Effect of calcium ion on triiodothyronine binding to kidney outer mitochondrial membrane in vitro

The effect of calcium ion on 3,5,3'-triiodothyronine (T3) binding to rat kidney outer mitochondrial membranes was examined in vitro. The outer mitochondrial membranes were prepared by using a discontinuous sucrose density gradient centrifugation. The membrane fraction, which is enriched with monoamine oxidase activity, contained specific binding sites for T3. Scatchard analysis of T3 binding to outer mitochondrial membranes gave an association constant (Ka) of 0.53 X 10(10)M-1. The binding of [125I]-T3 to the membranes was inhibited by the addition of CaCl2(0.25 X 10(-4)--2.5 X 10(-3)M). 50% inhibition was obtained by 0.75 X 10(-4)M CaCl2 in the presence of 0.1 mM EGTA. When outer mitochondrial membranes were solubilized with Triton X-100, four main T3 binding activities were isolated by a gel filtration study. On the other hand, the binding of [125I]-T3 to the solubilized T3 receptors derived from outer mitochondrial membranes was not strongly inhibited by calcium. When outer mitochondrial membranes were preincubated in the presence of 1 mM calcium, the number of T3 binding sites in the membranes was decreased, and this was associated with an increase in the number of T3 binding sites in the supernatants of the incubation mixture. Scatchard analysis showed that the number of T3 binding sites in the membranes is decreased by calcium ion without any change in the association constant. In studies with gel filtration of receptors which are released by Ca2+ from outer mitochondrial membranes, three main T3 binding activities were isolated. Mg2+, Mn2+, Zn2+ and Cu2+ did not affect T3 binding to outer mitochondrial membranes. The results indicate that calcium ion regulates T3 binding to the outer mitochondrial membrane through the release of T3 receptors from the membranes.     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both pro-mitochondria and mitochondria obtained from O₂-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N₂) to elaidic (O₂)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O₂-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.     

Effects of neuroleptics on 3H-haloperidol and 3H-cis(Z)-flupenthixol binding and on adenylate cyclase activity in vitro.

The subcellular localization of dopamine-sensitive adenylate cyclase was studied in rat brain striatum and compared to the distribution of dopamine binding sites. The highest specific activity of adenylate cyclase activities sensitive to dopamine was associated almost exclusively with synaptic membranes (mithchondrial fraction; P₂). Using [³H] haloperidol and [³H] apomorphine as markers for the dopamine receptor, specific binding was observed in both the mitochondrial (P₂) and microsomal (P₃) fractions. Data for the mitochondrial fraction revealed a heterogeneity of binding sites. Two saturable sites for [³H] haloperidol were observed with K_d values of 2.5nM and 12.5nM respectively. Overall, the localization of multiple binding sites in the crude synaptosomal fraction correlates well with the localization of dopamine-sensitive adenylate cyclase in this fraction.     

Subcellular distribution of [H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE₂ binding sites in four subcellular fractions (F1–F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of HPGE₂ to fractions F2–F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE₂ binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Subcellular distribution of [3H]-prostaglandin E2 binding sites in porcine gastric mucosa

The distribution of PGE₂ binding sites in four subcellular fractions (F1–F4) from porcine fundic mucosa obtained by gradient centrifugation was examined. Binding of HPGE₂ to fractions F2–F4 was specific, dissociable, saturable and pH dependent. A significant degree of specific binding was not evident in F1. The Scatchard analysis of binding to F2 and F3 revealed heterogenous populations of binding sites with similar dissociation constants but greater concentrations of binding sites than was evident in the initial 30,000 xg homogenate protein. A single class of low affinity binding sites was evident in F4. The ratio of total: nonspecific binding was approximately equal in F2 and F3. The ratio was considerably smaller in F4. The activity of 5' nucleotidase the marker enzyme for plasma membranes followed this ratio. There was no correlation between the binding ratio and marker enzyme activities for mitochondrial membranes and endoplasmic reticulum. These data suggest that high affinity PGE₂ binding sites occur predominantly on the plasma membrane from gastric mucosal tissue.     

Identification of the initial binding sites of αs2-casein f(183-207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide α_s2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of α_s2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the α_s2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.     

Substance P: Regional distribution and specific binding to synaptic membranes in rabbit central nervous system

Regional distribution of endogenous substance P and the specific [³H]substance P binding to synaptic membranes in rabbit central nervous system were investigated. The highest level of substance P was found in mesencephalon, followed by diencephalon, corpus striatum, hippocampus, pons-medulla and cortex. In spinal cord, much higher amount of substance P existed in dorsal half than in ventral half. Most of the substance P present in the areas enriched in substance P was located in crude mitochondrial P₂ fractions containing nerve endings. A saturable, high affinity, specific binding of [³H]substance P in synaptic membranes was found. The apparent maximal number of binding sites was 95.7 fmole/mg protein, while the dissociation constant (K_D) was 2.74 nM. The binding was displaced by substance P sequence fragments and the related peptides with relative potencies generally parallelizing their pharmacological activities. The distribution of such specific binding generally correlated with endogenous substance P. The presence of such binding sites for substance P in synaptic membranes suggests a possible role for substance P as a transmitter or modulator of neural function.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.     

Influence of Ca2+ on the isolation from rat brain mitochondria of a fraction enriched of boundary membrane contact sites

Data have been obtained suggesting that the complex porin-hexokinase of brain mitochondria may be related to the contact sites between the outer and inner membrane. In the attempt to isolate from brain mitochondria the inner and outer membranes and the boundary membrane contacts, a procedure was developed based on swelling and shrinking of the organelles, followed by sonication and reverse discontinuous density gradient centrifugation. Three fractions were obtained by this technique, which were identified by measuring the relative specific activities of marker enzymes, namely succinate-cytochrome c reductase; NADH-cytochrome c reductase (rotenone insensitive); hexokinase and glutathione transferase, for the inner and outer membranes and contact sites, respectively. The fraction which contains the contact sites is characterized by the highest specific activity of hexokinase and glutathione transferase and by the highest calcium binding capacity; physiological concentrations of this cation produces a sharper separation of this fraction. Results indicate that both the porin-hexokinase gating system of the outer membrane and the calcium transporting complex of the inner membrane are present in the fraction which contains the contact sites.     

The mitochondrial import machinery: preprotein-conducting channels with binding sites for presequences

Mitochondrial preproteins with amino-terminal presequences must cross two membranes to reach the matrix of the organelle. Both outer and inner membranes contain hydrophilic high-conductance channels that are responsible for selective translocation of preproteins. The channels are embedded in dynamic protein complexes, the TOM complex of the outer membrane and the TIM23 complex of the inner membrane. Both channel-forming proteins, Tom40 and Tim23, carry specific binding sites for presequences, but differ in their pore size and response to a membrane potential. Studies with the TOM machinery show that other subunits of the translocase complex also provide specific binding sites for preproteins, modulate the channel activity and are critical for assembly of the channel.     

The distribution of anionic sites on the surfaces of mitochondrial membranes. Visual probing with polycationic ferritin

Polycationic ferritin, a multivalent ligand, was used as a visual probe to determine the distribution and density of anionic sites on the surfaces of rat liver mitochondrial membranes. Both the distribution of bound polycationic ferritin and the topography of the outer surface of the inner mitochondrial membrane were studied in depth by utilizing thin sections and critical-point dried, whole mount preparations for transmission electron microscopy and by scanning electron microscopy. Based on its relative affinity for polycationic ferritin, the surface of the inner membrane contains discrete regions of high density and low density anionic sites. Whereas the surface of the cristal membrane contains a low density of anionic sites, the surface of the inner boundary membrane contains patches of high density anionic sites. The high density anionic sites on the inner boundary membrane were found to persist as stable patches and did not dissociate or randomize freely when the membrane was converted osmotically to a spherical configuration. The observations suggest that the inner mitochondrial membrane is composed of two major regions of anionic macromolecular distinction. It is well-known that an intermembrane space exists between the two membranes of the intact mitochondrion; however, a number of contact sites occur between the two membranes. We determined that the outer membrane, partially disrupted by treatment with digitonin, remains attached to the inner membrane at these contact sites as inverted vesicles. Such attached vesicles show that the inner surface of the outer membrane contains anionic sites, but of decreased density, surrounding the contact sites. Thus, the intermembrane space in the intact mitochondrion may be maintained by electronegative surfaces of the two mitochondrial membranes. The distribution of anionic sites on the outer surface of the outer membrane is random. The nature and function of fixed anionic surface charges and membrane contact sites are discussed with regard to recent reports relating to calcium transport, protein assembly into mitochondrial membranes, and membrane fluidity.     

Role of membrane contact sites in protein import into mitochondria

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.     

Lack of stimulation of renal (Na+ + K+)-ATPase by thyroid hormones in the rabbit

The effect of l-3,5,3′-triiodothyronine (T₃) and thyroxine (T₄) on (Na⁺ + K⁺)-ATPase activities was examined in rabbit kidneys because in this tissue almost 80% of the metabolism is connected to active sodium transport. T₃-receptor concentrations were estimated as 0.62 and 0.80 pmol/mg per DNA in the cortex and outer medulla, respectively. A dose of 0.5 mg T₃/kg body weight for 3 days increased basal metabolic rate by almost 60%, and the mitochondrial 1-α-glycerophosphate dehydrogenase activity was increased by 50% in both the cortex and medulla. (Na⁺ + K⁺)-ATPase activity in the liver was raised by almost 50%. However, no changes in (Na⁺ + K⁺)-ATPase activities or binding sites for [³H]ouabain in either the kidney cortex or medulla could be observed. T₄ at 16 mg/kg daily for 14 days was also without effect on renal (Na⁺ + K⁺)-ATPase activities. Furthermore, the response to T₃ was absent at high sodium excretion rates induced by unilateral nephrectomy and extracellular volume expansion. Thus, despite stimulation of basal metabolic rate and renal 1-α-glycerophosphate dehydrogenase activity by T₃ and T₄, the (Na⁺ + K⁺)-ATPase activity in the rabbit kidney is identical in euthyroid and hyperthyroid states. However, thyroid hormones prevent the normal natriuretic response to extracellular volume expansion.     

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The precursor of cytochrome b₂ (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b₂ precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.     

Mitochondrial protein import: differential recognition of various transport intermediates by antibodies

The precursors of the mitochondrial proteins ADP/ATP carrier (AAC) and F1-ATPase subunit beta (F1 beta) were accumulated at the stages of binding to receptor sites on the mitochondrial outer membrane, or in contact sites between outer and inner membranes. Specific antibodies raised against the mature proteins were added to the isolated mitochondria and efficiently bound to these translocation intermediates. Further movement of the precursors to consecutive steps along their import pathway was thereby inhibited. Controls showed that precursor proteins which were inserted into or translocated across the outer membrane were not recognized by the antibodies unless the mitochondrial membranes were disrupted. We conclude that the trapped translocation intermediates have antigenic sites exposed to the outside of the outer membrane.     

Effect of aging on the composition of mitochondrial membranes from potato slices

The phospholipid content of mitochondrial membranes from slices of potato tuber (Solanum tuberosum) remains stable during aging. The phospholipid compositions of whole mitochondria and inner membranes do not vary during aging whereas the concentrations of phosphatidylinositol and phosphatidyl-glycerol in outer membranes are slightly amplified. The saturation of outer membrane fatty acids is slightly increased during aging. Gel electrophoresis of mitochondrial membrane proteins show slight variations of one polypeptide in outer membranes and of three polypeptides in inner membranes. These results suggest parallel variations of lipids and proteins in membranes during aging, in marked contrast with the large modifications observed in mitochondrial activities.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Dextran causes aggregation of mitochondria and influences their oxidoreductase activities and light scattering

It has been reported that dextrans diminish the intermembrane space of mitochondria, increase the number of contact sites between the inner and the outer mitochondrial membranes, decrease the outer membrane permeability to adenosine 5(')-diphosphate, and change the kinetic properties of mitochondrial kinases. In the present work the influence of dextran M40 (5% w/v) on the oxidoreductase activities of the inner and outer membranes of mitochondria, the interaction of cytochrome c with mitochondrial membranes, and the light scattering by rat liver mitochondria were studied. No influence of dextran on the release of cytochrome c from mitochondria or its interaction with mitochondrial membranes was observed. Decreases in the NADH-oxidase (to 80+/-2% of the control), NADH-cytochrome c reductase (to 26+/-2%), succinate-cytochrome c reductase (to 70+/-5%), and NADH-ferricyanide reductase (to 75+/-3%) activities induced by dextran, which may be due to the mitochondrial aggregation, were observed. The formation of aggregates was registered by light scattering, confirmed by light microscopy, and explained within the framework of the Gouy-Chapman theory of the electrical double layer. The observed mitochondrial aggregation seems to be useful also for understanding the mechanisms of mitochondrial condensation and perinuclear clustering during apoptosis.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.