Activity of the inner and outer membrane oxidative enzymes in brown adipose tissue mitochondria.

1. The specific activity of cytochrome-oxidase, succinate-cytochrome c reductase and su-cinate-oxidase of brown adipose tissue mitochondria of 17-day-old rats was found to be twice as high in brwon adipose tissue mitochondria as in the liver. The specific activity of rotenone-sensitive NADH-cytochrome c reductase and NADH-oxidase was found to be six times higher in brown adipose tissue mitochondria than in the liver. 2. Brown adipose tissue mitochondria have extremely low activity of outer membrane enzymes. When compared with liver the specific activity of rotenone-insensitive NADH-cytochrome c reductase was found to be seven times lower, the specific activity of monoamineoxidase up to 30 times lower according to the substrate used. 3. The optimum conditions for the determination of both NADH-cytochrome c reductases in brown adipose tissue mitochondria were more specified on the base of the following findings: (a) the outer membrane rotenone-insensitive NADH-cytochrome c reductase is strongly inactivated by freezing-thawing, (b) freezing-thawing, alone is insufficient to release completely maximal activity of rotenone-sensitive NADH-cytochrone c reductase, freezing-thawing activite can be further potentiated by e.g. trypsin treatment. 4. The activities of the outer membranes of brown-adipose tissue mitochondria are discussed with regards to the structural integrity of the outer membrane, the activities of the inner membrane enzymes are discussed with regards to the functional specifity of the tissue.     

Model of the outer membrane potential generation by the inner membrane of mitochondria.

Voltage-dependent anion channels in the outer mitochondrial membrane are strongly regulated by electrical potential. In this work, one of the possible mechanisms of the outer membrane potential generation is proposed. We suggest that the inner membrane potential may be divided on two resistances in series, the resistance of the contact sites between the inner and outer membranes and the resistance of the voltage-dependent anion channels localized beyond the contacts in the outer membrane. The main principle of the proposed mechanism is illustrated by simplified electric and kinetic models. Computational behavior of the kinetic model shows a restriction of the steady-state metabolite flux through the mitochondrial membranes at relatively high concentration of the external ADP. The flux restriction was caused by a decrease of the voltage across the contact sites and by an increase in the outer membrane potential (up to +60 mV) leading to the closure of the voltage-dependent anion channels localized beyond the contact sites. This mechanism suggests that the outer membrane potential may arrest ATP release through the outer membrane beyond the contact sites, thus tightly coordinating mitochondrial metabolism and aerobic glycolysis in tumor and normal proliferating cells.     

Visualization of domain formation in the inner and outer leaflets of a phospholipid bilayer

Large vesicles (5-10-micron in diameter) were formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera and digital image processor. When such vesicles contained a fluorescent phosphatidic acid (PA) and were exposed to 2 mM CaCl2 or 0.5 mM PrCl3, it was possible to visualize PA-enriched domains within the vesicles. Calcium-induced domain formation was reversible in the presence of 4 mM EGTA. Vesicles were formed containing fluorescent PA on either the inner or outer leaflet of the bilayer and the patching and dissolution of patching were studied under conditions where calcium was present on the outside of the vesicle and where calcium was distributed across the bilayer. In addition, vesicles were formed with two different fluorescent PA's, one on the inner leaflet and a different one on the outer leaflet of the bilayer. The results of the experiments show that in vesicles formed primarily with naturally occurring phospholipids such as egg phosphatidylcholine or brain phosphatidylethanolamine, there was no coordinate action of the two leaflets of the bilayer. An exception to this was found, however, if the vesicles were formed in the presence of primarily dioleoyl phospholipids (greater than 95 mol %). In these vesicles there was a coordinate or coupled response to calcium by the two leaflets of the bilayer. In most cases, however, the two leaflets of the bilayer showed independent or uncoupled domain formation.     

Glucagon-stimulated adenylate cyclase detects a selective perturbation of the inner half of the liver plasma-membrane bilayer achieved by the local anaesthetic prilocaine.

Prilocaine can increase the fluidity of rat liver plasma membranes, as indicated by a fatty acid spin-probe. This led to the activation of the membrane-bound fluoride-stimulated adenylate cyclase activity, but not the Lubrol-solubilized activity, suggesting that increased lipid fluidity can activate the enzyme. With increasing prilocaine concentrations above 10 mM, the membrane-bound fluoride-stimulated activity was progressively inhibited, even though bilayer fluidity continued to increase and the activity of the solubilized enzyme remained unaffected. Glucagon-stimulated adenylate cyclase was progressively inhibited by increasing prilocaine concentrations. Prilocaine (10 mM) had no effect on the lipid phase separation occurring at 28 degrees C and attributed to those lipids in the external half of the bilayer, as indicated by Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and the order parameter of a fatty acid spin-probe. However, 10 mM-prilocaine induced a lipid phase separation at around 11 degrees C that was attributed to the lipids of the internal (cytosol-facing) half of the bilayer. It is suggested that prilocaine (10 mM) can selectively perturb the inner half of the bilayer of rat liver plasma membranes owing to its preferential interaction with the acidic phospholipids residing there.     

Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter.

The bacterial outer membrane transporter for vitamin B(12), BtuB, derives its energy for transport by interacting with the trans-periplasmic membrane protein TonB. This interaction with TonB occurs in part through an N-terminal segment in the BtuB sequence called the Ton box. In the present study, site-directed spin labeling of intact outer membrane preparations was used to investigate the conformation of the Ton box in wild-type BtuB and in two transport-defective mutants, L8P and V10P. In the wild-type protein, the Ton box is folded into the barrel of the transporter. The conformation of this segment is dramatically different in the transport-defective mutants L8P and V10P, where the Ton box is found to be flexible, and undocked from the transporter barrel with a greater exposure to the periplasm. In the wild-type protein, vitamin B(12) induces an undocking of the Ton box, but its addition to these transport defective mutants produces little or no change in the conformation of the Ton box. Proline substitutions at positions that do not alter transport do not alter the wild-type conformation of the Ton box; thus, the effect of substituting proline at positions 8 and 10 on the docked state of the Ton box appears to be unique. The failure of these mutants to execute the B(12) transport cycle may be a result of the altered conformation of the Ton box.     

Dual localization of the mitochondrial phospholipase A2: outer membrane contact sites and inner membrane.

Mitochondria were fractionated according to a procedure which allowed to get free outer and inner membrane plus two distinct contact sites between the two membranes. The data indicate that phospholipase A2 is localized in outer membrane contact sites and in inner membrane. The enzyme activity is twice higher in the contact site fraction than in the free membrane. The major fatty acids released are linoleic and docosahexanoic acids.     

Mutations in genes cpxA and cpxB alter the protein composition of Escherichia coli inner and outer membranes.

Mutations in chromosomal genes cpxA and cpxB altered the protein composition of the inner and outer bacterial membranes. Electrophoretic analyses of membrane proteins from isogenic strains differing only at their cpx loci and of spontaneous cpxA+ revertants of a cpxA cpxB double mutant showed that the alterations define a pattern that is uniquely attributable to the cpx mutations. Two major outer membrane proteins, the OmpF matrix porin and the murein lipoprotein, were deficient or absent from the outer membrane of mutant cells, whereas the quantities of two other major outer membrane proteins, the OmpC matrix porin and the OmpA protein, were not significantly altered. The cpx mutations did not generally alter the functional or chemical properties of the cell envelope. In the electron microscope, mutant cells appeared ovoid, but individual cells showed no surface irregularities to suggest gross defects in the cell envelope. These observations suggest that the primary effect of the mutations is to alter selectively the synthesis or translocation of certain envelope proteins.     

Peptidoglycan remodeling and conversion of an inner membrane into an outer membrane during sporulation

Two hallmarks of the Firmicute phylum, which includes the Bacilli and Clostridia classes, are their ability to form endospores and their "Gram-positive" single-membraned, thick-cell-wall envelope structure. Acetonema longum is part of a lesser-known family (the Veillonellaceae) of Clostridia that form endospores but that are surprisingly "Gram negative," possessing both an inner and outer membrane and a thin cell wall. Here, we present macromolecular resolution, 3D electron cryotomographic images of vegetative, sporulating, and germinating A. longum cells showing that during the sporulation process, the inner membrane of the mother cell is inverted and transformed to become the outer membrane of the germinating cell. Peptidoglycan persists throughout, leading to a revised, "continuous" model of its role in the process. Coupled with genomic analyses, these results point to sporulation as a mechanism by which the bacterial outer membrane may have arisen and A. longum as a potential "missing link" between single- and double-membraned bacteria.     

A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates.

Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in the outer mitochondrial membrane and the other (Tim) located in the inner membrane. These translocases reversibly cooperate during protein import. We have previously constructed a chimeric precursor (pPGPrA) consisting of an authentic mitochondrial precursor at the N terminus (Delta(1)-pyrroline-5-carboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, to protein A. When pPGPrA is expressed in yeast, it becomes irreversibly arrested during translocation across the outer and inner mitochondrial membranes. Consequently, the two membranes of mitochondria become progressively "zippered" together, forming long stretches in which they are in close contact (Schülke, N., Sepuri, N. B. V., and Pain, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7314-7319). We now demonstrate that trapped PGPrA intermediates hold the import channels stably together and inhibit mitochondrial protein import and cell growth. Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisubunit complex that contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and matrix-localized Hsp70. Further characterization of this complex may shed light on structural features of the complete mitochondrial import machinery.     

Selective release of plasma-membrane enzymes from rat hepatocytes by a phosphatidylinositol-specific phospholipase C.

When isolated hepatocytes are incubated with phosphatidylinositol-specific phospholipase C, three cell-surface enzymes show markedly different behaviour. Most of the alkaline phosphatase is released at very low values of phosphatidylinositol hydrolysis, whereas further phosphatidylinositol hydrolysis releases only a maximum of about one-third of the 5'-nucleotidase. Alkaline phosphodiesterase I is not released. If cells containing phosphatidyl[3H]inositol are similarly treated, then the released [3H]inositol is in the form of inositol phosphate: no evidence has been obtained for any covalent association between released [3H]inositol and alkaline phosphatase.     

Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.     

Amidination of the outer and inner surfaces of the human erythrocyte membrane

We have synthesized a novel imidoester, isethionyl acetimidate, which is unable to penetrate the membrane of the human erythrocyte. It has the same specificity for amino groups as ethyl acetimidate, which penetrates the membrane. Either reagent can be labeled with ³H or ¹⁴C and, thus, be used to convert amines to radioactive amidines. An erythrocyte membrane saturated with either compound functions nearly normally. Therefore, the membrane can be double labeled if the amino groups on the outer surface of a cell are saturated with isethionyl acetimidate (e.g. labeled with ¹⁴C) and the remaining active sites are saturated with ethyl acetimidate (labeled with ³H). Alternatively, the membrane can be isolated after saturation with [¹⁴C]isethionyl acetimidate and treated with [³H]isethionyl acetimidate. From quantitative experiments of this kind we conclude that there are more than ten times as many reactive amino groups in protein on the inner surface than on the outer surface of the membrane. Nearly all of the reactive amino groups in lipid are on the inner surface. The localization of individual polypeptides confirms and extends assignments made previously by other techniques; as many as four major components may span the membrane. The proteins and lipids react to the same extent with ethyl acetimidate in the intact cell as they do in isolated membranes; this implies that the isolation does not load to major structural rearrangements.     

Action of local anaesthetics on passive and energy-linked ion translocation in the inner mitochondrial membrane

The effect of dibucaine on passive and respiration-driven ion translocation and oxidative phosphorylation in submitochondrial particles from beef-heart has been studied.Dibucaine inhibited the nigericin-mediated H⁺/K⁺ exchange diffusion and the electrogenic, valinomycin-mediated K⁺ translocation in submitochondrial particles.The local anaesthetic exerted a direct stimulatory effect on the respiration-driven proton uptake and on the passive proton-diffusion reactions. The increase of the respiration-linked proton turnover caused by dibucaine was accompanied by uncoupling of oxidative phosphorylation. It is concluded that spontaneous noncoupled as well as ionophoremediated K⁺ translocation in mitochondria occurs across phospholipid bilayer regions of the membrane whilst other components of the membrane would be specifically involved in active and passive proton translocation across the membrane.The results indicate that polar groups of membrane phospholipids play an important role in energy conservation and transfer in the mitochondrial membrane.     

Externalization of phosphatidylserine from inner to outer layer may alter the effect of plant sterols on human erythrocyte membrane - The Langmuir monolayer studies

One of the factors, which can strongly modify the cell membrane composition, is disordering in membrane asymmetry, resulting from redistribution of lipids from inner to outer layer. Such a disturbance may affect the behavior of various biologically active compounds incorporating into membranes. In this contribution, the relationship between the amounts of phosphatidylserine (PS) in the model outer layer of human erythrocyte (RBC) membrane and the effect induced by a plant sterol (β-sitosterol) was verified. The experiments were performed on multicomponent Langmuir films imitating red blood cell (RBC) membrane, differing in the contents of PS (0%; 5% and 10%) into which the plant sterol was incorporated in various concentrations. The analysis of experimental results (surface pressure-area isotherms complemented with Brewster Angle Microscopy (BAM) proved that the presence of phosphatidylserine molecules, depending on their contents in the mixed monolayer mimicking RBC membrane, changes its properties and exerts influence on the effect of plant sterol on the model system. The addition of phytosterol into the monolayer that lacks or contains only 5% of PS was found to be of rather weak effect on the properties of the system. However, in the case of the model membrane containing the increased amount (10%) of PS, the incorporation of plant sterol strongly affects the interactions between molecules and caused thermodynamic destabilization of the monolayer imitating RBC membrane. These results allow one to suggest that externalization of phosphatidyserine to the outer membrane leaflet may differentiate the effect of plant sterols on cell membranes of various origins.     

Solutes alter the conformation of the ligand binding loops in outer membrane transporters

The binding and recognition of ligands by bacterial outer membrane transport proteins is mediated in part by interactions made through their extracellular loops. Here, site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy were used to examine the effect of stabilizing solutes on the extracellular loops in BtuB, the vitamin B12 transporter, and FecA, the ferric citrate transporter. EPR spectra from the extracellular loops of FecA and BtuB arise from dynamic backbone segments, and distance measurements made by double electron-electron resonance indicate that the second extracellular loop in BtuB samples a wide range of conformations. These conformations are dramatically restricted upon substrate binding. In addition, the EPR spectra from nitroxide labels attached to the extracellular loops in BtuB and FecA are highly sensitive to solutes, and at every site examined the motion of the label is significantly reduced in the presence of stabilizing osmolytes, such as polyethylene glycols. For the second extracellular loop in BtuB, the solute-induced structural changes are small, but they are sufficient to bring spin-labeled side chains into tertiary contact with other portions of the protein. The spectroscopic changes seen by SDSL suggest that high concentrations of stabilizing solutes, such as those used to generate membrane protein crystals, result in a more compact and ordered state of the protein than is seen under more physiological conditions.     

Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starch-binding sites and periplasmic starch-degrading enzymes.

Bacteroides thetaiotaomicron can utilize amylose, amylopectin, and pullulan as sole sources of carbon and energy. The enzymes that degrade these polysaccharides were found to be primarily cell associated rather than extracellular. Although some activity was detected in extracellular fluid, this appeared to be the result of cell lysis. The cell-associated amylase, amylopectinase, and pullulanase activities partitioned similarly to the periplasmic marker, acid phosphatase, when cells were exposed to a cold-shock treatment. Two other enzymes associated with starch breakdown, alpha-glucosidase and maltase, appeared to be located in the cytoplasm. Intact cells of B. thetaiotaomicron were found to bind 14C-starch. Binding was probably mediated by a protein because it was saturable and was decreased by treatment of cells with proteinase K. Results of competition experiments showed that the starch-binding proteins had a preference for maltodextrins larger than maltohexaose and a low affinity for maltose and maltotriose. Both the degradative enzymes and starch binding were induced by maltose. These findings indicate that starch utilization by B. thetaiotaomicron apparently does not involve secretion of extracellular enzymes. Rather, binding of the starch molecule to the cell surface appears to be a first step to passing the molecule through the outer membrane and into the periplasmic space.