raman spectra with satisfactory signal‐to‐noise ratios were obtained. Fourier transform IR spectra were also measured. The observed Raman and infrared wavenumbers were tentatively assigned to membrane proteins and phospholipids, respectively. CH stretching, phospholipid CC stretching and PO2− stretching vibrations are discussed. To examine the applicability of near‐infrared excited Fourier transform Raman spectroscopy to the study of the inhibitory effects of inorganic ions on membrane‐bound monoamine oxidase activity, measurements were also made on membrane preparations suspended in phosphate buffer (pH 7.0) at ambient temperature in the absence (control) and presence of NaCl, and studies were made of phospholipid CC stretching and PO2− stretching vibrations, which are relatively unaffected by H2O self‐absorption by near‐infrared radiation. It was found that the presence of NaCl decreased the intensity of the 1080 cm− 1 gauche form acyl chain CC stretching Raman band relative to those of the all‐trans CC stretching Raman band at 1126 and 1068 cm− 1. This result was different from that obtained with aqueous dispersions of phosphatidylcholine (PC), for which it has been reported previously that monovalent ions such as Na+ do not affect the Itrans/Igauche ratio.

Characterization of the inhibitory mechanism of 1-methyl-4-phenylpyridinium and 4-phenylpyridine analogs in inner membrane preparations.

We have investigated the mechanism of the inhibition of membrane-bound NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) and a series of its 4'-alkyl-substituted analogs of increasing hydrophobicity, as well as their neutral, desmethyl congeners. Comparison of hydrophobicity, as measured by partition coefficients, with the IC50 for the inhibition of NADH oxidase activity in mitochondrial inner membrane preparations shows a negative correlation, but the cationic inhibitors are more effective than the neutral analogs with similar hydrophobicity. The presence of 10 microM tetraphenylboron (TPB-) potentiates the inhibitory power of positively charged analogs up to 4'-pentyl-MPP+, while the neutral inhibitors are unaffected by TPB-. Without TPB-, the more hydrophilic analogs give incomplete inhibition, but the inclusion of TPB- permits the attainment of complete inhibition, accompanied by the appearance of sigmoidal titration curves. These data support the hypothesis that MPP+ analogs, like rotenone, are bound at two sites on the enzyme and occupancy of both is required for complete inhibition. TPB-, by forming ion pairs with the cationic analogs, facilitates their equilibration to both sites in membrane preparations. When present in molar excess over the MPP+ analog, TPB- partially reverses the inhibition by decreasing its concentration in the more hydrophilic binding site. The effect of temperature and of pH on the IC50 values for inhibition support the concept of dual binding sites, and the pH dependence of the inhibition reveals the participation of two ionized protein groups in the binding, one of which may be a thiol group.

Reaction of bulk protons with a mitochondrial inner membrane preparation: time-resolved measurements and their analysis.

The laser-induced proton pulse technique [Gutman, M. (1986) Methods Enzymol. 127, 522-538] was applied on suspensions of submitochondrial vesicles, and the exchange of protons between the bulk and the mitochondrial membranes was measured in the time-resolved domain with a submicrosecond resolution. The protons were discharged by photoexcitation of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) by a short laser pulse, and the reprotonation of the pyranine anion was monitored at 457.8 nm. In parallel, the protonation of the membrane was followed at 496.5 nm, looking at the transient absorbance of fluorescein, covalently attached to the M side of the membrane. The analysis of the relaxation dynamics was carried out by a simulation procedure that reconstructs the observed dynamics of the two chromophores. The analysis revealed the presence of the membrane indigenous buffering moieties. The low-pK buffer (pK 4.1) was present in a quantity of 100 +/- 20 nmol/mg of protein, and its kinetics indicate that it appears in multianionic clusters bearing a negative electric charge. The medium-pK buffer (pK 6.9) was present in a larger quantity (200 +/- 20 nmol/mg), and its kinetic parameter indicated clustering into positively charged domains. Both types of indigenous buffer reacted with the proton and pyranine anion in unhindered diffusion-controlled reactions. On the other hand, the exchange of protons between the indigenous buffer moieties was rather slow. No evidence was found for the presence of sites capable of retaining a proton, secluded from the bulk, for a time frame longer than 100 microseconds as required by the models of localized proton gradient.

Modulation of Membrane Phospholipid Fatty Acid Composition by Age and Food Restriction

Phospholipids from liver mitochondrial and microsomal membrane preparations were analyzed to further assess the effects of age and lifelong calorie restriction on membrane lipid composition. Results showed that the major phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol and cardiolipin did not vary significantly with age or diet. The fatty acid composition of the phospholipids was determined in PC and PE and ages of 6, 12 and 24 months. The data revealed characteristic patterns of age-related changes in ad libitum (AL) fed rats: membrane levels of long-chain polyunsaturated fatty acids, 22:4 and 22:5, increased progressively, while membrane linoleic acid (18:2) decreased steadily with age. Levels of 18:2 fell by approximately 40%, and 22:5 content almost doubled making the peroxidizability index increase with age. In addition, levels of 16:1 and 18:1 decreased significantly with age, indicating a possible change in delta 9-desaturase activity coefficient. Food restriction resulted in a significant increase in levels of essential fatty acids while attenuating levels of 22:4, 22:5, 22:6 and peroxidizability. We concluded that the membrane-stabilizing action of long-term calorie restriction relates to the selective modification of membrane long-chain polyunsaturated fatty acids during aging.

Repeated administration of diazepam reduces [ 3H]Ro 5-4864 binding in cerebral cortex of rats

Binding of [ 3H]Ro 5-4864 to mitochondrial membrane preparations of rat cerebral cortex was measured after repeated (5 days) IV administration of diazepam (10 mg/kg daily) and clonazepam (2.5 mg/kg daily). The B max value for [ 3H]Ro 5-4864 was significantly reduced in rats treated with diazepam (−49%) but not in those treated with clonazepam. These findings suggest the involvement of peripheral-type binding sites in the development of rapid tolerance to the sedative effects of benzodiazepines. A downregulation of [ 3H]Ro 5-4864 (−65%) was also observed after repeated administration of Ro 5-4864 (4 mg/kg daily), thus confirming that this compound behaves as an agonist at its own recognition sites.

Characterization of mitochondrial imidazoline-guanidinium receptive sites (IGRS) in liver.

Some imidazoline and guanidinium antihypertensive drugs display high affinity for a nonadrenergic membrane protein, the imidazoline-guanidinium receptive site (IGRS), which is insensitive to catecholamine and physically distinct from alpha 2-adrenoceptor. In the present report, we characterized IGRS in human and rabbit liver using [3H]idazoxan as radioligand. By performing subcellular fractionation, we showed a significant increase in [3H]idazoxan binding sites on membrane fractions enriched in cytochrome oxidase activity, a mitochondrial marker. A further enrichment in [3H]idazoxan binding (53-fold with respect to the homogenate) was found in a purified preparation of mitochondrial outer membranes. This localization of IGRS will facilitate the characterization of its functional activity in liver.

DNA topoisomerase I from calf thymus mitochondria is associated with a DNA binding, inner membrane protein

During purification of the type I DNA topoisomerase from calf thymus mitochondria, two polypeptides, p78 and p63, cofractionate with the enzymatic activity (Lazarus et al., (1987) Biochemistry 26, 6195–6203). The two polypeptides are released from a mitochondrial inner membrane preparation by nonionic detergent lysis and both adsorb strongly to a single-stranded DNA agarose column. We have attempted to characterize the relationship between these two polypeptides and have found the following: (i) the mitochondrial topoisomerase is active in free (monomer) and associated (heterodimer) form; (ii) the catalytic activity resides solely in p78, as adjudged by both the covalent linkage of the enzyme to substrate DNA and the ability of the enzyme to relax supercoils; (iii) at low ionic strength the enzyme is active in monomer form with p78 alone being sufficient for activity; (iv) in high salt, the high molecular weight species is a 140-kDa heterodimer composed of one p78 and one p63; and (v) the two polypeptides are not structurally related as digestion with V 8 protease results in distinct proteolytic fragment patterns. These results suggest that p63 may have an important role in the metabolism of the mitochondrial topoisomerase.

Food restriction in female Wistar rats: V. Lipid peroxidation and antioxidant enzymes in the liver

The activities of antioxidant enzymes as well as the levels of basal and enzyme induced peroxidation have been investigated in liver of female Wistar undernourished rats. Food restriction was applied starting from the age of 3.5 months by feeding the animals on every-other-day schedule (EOD). Diet restriction prevented the age-dependent increase of basal and enzyme induced lipid peroxidation in both mitochondrial and microsomal liver membrane preparations. The activities of antioxidant enzyme, i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) of liver decreased during aging in ad libitum fed rats. In the diet conditioned animals, a small increase of SOD and a complete recovery of CAT activities were observed. Present data support that food restriction improved the protection against peroxidation, and this may be in close relationship with the life prolonging effect of such a treatment.

Electron transfer properties of NADH: Ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON)

We report the electron transfer properties of the NADH: ubiquinone oxidoreductase complex of the respiratory chain (Complex I) in mitochondria of cells derived from LHON patients with two different mutations in mitochondrial DNA (mtDNA). The mutations occur in the mtDNA genes coding for the ND1 and ND4 subunits of Complex I. The NDI/3460 mutation exhibits 80% reduction in rotenone-sensitive and ubiquinone-dependent electron transfer activity, whereas the proximal NADH dehydrogenase activity of the Complex is unaffected. This is in accordance with the proposal that the ND1 subunit interacts with rotenone and ubiquinone. In contrast, the ND4/11778 mutation had no effect on electron transfer activity of the Complex in inner mitochondrial membrane preparations: also K m for NADH and NADH dehydrogenase activity were unaffected. However, in isolated mitochondria with the ND4 mutation, the rate of oxidation of NAD-linked substrates, but not of succinate, was significantly decreased. This suggests that the ND4 subunit might be involved in specific aggregation of NADH-dependent dehydrogenases and Complex I, which may result in fast (‘solid state’) electron transfer from the former to the latter.

Thermal Resistance of Mitochondrial Respiration: Hydrophobic Interactions of Membrane Proteins May Limit Thermal Resistance

The resistance of mitochondrial respiration and enzymatic activity to elevated temperatures differs among species in parallel with differences in their maximal body temperatures. To examine the causes of thermal disruption of mitochondrial respiration, we have measured rates of succinate-supported respiration and activities of the mitochondrial enzymes cytochrome c oxidase, succinic dehydrogenase, and malate dehydrogenase in mitochondria from the hydrothermal vent tube worm Riftia pachyptila and the coastal bivalve Solemya reidi. Intact mitochondria and mitochondrial membrane preparations were incubated for 10 min at temperatures between 0° and 70° C, and function was examined at 20° C (R. pachyptila) or 15° C (S. reidi). Incubation at similar elevated temperatures caused inactivation of respiration and caused the activities of several mitochondrial enzymes to either increase or decrease sharply. Fluorescence polarization measurements using the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene showed that no sharp changes in membrane fluidity occurred at the temperatures at which respiration and membrane-associated enzymes altered activity. Reducing bulk membrane fluidity by enrichment with saturated phospholipids had no effect on the thermal inactivation of membrane-associated enzymes. However, disruption of membrane hydrophobic interactions with detergents, 1-5 mM deoxycholate or 5-25 mM octyl glucoside, significantly reduced the temperatures at which loss of enzyme activity occurred. We conclude that disruption of hydrophobic interactions between membrane proteins and their closely associated lipids, or between membrane proteins themselves, is at least partially responsible for high temperature-induced loss of mitochondrial function.

Cofractionation of the 17-kD PK 14105 binding site protein with solubilized peripheral-type benzodiazepine binding sites

To examine the relationship between PKBS, a 17-kD protein covalently photolabeled by [ 3H]PK 14105, and its association with peripheral-type benzodiazepine binding sites, rat adrenal mitochondrial fractions were photolabeled with [ 3H]PK 14105, solubilized in digitonin, and subjected to anion-exchange chromatography over Q-Sepharose. The Chromatographic behavior of PKBS was evident as principally two major fractions, signified as Q-I and Q-II. Specific binding sites for [ 3H]Ro5-4864 and [ 3H]PK 11195 were also assayed and found to cofractionate with each other and in a manner which coincided with the photolabeled PKBS profile. The Q-I and Q-II fractions were further distinguished based on their different molecular sizes observed by gel filtration, yet both fractions were characterized as containing peripheral-type benzodiazepine recognition sites according to the following criteria. Scatchard analysis of both subpopulations revealed a single class of binding sites for [ 3H]Ro5-4864 with an apparent K D of 14 nM for Q-I and 22 nM for Q-II; these affinities were slightly lower than those found in mitochondrial membrane preparations used as the starting material for solubilization. The rank order of potency to inhibit [ 3H]Ro5-4864 binding in both subpopulations was PK 11195 > Ro5-4864 > diazepam > clonazepam, in connection with the pharmacological specificity of membrane-associated peripheral-type benzodiazepine binding sites. These studies provide direct biochemical evidence that the recognition sites for benzodiazepines and isoquinoline carboxamides cofractionate in unison with the 17-kD PKBS protein, demonstrating an intimate relationship between this protein and the binding domains for peripheral-type benzodiazepine ligands.

Topological Investigations. Study of the Trypsin Sensitivity of the iV-Acetylglucosaminyl and Mannosyl-Transferase Activities Located in the Outer Mitochondrial Membrane1

The trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransferase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosylation were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenylate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosylation pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane. Based on the activator effect of the trypsin on these enzyme activities, the results suggested two distinct orientations of their active sites. As regards the N-glycoprotein biosynthesis pathway through dolichol intermediates, the dolicholphosphoryl-mannose and dolichol-pyrophosphoryl-di-N-acetylchitobiose synthases would be oriented outside while the oligomannosyl-synthase and the oligomannosyl-transferase would be rather oriented inside in the outer membrane. The N-acetylglucosaminyl-transferase involved in the direct transfer of N-acetylglucosamine from its nucleotide donor to a proteinic acceptor would be oriented outside in the outer membrane.

Specific inhibition of ATP-ADP translocase in cardiac mitoplasts by antibodies against mitochondrial creatine kinase

Mitochondrial creatine kinase was purified from rat hearts and used to produce antibodies in chicken and rabbits. Antibodies were purified to a high degree of homogeneity by an affinity chromatography method. Chicken antibodies against mitochondrial creatine kinase inhibited this enzyme in rat-heart mitochondrial inner membrane and matrix preparation, and simultaneously blocked oxidative phosphorylation. Under these conditions respiratory chain activities remained unchanged, but adenine nucleotide translocase was inhibited. Removal of mitochondrial creatine kinase from the membrane by pretreatment with 0.15 M KCl and 20 mM ADP completely abolished the effect of antibodies against mitochondrial creatine kinase on oxidative phosphorylation. Noninhibitory antibodies from rabbit with high affinity to rat mitochondrial creatine kinase inhibited neither creatine kinase activity nor oxidative phosphorylation. These data show close and specific spatial arrangement of mitochondrial creatine kinase and adenine nucleotide translocase in mitochondria. It is supposed that there is a fixed orientation of these proteins in the cardiolipin domain in the membrane and that their interaction may occur by a frequent collision due to their lateral movement.

Proton flow through the intramembrane sector of the ATP synthase complex of bovine heart mitochondria

The mechanism of proton translocation across the mitochondrial F 0-F 1 ATP synthase was investigated by studying the electrical behaviour of the intramembrane sector, F 0, of the ATP synthase as well as by studying the role of dithiol groups of F 0 in proton transport. The conductance properties of F 0 were inferred by determining the current-voltage characteristics of inner mitochondrial membrane preparations in which the interaction between F 1 and F 0 had been selectively damaged so as to open F 0 channels. Such a study indicated that open F 0 channels have a gated response to the membrane potential so that proton flow through F 0 is inhibited at membrane potential values below approximately 100 mV, Specific oxidation of dithiol groups to disulphides was obtained by incubating purified, urea-extracted F 0 with diamide. It was found that, following such treatment, F 0 could no longer be reconstituted into an energy-coupled F 0-F 1 ATP synthase, as apparent from the loss of sensitivity to oligomycin of ATP hydrolysis by the reconstituted enzyme. Moreover, it was found that F 1 and/or the oligomycin sensitivity conferring protein protect F 0 essential dithiols from diamide deleterious oxidation.

Chapter 19 Aspects on the information handling by the central nervous system: focus on cotransmission in the aged rat brain

Publisher Summary This chapter discusses a study on a particular animal model—the aged brain of the rat—to evaluate the possible functional meaning of cotransmission. Aging is a progressive change in the morphological and biochemical features of the organism that takes place toward the end of adult life. Regional data on the effects of aging on two basic parameters of neural function—namely, Na + /K + ATPase, which is involved in the maintenance of membrane polarization, and protein phosphorylation, which is a biochemical system related to the intracellular decoding of extracellular signals—is reported. Cyclic adenosine monophosphate and Ca 2+ -induced protein phosphorylation was carried out. The studies were performed on crude mitochondrial membrane preparations obtained from dorsal cortex, striatum, limbic system, hippocampal formation, and spinal cord. It has been shown that the density of [ 3 H]ouabain binding sites shows a gradient with the highest density in the phylogenetically oldest regions of the central nervous system (CNS) and the lowest density in the most recent ones (cerebral cortex has the lowest value), while protein phosphorylation tends to have an opposite gradient.

Effect of a zinc-deficient diet on lipid peroxidation in liver and tumor subcellular membranes.

The effect of a zinc-deficient diet on lipid peroxidation in liver and tumor mitochondrial and microsomal membrane preparations from BALB/c mice was investigated. Mitochondrial and microsomal membranes from both tissues displayed increased rates of in vitro peroxidation, both enzymatic and nonenzymatic. Measurement of in vivo peroxidation, using diene conjugation as an index of measurement revealed slight increases in tissues from zinc-deficient animals that were not statistically significant. Serum lipoperoxides analyzed from all three groups revealed no significant differences. The results point to an alteration in the peroxidation potential of mitochondrial and microsomal membranes due to zinc deficiency which may be related to an alteration in fatty acid composition.

Adrenocortical cytochrome P-450 responsible for cholesterol side chain cleavage (P-450 scc) is phosphorylated by the calcium-activated, phospholipid-sensitive protein kinase (protein kinase C)

Purified bovine adrenocortical cytochrome P-450 scc (specific for cholesterol side chain cleavage in the inner mitochondrial membrane) was selectively phosphorylated in vitro by a Ca 2+-activated, phospholipid-sensitive protein kinase (protein kinase C) preparation, whereas cyclic AMP dependent and two cyclic nucleotide independent kinases were ineffective. Cytochrome P-450 scc incorporated a maximum of 4 mol of phosphate in the presence of protein kinase C within 15 min at 30°C, with apparent Km and Vmax of 0.14 μmol and 0.76 pmol/min, respectively. Serine and threonine were the two target aminoacids phosphorylated in a ratio of about 1:1. In the presence of 1 μM Ca 2+, a mixture of phosphatidylserine and diolein (or a potent tumor promoter phorbol ester) was required for optimal cytochrome P-450 scc phosphorylation. In addition, purified inner mitochondrial membrane preparations from adrenocortical mitochondria were found to contain protein kinase C activity. These findings, together with the previous demonstration that activators of protein kinase C such as a potent phorbol ester activates steroidogenesis of intact adrenocortical cells, suggest that phosphorylation of P-450 scc should be examined for its possible role in the regulation of adrenocortical functions.