Energy-dependent Fashion Research Articles

Calcium transport sensitive to ruthenium red in cytochrome oxidase vesicles reconstituted with mitochondrial proteins

We describe a calcium transport that is sensitive to ruthenium red in liposomes reconstituted with mitochondrial extracts. This system is able to build an internally negative membrane potential, which allows the electrogenic influx of Ca2+ and Sr2+. Proteins with molecular weights higher than 35 kDa were incorporated to the vesicles, and enhanced the accumulation of the cation in an energy-dependent fashion.

Stoichiometry of chargerin II (A6L) in the H +-ATP synthase of rat liver mitochondria

Previous studies suggested that the hydrophobic protein chargerin II, which is encoded in the A6L of mitochondrial DNA, may have a key role in the energy transduction by mitochondrial H +-ATP synthase because an antibody against chargerin II inhibited ATP synthesis and ATP-PI exchange, in an energy-dependent fashion. In the present work, the contents of chargerin II in the H +-ATP synthase purified from rat liver mitochondria and in submitochondrial particles were determined by radioimmunoassay. Results showed that the H +-ATP synthase contained chargerin II in a molar ratio of one to one. This is the first report on the stoichiometry of the A6L-product in mitochondrial H +-ATP synthase.

Orientation of chargerin II (A6L) in the ATP synthase of rat liver mitochondria determined with antibodies against peptides of the protein

Previous studies suggested that the hydrophobic protein chargerin II, which is encoded in the unidentified reading frame A6L of mitochondrial DNA (URFA6L), may have a key role in the energy transduction by mitochondrial ATP synthase because an antibody against chargerin II inhibited ATP synthesis and ATP-Pi exchange, in an energy-dependent fashion. In the present work, the orientation of chargerin II in F o of the ATP synthase of rat liver mitochondria was examined using antibodies against peptides of chargerin II. Results showed that its N-terminal region (about 8 amino acid residues) was exposed on the surface of the C-side of F o, but its C-terminal and charge-cluster regions were buried in F o.

Effect of NaCl and urea concentration comparable to renal medulla on superoxide production by human polymorphonuclear leukocytes.

The influence of hyperosmolarity on superoxide production by polymorphonuclear leukocytes (PMNL) was examined using NaCl and urea as osmotic substances. Superoxide production was inhibited in a hyperosmotic environment produced by high concentrations of these substances with the following IC50: 440 +/- 75 (SD) mOsm/kg for NaCl and 660 +/- 100 for urea. In the case of NaCl, this inhibition was time-dependent and abolished at 4 degrees C. Since PMNL pump out Na+ ion for maintenance of cellular volume in an energy dependent fashion, it was suggested that the inhibition of superoxide production was due to the exhaustion of energy stores. On the other hand, urea inhibition was almost immediate and remained even when preincubation was performed at 4 degrees C. Because the transport of urea through the cell membrane is known to be energy independent, these findings suggested that urea was either an inhibitor of the NADPH oxidase or a scavenger of superoxide anion.

Growth hormone binding to cultured human breast cancer cells.

The role of pituitary hormones in the pathogenesis of human breast cancer is unclear, although hypophysectomy is of therapeutic benefit in some patients with advanced breast cancer. Agents that lower serum PRL are of little value in the treatment of breast cancer, suggesting that other pituitary hormones may be important in the control of the growth of human breast cancer in vivo. Since human (h) GH is lactogenic in rodents, we investigated the binding of [125I]hGH and [125I]hPRL to the cultured human breast cancer cell lines T-47D and MCF-7. Both [125I]hGH and [125I]hPRL bound to a saturable binding site with high affinity (Ka = 0.94-1.70 X 10(9) M-1) and low capacity (4140-6560 sites/cells) in the two cell types. hGH and hPRL were mutually competitive, indicating that both hormones bound to the same receptor site. After binding of [125I]hGH to cell monolayers, the hormone was rapidly internalized in a time-, temperature-, and energy-dependent fashion. Lysosomotropic agents inhibited degradation of [125I]hGH and enhanced specific binding. Preincubation of MCF-7 cells with either hGH or hPRL resulted in loss of hGH/hPRL-binding sites, although hGH was consistently more potent in inducing down-regulation of the receptor. On the basis of these observations we suggest that hGH is a potent ligand for the lactogenic receptor in human breast cancer cells in vitro and may be important in the pathogenesis, growth, and metastasis of human breast cancer.

Triphenyltetrazolium and its derivatives are anisotropic inhibitors of energy transduction in oxidative phosphorylation in rat liver mitochondria

Triphenyltetrazolium and its derivatives inhibited energy transduction in mitochondria but not in submitochondrial particles, which are inside-out relative to the membranes of mitochondria. Triphenyltetrazolium incorporated into the inside of submitochondrial particles inhibited ATP synthesis in the particles. Triphenyltetrazolium also inhibited the reduction of NAD by succinate coupled with oxidation of succinate by O 2 and hydrolysis of ATP. Energization of mitochondrial inner membranes with succinate and with ATP induced sites on the membranes for triphenyltetrazolium and its derivatives. The maximum amounts of energy-dependent binding sites for triphenyltetrazolium on membranes energized with succinate and ATP, respectively, were 14 and 4 nmol / mg protein. Triphenyltetrazolium also induced H + ejection from the energized membranes. The maximum amounts of H + ejection from membranes energized with succinate and ATP, respectively, were 4 and 2.4 nmol / mg protein. Triphenyltetrazolium also decreased the membrane potential up to about half the control value and caused shrinkage of mitochondria in an energy-dependent fashion. Comparison of the Hammett's sigma constants of triphenyltetrazolium derivatives with various substituents on the 3-benzene ring showed that lower concentrations of triphenyltetrazolium derivatives with a stronger positive charge were required for inhibition of energy transduction. The present findings show that triphenyltetrazolium and its derivatives act as anisotropic inhibitors of energy transduction by binding to negative charges created on the outer side (C-side) of energized mitochondria, and that the positive charge of these inhibitors is one of important factors for their inhibitory activity. These negative charges may be an essential part of the H + pump.

Phosphate dependent, ruthenium red insensitive Ca 2+ uptake in mung bean mitochondria

Energy linked Ca 2+ uptake into mung bean mitochondria has been studied. Using arsenazo III as a monitor of extramitochondrial Ca 2+, we observe a respiration-linked uptake of Ca 2+ which requires phosphate and is insensitive to ruthenium red. The rate of uptake is of the order of 5 nmol/mg protein/min. Acetate, sulphate and thiosulphate are unable to support Ca 2+ uptake. The results suggest that although plant mitochondria accumulate Ca 2+ in an energy dependent fashion, it is not via a simple electrophoretic uniport mechanism.

Anisotropic inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by tetraphenylarsonium.

Tetraphenylarsonium (TPA+) inhibited energy transduction in oxidative phosphorylation in mitochondria but not in submitochondrial particles, which are inside-out relative to the membranes of mitochondria. TPA+ incorporated into the inside of submitochondrial particles inhibited ATP synthesis in the particles. TPA+ also inhibited the reduction of NAD by succinate coupled with oxidation of succinate by O2 and hydrolysis of ATP. Energization of mitochondrial inner membranes with succinate and with ATP induced binding sites on the membranes for TPA+. The amounts of energy-dependent binding sites for TPA+ on mitochondria energized with succinate and with ATP, respectively, were 90 and 13 nmol/mg of protein. TPA+ also caused shrinkage of mitochondria energized with succinate and with ATP in an energy-dependent fashion. The energy-dependent binding of TPA+, TPA+-induced H+-ejection, TPA+-induced shrinkage of mitochondria, and TPA+-induced inhibition of energy transduction occurred in parallel. The present findings show that TPA+ inhibits energy transduction by binding to negative charges created on lipophilic domains near the surface of the outer side (C-side) of the mitochondrial inner membranes, and that it has no inhibitory activity on the inner side (M-side) of the membranes.

Characterization of the energy-dependent calcium binding of a mitochondrial fraction isolated from bovine intrapulmonary vein.

A mitochondrial-enriched fraction was isolated from bovine intrapulmonary vein; this fraction had a three- to four-fold enrichment factor for succinic dehydrogenase, a mitochondrial marker enzyme, and bound calcium in an azide-sensitive, energy-dependent fashion using either ATP or succinate as substrate. The calcium binding was sensitive to the concentrations of H+, ATP, Ca2+, and mitochondrial fraction. Binding and accumulation were time dependent, reaching at 10–12 min a plateau which was maintained over the time interval studied, i.e. 18 min. At the plateau, maximal binding was 221 ± 8.64 and maximal accumulation was 285 ± 20.2 nmol Ca2+/mg protein. The mitochondrial-enriched fraction contained an azide-sensitive, Mg2+-dependent, Ca2+-stimulated ATPase (Mg2+ + Ca2+ = 25.59 ± 1.03, Ca2+ = 3.96 ± 1.06 μmol P1/mg protein per hour). This study is the first to report the isolation from intrapulmonary vein of a subcellular organelle capable of binding calcium in an energy-dependent fashion; furthermore, it confirms and extends the observations of others that mitochondria may contribute to intracellular calcium homeostasis in vascular smooth muscle.

The mobilization of arachidonic acid in platelets exposed to thrombin or ionophore A23187. Effects of adenosine triphosphate deprivation.

In studies conducted with human gel-filtered platelets, we have found: (a) that the release of serotonin and transfer of [3H]arachidonic acid from phosphatidylcholine and phosphatidylinositol to plasmalogen phosphatidylethanolamine which are associated with the activation of platelets by thrombin are both strongly dependent upon the presence of metabolic ATP; (b) that serotonin release and arachidonic acid mobilization in labeled phosphatides are promoted by the calcium ionophore A-23187 in media free of calcium ions; (c) that inhibitors of ATP synthesis, while leading to impairment of the release reaction induced by ionophore, do not inhibit ionophore-stimulated mobilization of arachidonic acid. We conclude that the activation of phospholipase A2 responsible for freeing arachidonic acid from platelet phosphatides is solely dependent upon the increased cytoplasmic levels of calcium ions promoted by either ionophore or, in an energy-dependent fashion by thrombin. Phospholipase activation is not a function of latent hydrolytic activity made available by the release reaction.

Influence of insulin on amino acid uptake by lung slices.

We examined the transport of amino acids by rat lung slices using mainly 14C-labeled alpha-aminoisobutyric acid ([14C]AIB) as a nonmetabolized amino acid. We found that [14C]AIB is accumulated by the lung in an energy-dependent fashion against a concentration gradient. The uptake is saturable, stereospecific, and follows Michaelis-Menten kinetics suggesting enzyme or carrier mediation across the plasma membrane. Insulin increases the uptake of [14C]AIB and insulin plus glucose increases its uptake even more. The diffusion constant (KD) in the presence of glucose, insulin, or glucose plus insulin is the same, 0.29 h-1; the Vmax is also the same, 83.0 mmol-1-1-h-1, under these three conditions. The apparent Km is 14.0 mM with glucose, 9.0 mM with insulin, and 4.0 mM in the presence of glucose and insulin. We conclude that the uptake of [14C]AIB is increased by insulin, and insulin plus glucose, and, based on this kinetic analysis, this is due to an increased affinity of the transport sites for AIB (decreased Km, unchanged Vmax, and KD).

Biogenesis of mitochondria. The effects of altered membrane lipid composition on cation transport by mitochondria of Saccharomyces cerevisiae.

1. The fatty acid composition of the membrane lipids of a fatty acid desaturase mutant of Saccharomyces cerevisiae was manipulated by growing the organism in a medium containing defined fatty acid supplements. 2. Mitochondria were obtained whose fatty acids contain between 20% and 80% unsaturated fatty acids. 3. Mitochondria with high proportions of unsaturated fatty acids in their lipids have coupled oxidative phosphorylation with normal P/O ratios, accumulate K(+) ions in the presence of valinomycin and an energy source, and eject protons in an energy-dependent fashion. 4. If the unsaturated fatty acid content of the mitochondrial fatty acids is lowered to 20%, the mitochondria simultaneously lose active cation transport and the ability to couple phosphorylation to respiration. 5. The loss of energy-linked reactions is accompanied by an increased passive permeability of the mitochondria to protons. 6. Free fatty acids uncouple oxidative phosphorylation in yeast mitochondria and the effect is reversed by bovine serum albumin. 7. The free fatty acid contents of yeast mitochondria are unaffected by depletion of unsaturated fatty acids, and free fatty acids are not responsible for the uncoupling of oxidative phosphorylation in organelles depleted in unsaturated fatty acids. 8. It is suggested that the loss of energy-linked reactions in yeast mitochondria that are depleted in unsaturated fatty acids is a consequence of the increased passive permeability to protons, and is caused by a change in the physical properties of the lipid phase of the inner mitochondrial membrane.

The energy-dependent accumulation of phosphate by blowfly mitochondria and its effect on the rate of pyruvate oxidation

During an investigation of the kinetic properties of the NAD-linked isocitrate dehydrogenase (NAD-IDH) of mitochondria derived from the thorax of Caliphora vomitora (a species of blowfly) it became apparent that this enzyme was inhibited by ATP and NADH 2 and was activated by ADP, Mg 2+, isocitrate and phosphate. A particularly striking feature was the dependence on phosphate. A plot of activity versus concentration resulted in a sigmoidal curve. At 5mM phosphate the enzyme produced 4–5% of its maximal activity and at 30mM 80%. Sacktor & Hurlbut (1966) have reported that at rest the phosphate content of whole blowfly thorax is 6.8μmoles/gm. wet wt. and in activity 7.5μmoles/gm. In this paper it is shown that isolated blowfly mitochondria are able to accumulate phosphate in a reversible energy-dependent fashion. The concentration in the intramitochondrial water can be as high as 4 times that in the suspending medium. The significance of this finding is illustrated in terms of the kinetic and steady-state response of O 2 uptake and respiratory carriers of intact mitochondria to ADP, phosphate and uncoupling agents. The conclusion is drawn that NAD-IDH activity is the prime controlling factor in the rate of O 2 uptake during pyruvate oxidation by blowfly flight-muscle mitochondria.