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Structure of paracrystalline arrays on outer membranes of rat-liver and rat-heart mitochondria.

Crystalline arrays are induced in outer membranes of rat-liver and rat-heart mitochondria by phosphotungstate and silicotungstate. The basic structure of the arrays has been determined by correlation averaging of electron microscopic images of side views of tubular arrays and en face views of planar arrays. The arrays consist of rows of bilobed projecting subunits and are similar (in lattice parameters and projected subunit dimensions) to periodic arrays of ion transport ATPases, e.g., arrays of Ca(2+)-ATPase induced by vanadate in sarcoplasmic reticulum. Hexokinase-labeled colloidal gold particles do not specifically decorate the arrays, suggesting that the hexokinase receptor (VDAC channel) is not a component of the arrays.

Animals↗

PubMed1983

Subunit interaction in the mitochondrial H+-translocating ATPase. The role of oligomycin sensitivity conferral protein and coupling factor 6 in ATPase binding and Pi-ATP exchange in mitochondrial membranes.

Oligomycin sensitivity conferral protein, in the absence of coupling factor 6 (F6), is able to bind the ATPase to mitochondrial membranes with an apparent association constant of 10(6) M-1. The F6-dependent ATPase binding has an apparent association constant 1 to 2 orders of magnitude lower than that obtained with oligomycin sensitivity conferral protein. The oligomycin sensitivity conferral protein-dependent, membrane-bound ATPase activity is sensitive to rutamycin while the F6-dependent, membrane-bound ATPase activity is insensitive to rutamycin. F1-ATPase and Type II ATPase require F6 in addition to oligomycin sensitivity conferral protein and FB to reconstitute 32Pi-ATP exchange activity in silicotungstic acid particles. This F6 requirement for the 32Pi-ATP exchange is not related to the F6 effect on the ATPase binding. The Type I ATPase and therefore the 26,500-dalton subunit associated with it requires F6 and FB to reconstitute 32Pi-ATP exchange activity in silicotungstic acid particles. Oligomycin sensitivity conferral protein can be interchanged with the 26,500-dalton ATPase binding protein in the binding of the ATPase and the 32Pi-ATP exchange.

Adenosine Triphosphatases↗

PubMed1988

Pathological findings of the sural nerve in mitochondrial encephalomyopathy.

We examined the muscle and peripheral nerve of a 55-year-old woman with familial mitochondrial encephalomyopathy. In the gastrocnemius muscle, many ragged red fibers and mitochondria containing paracrystalline inclusions in those fibers were observed by light and electron microscopy, respectively. Histopathological studies of the sural nerve revealed a marked decrease in the number of large myelinated fibers. Electron microscopic studies showed an accumulation of glycogen particles and mitochondria containing abnormal, structurally obscure cristae in the Schwann cell cytoplasm. These results suggest that the cause of loss of the large myelinated fibers may be some disturbance of metabolism in the Schwann cells due to mitochondrial dysfunction.

Biopsy↗

PubMed1982

Reversible inhibition of the mitochondrial ubiquinol-cytochrome c oxidoreductase complex (complex III) by ethoxyformic anhydride.

The mitochondrial ubiquinol-cytochrome c oxidoreductase (complex III) is inhibited by ethoxyformic anhydride (EFA). The inhibition is readily reversed by hydroxylamine, suggesting the involvement of essential histidyl or possibly tyrosyl residues. The spectrum of ethoxyformylated complex III in the UV region showed a peak at 238 nm, indicative of N-(ethoxyformyl)histidine. Addition of hydroxylamine caused a large decrease of the 238-nm peak, which amounted to 16 mol of (ethoxyformyl)histidine/mol of cytochrome c1. Hydroxylamine addition to ethoxyformylated complex III also caused a small change at about 280 nm, which could be due to reversal of 1.6 O-ethoxyformylated tyrosyl residues/mol of cytochrome c1. Among many inhibitors of the cytochrome bc1 region of the respiratory chain, EFA is the only reagent known to cause reversible inhibition by covalent modification of amino acid residues. The inhibition site of EFA was determined to be between cytochromes b-562 and c1. However, unlike antimycin, which also inhibits in the same region, EFA did not promote the reduction of cytochrome b-566 in particles treated with substrates. In addition, it was found that EFA inhibits proton translocation in the cytochrome bc1 region and is a more effective electron transport inhibitor when added to reduced particles as compared to oxidized particles. These results together with the strong possibility that the EFA target is a histidyl or possibly a tyrosyl residue have been discussed in relation to the mechanism of proton translocation by complex III.

Anaerobiosis↗

PubMed1989

Intramitochondrial fatty acylation of a cytoplasmic imported protein in animal cells.

Mitochondrial digitonin particles from mouse liver (and also from other tissues) incorporate [3H]myristic acid into a 52-kilodalton (kDa) protein in an energy-dependent manner. The 52-kDa N-myristylated protein is located inside the mitochondrial inner membrane since it is protected against proteolytic degradation in intact mitoplasts. Disruption of mitochondrial inner membrane by sonication results in severalfold higher labeling of the 52-kDa protein, further confirming that the enzyme system for protein fatty acylation as well as the 52-kDa target protein are compartmentalized inside the mitochondrial inner membrane matrix. The results of in vitro labeling of submitochondrial fractions suggest that both the 52-kDa target protein and the enzyme system for fatty acylation are in the matrix fraction, although the N-myristylated protein is found loosely associated with the inner membrane. Finally, immunoprecipitation of cytoplasmic free polysome translation products and in vitro transport of proteins into isolated mitochondria show that the 52-kDa protein is of cytoplasmic translation origin. These results demonstrate that the intramitochondrial N-myristylation of the 52-kDa protein is not translationally linked.

Acylation↗

PubMed1979

Radiochemical synthesis and photochemical properties of the uncoupler 2-azido-4-nitrophenol, a versatile photoaffinity labeling reagent.

2-Amino-4-nitrophenol was tritiated in an acid-catalyzed hydrogen exchange reaction. Radioactive 2-azido-4-nitrophenol with a specific radioactivity up to 21 mCi/mmol was synthesized from 2-amino-4-nitrophenol by diazotization and azide coupling. The photochemical properties of the uncoupler, 2-azido-4-nitrophenol, were studied as free solute and as ligand bound to uncoupler binding sites in bovine serum albumin and mitochondria. Based on product analyses, irradiation of free or bound 2-azido-4-nitrophenolate with visible light results in the formation of nitrene intermediates with a singlet to triplet ratio of 6:1 to 9:1. 2-Azido-4-nitrophenolate and bovine serum albumin form a strong 1:1 complex (KD = 0.7 micron) which can be converted into a photoproduct with a covalent bond between the label and the protein. The acid dissociation constant of the protein-bound 2-amino-4-nitrophenol moiety is strongly pH dependent. Photoaffinity labeling of mitochondria by 2-azido-4-nitrophenolate follows a pattern expected from equilibrium binding studies using normal and lipid-depleted particles: polypeptides were found to bear 90-95% of the radioactive label, and 5-10% of the latter was bound to phospholipids. Two polypeptides (approximately 56 000 and 31 000 daltons) were associated with 60% of the label, indicating a high degree of specific photochemical labeling.

Affinity Labels↗

PubMed1979

Localisation of adenine nucleotide-binding sites on beef-heart mitochondrial ATPase by photolabelling with 8-azido-ADP and 8-azido-ATP.

1. In addition to the previously studied 8-azido-ATP, 8-azido-ADP is a suitable photoaffinity label for beef-heart mitochondrial ATPase (F1). 2. Photolysis at 350 nm of 8-azido-ADP in the presence of isolated F1 leads to inactivation of ATPase activity. Both ATP and ADP (but not AMP) protect against the inactivation. 3. In the absence of Mg2+, 8-azido-ADP binds almost equally to the alpha and beta subunits of F1, whereas in the presence of Mg2+ the alpha subunits are predominantly labelled. 4. The ATPase activity is completely inhibited when two molecules of 8-azido-ADP are bound per molecule F1. 5. 8-Azido-ATP and ATP are competitive substrates for F1, indicating that in the presence of Mg2+ 8-azido-ATP binds to the same site as ATP. 6. The amount of tightly bound nucleotides in F1 is not significantly changed upon incubation with 8-azido-ATP either in the light or the dark. 7. 8-Azido-ATP is also a suitadrial particles, photolabelling leading to inactivation of ATPase activity. 9. Oxidative phosphorylation and the ATP-driven reduction of NAD+ by succinate are also inhibited by photolabelling Mg-ATP particles with 8-azido-ATP. 10. In contrast to the uncoupled ATPase activity, where the two ATP-binding sites do not interact, cooperation between the two sites is required for ATP hydrolysis coupled to reduction of NAD+ by succinate.

Adenosine Diphosphate↗

PubMed1991

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.

Alanine↗

PubMed1985

Variation in protein lateral diffusion coefficients is related to variation in protein concentration found in mitochondrial inner membranes.

The electrophoretic freeze-fracture electron microscopy method (Sowers, A.E. and Hackenbrock, C.R. (1984) Proc. Natl. Acad. Sci. USA 78, 6246-6250) for measuring the lateral diffusion coefficient of integral proteins was applied to a large population of spherical-shaped mitochondrial inner membranes. Membrane integral protein concentration was estimated by determining the intramembrane particle concentration. Analysis of the data reveals that: (a) the radii of the spherical inner membranes in the selected population ranged from 0.22 to 1.2 micron, (b) the intramembrane particle concentrations ranged from 2300 to 6400 per micron2, and (c) the calculated lateral diffusion coefficients of the intramembrane particles ranged from 1.3 X 10(-10) to 3.35 X 10(-9) cm2/s. The data clearly show a naturally occurring large range in protein concentration in the mitochondrial inner membrane and an inverse correlation of lateral diffusion coefficient with the membrane protein concentration. This study is the first to show that the lateral diffusion coefficient of integral proteins in a native membrane varies as the membrane protein concentration.

Diffusion↗

PubMed2003

Isolation of the mitochondrial nucleoids from yeast Kluyveromyces lactis and analyses of the nucleoid proteins.

Mitochondrial (mt) nucleoids were isolated from yeast Kluyveromyces lactis with morphological intactness. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed more than 20 proteins that are associated with the mt-nucleoids. However, the protein profile of the mt-nucleoids of K. lactis was significantly different from that of the mt-nucleoid proteins from Saccharomyces cerevisiae. SDS-DNA PAGE, which detected an Abf2p, a major mitochondrial DNA-binding protein, among the mt-nucleoid proteins of S. cerevisiae on a gel, detected only a 17-kDa protein in the K. lactis mt-nucleoid proteins. The 17-kDa protein was purified as homogeneous from the mt-nucleoids by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography. The 17-kDa protein introduced a negative supercoil into circular plasmid DNA in the presence of topoisomerase I, as does S. cerevisiae Abf2p, and it packed K. lactis mtDNA into nucleoid-like particles in vitro. These results, together with the determination of the N-terminal amino acid sequence, suggested that the 17-kDa protein is an Abf2p homologue of K. lactis and plays structural roles in compacting mtDNA in cooperation with other nucleoid proteins.

Amino Acid Sequence↗

PubMed1985

Analysis of cytoplasmic 19 S ring-type particles in Drosophila which contain hsp 23 at normal growth temperature.

Cytoplasmic 19 S particles were isolated from postpolysomal supernatants of 25 degrees C Drosophila embryos and culture cells. The particles were purified by salt extraction and sucrose gradient centrifugation. Electron microscopic investigation showed that the 19 S particles possess a ring-shaped morphology with an outer diameter of 12 nm and a hollow core of 3 nm. Biochemically the particles are characterized by a group of 16 polypeptides within the molecular weight range of 35 to 23 kDa, and small RNA molecules in the size range of 200 to 60 nucleotides. The RNP character of the particles is also shown by their buoyant density in Cs2SO4 of rho = 1.29 g/cm3 and their susceptibility to uv crosslinking and density in CsCl of rho = 1.38 g/cm3. Antibodies were raised against the proteins of the 19 S particles isolated from 25 degrees C cells and tested by immunoblotting after one- and two-dimensional gel-electrophoresis. Two of the antibodies raised cross react with the small heat-shock proteins hsp 28/27 and hsp 23. Comparative protease V8 cleavage of hsp 23 and the 23-kDa particle protein demonstrates that these two proteins are identical and that the small hsp of Drosophila must be a genuine part of the 19 S cytoplasmic ring-shaped complexes at normal growth temperature. The data support the idea of a general developmental role of some of the so-called heat-shock proteins.

Animals↗

PubMed1987

Electron-microscopic studies on location of SH-groups in mitochondrial F1-ATPase using a ferritin label.

A new approach has been suggested for electron-microscopic study of the structure of mitochondrial F1-ATPase based on ferritin labeling. By means of sequential treatment with 2-iminothiolane and Nbs2 we obtained a modified ferritin (NbsSPrCNH-Ft) able to react with SH-groups of proteins and to form conjugates in which the protein and ferritin are bound by disulfide bonds. An electron-microscopic investigation of the negatively stained preparations of mitochondrial F1-ATPase, preincubated with modified ferritin, revealed such enzyme-ferritin conjugates. In case of modified ferritin, containing 360 mol SH-groups per mol protein, and F1-ATPase, pretreated with N-ethylmaleimide and then with dithiothreitol, conjugates were obtained in which ferritin molecules are bound to several (as many as four) of the six protein masses, comprising a bilayer molecule of the enzyme. Taking into consideration the biochemical data on the location of accessible SH-groups (only in alpha, gamma or epsilon subunits), it is inferred from the results obtained that one of the protein masses is a complex between beta subunit and at least one of the minor subunits located partially on the molecule's external side. This indicates the nonequivalence of different copies of the major subunits. Averaged images of the particles of the F1-F0 complex from bovine heart mitochondria and bacteria Micrococcus lysodeicticus were obtained. It was found that F0 component is bound to two adjacent protein masses of the F1-ATPase molecule. It is suggested that this binding may be due the nonequivalency of single-type major subunits.

Animals↗

PubMed1981

Alterations in density and size distribution of intramembrane particles in the inner membrane of mitochondria from chloramphenicol-fed mice.

The diameters and number per unit area (density) of intramembrane particles in mitochondrial inner membranes isolated from livers of chloramphenicol-fed and control mice were assessed by freeze-fracture electron microscopy. In addition, the levels of cytochrome hemes aa3, b, and c + c1 of the mitochondria were measured spectrophotometrically. The results show that CAP caused (1) a portion of the mitochondria to develop much larger than normal matrix volumes and inner membrane surface areas, (2) a decrease in the average diameter of intramembrane particles on both the convex and concave fracture faces of all membranes regardless of the membrane surface area, (3) a decrease in intramembrane particle density on only those membranes with a greater than normal surface area, and (4) a 75% decrease in heme aa3, a 25% decrease in heme b, and no change in heme c + c1 levels. It is concluded that intramembrane particles represent membrane integral proteins and that the components of integral proteins which originate from mitochondrial translation are located in the hydrophobic region of the bilayer and occupy volumes shared equally by each bilayer leaflet. In addition the mechanism by which CAP induces a portion of the mitochondria to develop larger than normal sizes most likely includes a net lipid insertion into the bilayer which exceeds the net protein insertion during the CAP-feeding interval.

Animals↗

PubMed1993

Enhanced enzymatic degradation of radical damaged mitochondrial membrane components.

The location of a protein (soluble or membrane-bound) influences the extent of oxidative damage caused by free radicals. It has been established that after radical attack, soluble proteins can become more susceptible to hydrolysis by individual proteinases than native proteins. We have now examined the hydrolytic susceptibility following radical attack of a protein that is located within a membrane environment, mitochondrial monoamine oxidase (MAO). After exposure to oxygen radicals generated by gamma irradiation, hydrolysis of sub-mitochondrial particles (SMP) containing MAO was increased in three respects. First, the generation of small fragments of MAO by the proteinases elastase and trypsin, was enhanced. Second, the generation by these enzymes and by phospholipase A2 of non-sedimentable membrane fragments containing MAO was also increased. Third, autolysis of SMP was enhanced. Hence, proteins located within membranes may become more susceptible to enzymatic degradation following oxidative damage.

Animals↗

PubMed1986

Solubilization and reassembly of the mitochondrial benzodiazepine receptor.

We have solubilized and reassembled the peripheral-type benzodiazepine receptor, a component of the mitochondrial outer membrane, from rat adrenal gland mitochondria. The ligand binding site of this receptor undergoes denaturation during solubilization in digitonin, Triton X-100, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at detergent concentrations above 0.1%, which is evident from the loss of high-affinity binding of [3H]PK11195, a ligand selective for the mitochondrial benzodiazepine receptor. The conformation of the binding site for PK11195 can be stabilized during solubilization in sodium cholate by relatively low concentrations of supplementary soybean lipid. Drug displacement studies demonstrate that the pharmacological properties of the receptor are preserved under these conditions. Electron micrographs of the solubilized preparation show a heterogeneous population of many small particles (less than 100 A) and some larger membranous aggregates (up to 500 A). Sucrose gradient centrifugation indicates that these lipoprotein complexes are of high buoyant density. They can be incorporated in liposomes via cholate dialysis in the presence of additional supplementary lipid. The behavior of the mitochondrial benzodiazepine receptor during solubilization and reassembly suggests that it is an integral protein of the outer membrane.

Adrenal Glands↗

PubMed1983

Effects of 2-hydroxy-3-undecyl-1,4-naphthoquinone on respiration of electron transport particles and mitochondria: topographical location of the Rieske iron-sulfur protein and the quinone binding site.

2-Hydroxy-3-undecyl-1,4-naphthoquinone is a quinone analogue that inhibits mitochondrial respiration in the cytochrome b-c1 region with an apparent Ki of 2.5 X 10(-7) M. In electron transport particles, it prevents the reduction of cytochrome c1 by succinate but not its oxidation by oxygen and prevents oxidation of cytochrome b but not its reduction by succinate. The analogue increases the amount of steady-state cytochrome b reduction in actively respiring particles. It inhibits oxidant-induced reduction of cytochrome b in the presence of antimycin. Inhibition of succinate oxidase activity in electron transport particles is independent of the pH of the suspending medium while at pH values above 8 with mitochondria, inhibition decreases. Since the apparent pK of the bound quinone is pH 6.6, the pH dependency of the inhibition is likely due to the pK of the Rieske iron-sulfur center (pH 8). The Rieske center and thus the quinone binding site are located on the cytoplasmic face of the inner membrane.

Animals↗

PubMed1984

Identification of the 29,000-dalton protein and its relevance to oligomycin-sensitive 32Pi-ATP exchange in bovine heart electron transport particles.

There have been several reports on the involvement of a 29,000-dalton protein in the regulation of ATP synthesis and 32Pi-ATP exchange (Zimmer, G., Mainka, L., and Heil, B. M. (1982) FEBS Lett. 150, 207-210). The present communication demonstrates that incubation of electron transport particles with 50 microM copper-o-phenanthroline results in reversible loss of 32Pi-ATP exchange but not of oligomycin-sensitive ATPase. Dependence of the inhibition on oxygen, its prevention by EDTA, ATP, or 2-mercaptoethanol, and subsequent restoration of the activity by 2-mercaptoethanol point to a thiol-disulfide interchange as the cause of inhibition. Analysis of copper-o-phenanthroline-treated samples by polyacrylamide gel electrophoresis conducted under nonreducing conditions shows four major changes. There is a decrease in the staining intensity of two bands with molecular weights of 34,000 and 29,000 with concomitant appearance of two new bands with molecular weights of 28,000 and 58,000-60,000. The 34,000-dalton band is tentatively identified as the phosphate transport protein. The 28,000-dalton component is formed by intramolecular and the 58,000-60,000-dalton component by intermolecular cross-linking of the 29,000-dalton protein. Pretreatment of electron transport particles with 2 mM N-ethylmaleimide does not affect 32Pi-ATP exchange or its inhibition by copper-o-phenanthroline but prevents cross-linking of the 34,000- and 29,000-dalton proteins. Evidence is presented to demonstrate that the purified H+-ATPase preparation has a single 29,000-dalton protein, identical to the adenine nucleotide translocase, and that it is not essential for 32Pi-ATP exchange or oligomycin-sensitive ATPase.

Adenosine Triphosphatases↗

PubMed1987

Chronic ethanol consumption affects filipin-cholesterol complexes and intramembranous particles of synaptosomes of rat brain cortex.

To assess the effect of ethanol on the planar distribution of cholesterol as well as on the surface architecture of presynaptic terminals of rats, synaptosomes isolated from cerebral cortex of rats chronically exposed to alcohol were incubated with filipin, a cytochemical marker for beta-hydroxycholesterol, and analyzed using both conventional (qualitative and quantitative) and freeze-fracture electron microscopy. Synaptosomes incubated in the absence of filipin were used as cytochemical controls. Biochemical determination indicates a 12% increase of cholesterol in synaptosomal membranes from alcohol treated rats. This increase was confirmed by a significant increment in the number of filipin-cholesterol complexes. Synaptosomes of treated rats showed a reduction in the total number of synaptic vesicles (SV) as well as a decrease in the density and total number of intramembranous particles (IMP) per synaptosome. In control rats, most synaptosomal IMP were distributed in clusters whereas in those of rats exposed to alcohol they were distributed at random. These changes in distribution of IMP were also observed in presynaptic terminals analyzed "in situ." These findings indicate that ethanol acts on the presynaptic terminals. The variations in cholesterol content as well as in the density and distribution of IMP appear to be related to alcohol-induced changes in the physicochemical properties of components of the synaptosomal membrane.

Alcoholism↗