Effect of lysolecithin treatment on the structure and functions of the mitochondrial inner membrane

Lysolecithin treatment of electron transport particles (ETP) generated membrane fragments capable of catalyzing ATP-³²P_i exchange, which was resistant to the uncoupling action of Valinomycin plus Nigericin or Valinomycin plus Monensin A in the presence of K⁺. Electron micrographs of ultrathin, positively stained sections of lysolecithin treated ETP were virtually devoid of circular patterns characteristic of closed vesicles. The results suggest that the closed vesicular structure of the mitochondrial inner membrane demanded by the chemiosmotic hypothesis of energy transduction (1) may not be essential for the ATP-³²P_i exchange reaction.     

Accessibility of lysolecithin in catecholamine secretory vesicles to acyl CoA:lysolecithin acyl transferase

Lysolecithin (monoacylglycerophosphorylcholine) accounts for 13 to 20% of the lipid phosphorous of the bovine adrenal catecholamine secretory vesicles (chromaffin granules). We have incubated purified vesicles with [1-¹⁴C] oleyl coenzyme A and rat liver microsomes containing acyl coenzyme A: monoacylglycerophosphorylcholine acyl transferase to determine the accessibility of the granule membrane lysolecithin to another membrane. No acylation of lysolecithin occurs when the chromaffin granules are intact. The accessibility of the granule membrane lysolecithin increases markedly when the vesicles are broken.     

SIZE AND SHAPE TRANSFORMATIONS CORRELATED WITH OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA : II. Structural Changes in Mitochondrial Membrane Fragments

It has been demonstrated that the nature of the physical change in mitochondrial membrane fragments associated with the action of the respiratory enzymes is likely one of shape or symmetry rather than size. The findings suggest that in the state of decreased scattering the macromolecules may be present in an extended physical state. Conditions favorable for phosphorylation may give rise to a folding or contraction of the molecular complex to a more symmetrical structure. Since earlier studies have shown that there is a compulsory relationship between the integrity of systems operative in oxidative phosphorylation and scattering changes, experiments of this type may lead to values for the minimal size of a phosphorylating unit, which at present is estimated to be 2.1 x 10⁶ from light-scattering studies.     

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.     

Asymmetric distribution of phospholipids in membranes from Mycobacterium phlei

The distribution of phospholipids in the membranes of Mycobacterium phlei has been studied by the use of phospholipase C and trinitrobenzenesulfonic acid. In inverted membrane vesicles, whose external surface apparently corresponds topologically to the cytoplasmic surface of the membrane in intact cells, 80% of the phosphatidyl ethanolamine, 24% of diphosphatidyl glycerol, and 13% of phosphatidyl inositol are accessible to cleavage by phospholipase C. These results are in agreement with the finding that 70–75% of phosphatidyl ethanolamine in the membrane is accessible to chemical modification by trinitrobenzenesulfonic acid or dimethylsuberimidate at 4 °C. It can be inferred that in the inverted membrane the majority of phosphatidyl ethanolamine is present on the outer half of the lipid bilayer while inner half constitutes primarily other phospholipids namely phosphatidyl inositol and diphosphatidyl glycerol. Phospholipase C treatment of ETP membranes selectively impairs the active transport of Ca²⁺ without affecting the generation of a proton gradient, respiration, and coupled phosphorylation.     

Reversible activation/inactivation of the deacylation/acylation cycle in rat liver microsomes

Rat liver microsomes incorporate [¹⁴C]palmitoyl CoA into membrane phospholipids via the deacylation/acylation cycle. This activity is reversibly inactivated/activated by treatment of the microsomes with ATP, MgCl₂, and 105,000g supernatant or with 105,000g supernatant alone. These observations suggest that the acylation cycle is controlled by a mechanism involving phosphorylation/dephosphorylation. As the pool of lysolecithin in the membranes is not altered by conditions increasing incorporation of palmitoyl CoA into phospholipid, it is probable that the site of regulation of deacylation/acylation is at the acyltransferase rather than the phospholipase.     

ACYLATION OF LYSOPHOSPHATIDES BY PLASMA MEMBRANE FRACTIONS OF RAT LIVER

The plasma membrane fraction of rat liver was isolated and incubated with labeled lysophosphatides in the presence of cofactors; the acylation of lysolecithin to lecithin by the fraction was compared to that of the rough and smooth microsomes. The purity of the isolated fractions was ascertained by enzyme markers and electron microscopy, and the maximal contamination of the plasma membrane fraction by microsomes did not exceed 20%. Under conditions at which the reaction was proportional to the amount of enzyme used, the plasma membrane had a specific activity similar to that of the smooth and rough microsomes. With doubly labeled lysolecithin (containing palmitic acid-¹⁴C and choline-³H) it was shown that the lecithin formed retained the same ratio of the two labels, which indicated that lysolecithin was converted to lecithin through an acylation reaction. The newly formed lecithin was shown to be bound to the plasma membrane fraction; this suggested that it is incorporated into the structure of the membrane itself.     

Tyrosine phosphorylation of clathrin heavy chain under oxidative stress

In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking.     

The inhibition of NADH oxidase by the lower homologs of coenzyme Q.

The Coenzyme Q homologs having short isoprenoid chains are much less efficient than the higher homologs in restoring NADH oxidation in pentane-extracted lyophilized beef heart mitochondria; they have however high restoring activity for succinate oxidation. The same pattern is observed in pentane extracted submitochondrial particles ETP only if the quinones are added to detergent-treated membranes, showing that in ETP there is a decreased accessibility of the long chain quinones in comparison with the lower homologs. In intact mitochondria and ETP, CoQ₃ inhibits NADH oxidation while leaving succinate oxidation unaffected; the inhibition of NADH oxidation by CoQ₃ is not reversed by serum albumin but is reversed by CoQ₇, particularly when the membrane has been previously “opened” with deoxycholate. CoQ₃ may accept electrons from NADH in cyanide-inhibited ETP, allowing coupling at the first phosphorylation site as shown by the quenching of the fluorescence of atebrine. The mechanism of CoQ₃ inhibition is probably related to its insufficient rate of reoxidation by the following segment of the respiratory chain when it has been reduced by NADH dehydrogenase.     

Nitric oxide increases oxidative phosphorylation efficiency

We have studied the effect of nitric oxide (NO) and potassium cyanide (KCN) on oxidative phosphorylation efficiency. Concentrations of NO or KCN that decrease resting oxygen consumption by 10–20% increased oxidative phosphorylation efficiency in mitochondria oxidizing succinate or palmitoyl-L-carnitine, but not in mitochondria oxidizing malate plus glutamate. When compared to malate plus glutamate, succinate or palmitoyl-L-carnitine reduced the redox state of cytochrome oxidase. The relationship between membrane potential and oxygen consumption rates was measured at different degrees of ATP synthesis. The use of malate plus glutamate instead of succinate (that changes the H⁺/2e⁻ stoichiometry of the respiratory chain) affected the relationship, whereas a change in membrane permeability did not affect it. NO or KCN also affected the relationship, suggesting that they change the H⁺/2e⁻ stoichiometry of the respiratory chain. We propose that NO may be a natural short-term regulator of mitochondrial physiology that increases oxidative phosphorylation efficiency in a redox-sensitive manner by decreasing the slipping in the proton pumps.     

Action of the herbicides neburon and siduron on membrane permeabilities in potato tuber mitochondria

The herbicides neburon and siduron are uncouplers of oxidative phosphorylation in potato tuber (Solanum tuberosum L. cv. Bintje) mitochondria. Their effect on the ion permeabilities of the mitochondrial membrane was investigated using the acid-base pulse technique, swelling experiments and integrity tests. Both herbicides permeabilize the membrane to H⁺ ions. They have no action on the permeabilities of K⁺ and Fe(CN)^3?₆. The swelling observed with Ca²⁺ was better interpreted as an effect on membrane structure than as a true swelling. Diuron, a parent compound that does not uncouple oxidative phosphorylation, does not act on Ca²⁺-induced apparent swelling.     

A theoretical treatment of the turnover of protein-bound phosphate in the presence of both protein kinase and phosphatase activities

Preparations of membrane fragments from brain have previously been shown to contain tightly bound protein kinase and phophatase enzymes which, together, are responsible for the turnover of protein-bound phosphate in the membrane.An equation has now been derived which describes the time-course of the phophorylation of the membrane-bound proteins in terms of the activities of the kinase and phosphatase enzymes and the initial state of phosphorylation of the membrane proteins. The use of this equation makes it possible to define the effects of substances or treatments which alter the overall rate of protein phosphorylation and to show whether kinase activity, phosphotase activity, or initial state of protein phosphorylation is being changed.Treatment of membrane fragmetns with NaI is found to decrease both protein kinase and phosphatase activities. Na⁺ decreases overall protein phosphorylation solely by decreasing phosphotase activity and cyclic AMP stimulates protein phosphorylation by an action on kinase activity alone.It has been deduced that if there is more than one type of site for protein phosphorylation in cerebral membrane fragments these should react with the kinase at equal rates and with the phosphatase at equal rates.It is hoped that the treatment given in this paper may prove generally applicable to situatiions where the rate of enzymic reaction is controlled by the concentration of substrate.     

Resolution and reconstitution of the succinoxidase pathway of Mycobacterium phlei

Alkaline treatment of the electron transport particles of Mycobacterium phlei resulted in a loss of oxidation and coupled phosphorylation with succinate and NAD⁺-linked substrates but not with ascorbate-TPD as the electron donor. Furthermore, alkaline treatment of the electron transport particles resulted in dissociation of succinic dehydrogenase from the membrane vesicles. However, the membrane retained the menaquinone MK₉(II-H), cytochromes b, c₁ + c, and a + a₃. Restoration of oxidation and coupled phosphorylation with succinate was found to occur on addition of a succinic dehydrogenase preparation to the resolved particles. Silicotungstate treatment of ETP yielded particles deficient in succinie dehydrogenase. Furthermore, membrane-bound or solubilized-latent ATPase was inactivated in the presence of low concentration of silicotungstate. The addition of a soluble succinic dehydrogenase to the silicotungstate-treated particles resulted in the restoration of only oxidation.     

An energetic profile of Corynebacterium glutamicum underpinned by measured biomass yield on ATP

The supply and usage of energetic cofactors in metabolism is a central concern for systems metabolic engineering, particularly in case of energy intensive products. One of the most important parameters for systems wide balancing of energetic cofactors is the ATP requirement for biomass formation Y_ATP/Biomass. Despite its fundamental importance, Y_ATP/Biomass values for non-fermentative organisms are still rough estimates deduced from theoretical considerations. For the first time, we present an approach for the experimental determination of Y_ATP/Biomass using comparative ¹³C metabolic flux analysis (¹³C MFA) of a wild type strain and an ATP synthase knockout mutant. We show that the energetic profile of a cell can then be deduced from a genome wide stoichiometric model and experimental maintenance data. Particularly, the contributions of substrate level phosphorylation (SLP) and electron transport phosphorylation (ETP) to ATP generation become available which enables the overall energetic efficiency of a cell to be characterized. As a model organism, the industrial platform organism Corynebacterium glutamicum is used. C. glutamicum uses a respiratory type of energy metabolism, implying that ATP can be synthesized either by SLP or by ETP with the membrane-bound F₁F_O-ATP synthase using the proton motive force (pmf) as driving force. The presence of two terminal oxidases, which differ in their proton translocation efficiency by a factor of three, further complicates energy balancing for this organism. By integration of experimental data and network models, we show that in the wild type SLP and ETP contribute equally to ATP generation. Thus, the role of ETP in respiring bacteria may have been overrated in the past. Remarkably, in the genome wide setting 65% of the pmf is actually not used for ATP synthesis. However, it turns out that, compared to other organisms C. glutamicum still uses its energy budget rather efficiently.     

Electron flow at the polarized mercury-water interface in the presence of membrane fragments rich in Na+−K+-Activated ATPase

Summary Measurements of interfacial electron flow indicate that membrane fragments rich in Na⁺–K⁺-ATPase are capable of absorbing and releasing electrons in the form of random currents at an electrode surface. The electron transporting system, which functions in the presence or absence of substrate and activating ions, may be part of or in contact with the enzyme system, but it is not related to the ATPase activity. The observed electron transport at an electrode surface resembles physiological electron transport processes in being reversible, in extending over the same range of potential, and in being affected by some of the chemicals that interfere with electron transport and oxidative phosphorylation in mitochondria. Our experiments do not provide sufficient evidence to identify the substances that are responsible for the random currents, but the results suggest that the electro-active substances are similar to those which are involved in the reactions at the second phosphorylation site in mitochondria. Experiments with this technique provide a new approach to the study of the mechanism of biological electron transport processes and their possible relation to ATP synthesis and hydrolysis.Supported by U.S. Public Health Service Research Career Development Award K3-GM-8158.     

Thiophosphate labelling of mitochondria — Lack of evidence for an acyl-phosphate intermediate in oxidative phosphorylation

In the presence of substrate and oxygen, aurovertin-inhibited rat-liver mitochondria incorporate 0.27±0.02 nanomoles of [³⁵S] thiophosphate per mg protein into an acid-precipitable fraction. This incorporation is not prevented by uncouplers of oxidative phosphorylation. Furthermore, there is no significant difference in thiophosphate incorporation by aurovertin-or oligomycin-inhibited mitochondria. Since acyl-phosphate intermediates in other energy-transducing membrane systems are stable to acid precipitation and since acyl thiophosphates are anticipated to be more stable than acyl phosphates, these results support previous indications that acyl phosphates do not participate as intermediates in oxidative phosphorylation.     

LOCALIZATION OF Na+, K+-ATPASE AND OTHER ENZYMES IN TELEOST PSEUDOBRANCH : II. Morphological Characterization of Intact Pseudobranch, Subcellular Fractions, and Plasma Membrane Substructure

The pseudobranch of the pinfish Lagodon rhomboides is an unusually homogeneous and structurally simple tissue, well suited to cell fractionation studies. Its principal cell type, closely related to the chloride cells of teleost gill, is characterized by numerous mitochondria in close association with abundant tubular invaginations of the plasma membrane. Other cytoplasmic organelles are rarely encountered. In broken fresh pseudobranch cells negatively stained with ammonium molybdate, a 40 Å particulate layer was observed on the intracellular surface of the tubular plasma membrane fragments. Nuclear (N), mitochondrial-light mitochondrial (M+L), and microsomal (P) fractions, obtained by differential centrifugation, were characterized by examination of fixed, embedded pellets and unfixed preparations negatively stained with ammonium molybdate and potassium phosphotung-state. Mitochondria, in orthodox configuration and retaining their outer membranes, were observed in M+L and N. Significant amounts of tubular, sheetlike, or vesicular membrane fragments were observed in all three fractions. Many such fragments, when negatively stained, showed the 40 Å particulate surface layer characteristic of plasma membrane invaginations, and in some cases 20-Å projections could be resolved on the opposite (extracellular) surface. Since these morphological observations, together with previously presented biochemical data, suggest a plasma membrane localization of Na⁺, K⁺-ATPase, the possible association of the enzyme with membrane projections is discussed.     

THE MECHANISM OF ACTION OF β-BUNGAROTOXIN

—β-Bungarotoxin, a presynaptically-acting polypeptide neurotoxin, caused an efflux from synaptosomes of previously accumulated γ-aminobutyric acid and 2-deoxy-d -glucose. The toxin-induced efflux of γ-aminobutyric acid occurred by a Na⁺ -dependent process while that of 2-deoxyglucose was Na⁺ -independent. These effects were also produced by treating synaptosomes with low molecular weight compounds, including fatty acids, that inhibit oxidative phosphorylation. After incubation with β-bungarotoxin, synaptosomes exhibited increased production of ¹⁴CO₂ from [U-¹⁴C]glucose and decreased ATP levels. β-Bungarotoxin treatment of various subcellular membrane fractions caused the production of a factor that uncoupled oxidative phosphorylation when added to mitochondria. Mitochondria from toxin-treated brain tissue exhibited a limitation in the maximal rate of substrate utilization. We conclude that β-bungarotoxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. This inhibition accounts for the observed β-bungarotoxin effects on synaptosomes and at neuromuscular junctions. We suggest that the effects on energy metabolism result from a phospholipase A activity found to be associated with the toxin.     

The membrane of the catecholamine storage vesicles of the adrenal medulla

Summary Ultrastructure and biochemical properties—catecholamine uptake and release and ATPase activity—of membrane preparation obtained from bovine adrenal medulla vesicles were studied.Destruction of vesicular organisation by sonication of catecholamine loaded membranes leads to the liberation of catecholamine, demonstrating that the latter is stored within the vesicular space rather than bound to the membrane.Four bands were obtained by sucrose gradient centrifugation, exhibiting distinct differences in ATPase activity and ability to take up catecholamine. The maior portion of material—fraction III—equilibrates in a position corresponding to 0.8 M sucrose. This fraction, although containing only 1/6 of catecholamine of the fraction I, displays the highest uptake of ¹⁴C-noradrenaline and a 4 fold higher ATPase activity than fraction I.Fraction III and IV contain several open membrane fragments and collapsed structures and a small proportion of disrupted mitochondria. Correlative estimations of succinate dehydrogenase- and ATPase-activity indicate that differences in ATPase activity of the fractions are only to a minor extent due to mitochondrial impurities.Negatively stained images reveal the great plasticity of the membrane of catecholamine storage vesicles, which is probably the basis of their ability to undergo the structural changes during the secretory cycle in the intact cell.     

Oxidative stress alters the regulatory control of p66 and Akt in PINK1 deficient cells

Mitochondrial dysfunction is involved in the underlying pathology of Parkinson’s Disease (PD). PINK1 deficiency, which gives rise to familial early-onset PD, is associated with this dysfunction as well as increased oxidative stress. We have established primary fibroblast cell lines from two patients with PD who carry mutations in the PINK1 gene. The phosphorylation of Akt is abrogated in the presence of oxidative stressors in the complete absence of PINK1 suggesting enhanced apoptotic signalling. We have found an imbalance between the production of reactive oxygen species where the capacity of the cell to remove these toxins by anti-oxidative enzymes is greatly reduced. The expression levels of the anti-oxidant enzymes glutathione peroxidase-1, MnSOD, peroxiredoxin-3 and thioredoxin-2 were diminished. The p66^Shc adaptor protein has recently been identified to become activated by oxidative stress by phosphorylation at residue Ser36 which then translocates to the mitochondrial inner membrane space. The phosphorylation of p66^Shc at Ser36 is significantly increased in PINK1 deficient cell lines under normal tissue culture conditions, further still in the presence of compounds which elicit oxidative stress. The stable transfection of PINK1 in the fibroblasts which display the null phenotype ameliorates the hyper-phosphorylation of p66^Shc.