Effect of cyclosporine A and oligomycin on non-specific permeability of the inner mitochondrial membrane

The effect of oligomycin and cyclosporine A on the induction of non-specific permeability of the inner mitochondrial membrane by Ca²⁺ was under study. Both oligomycin and cyclosporine A were able to prevent the activation of non-specific permeability, but cyclosporine A was the only agent which could restore initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was shown not to be mediated through redistribution of Ca²⁺ between different mitochondrial subpopulations     

Kinetics of interaction between the H+-translocating component of the mitochondrial ATPase complex and oligomycin or dicyclohexylcarbodiimide

Kinetics of interaction between the H+-translocating component of the mitochondrial ATPase complex and oligomycin or dicyclohexylcarbodiimide were studied in beef heart submitochondrial particles, and the results suggest that the two inhibitors have different binding sites with respect to the membrane and to F1. Oligomycin seems to be bound to a subunit or a part of a subunit in F0, which is localized superficially, and which is influenced by F1, since the presence of F1 considerably lowers the rate of inhibition. The oligomycin binding site further seems to be influenced by the different conformational states of F1 occurring during the catalytic cycle of the enzyme. The binding site of DCCD on F0, on the other hand, seems to be deeply embedded in the membrane and not influenced by F1.     

Energy dependent changes in the cytochromes of the mitochondrial respiratory chain

In intact mitochondria a stoichiometric coupling exists between cytochrome a₃ and the hydrolysis of adenosine triphosphate (ATP). In each case the modification of one cytochrome a₃ (measured as a spectral change) is coupled to the hydrolysis of one ATP molecule. When both cytochromes a₃ and a are reduced the measured equilibrium constant is 0.06 m^?1 but this constant is 10³ M^?1 when both cytochromes are oxidized. When the sixth ligand for cytochrome a₃ is an externally added ligand (HCN, H₂S, CO, NO) the equilibrium constant is different for each ligand, suggesting that the ATP induced modification is of the fifth ligand but that it is energetically dependent on the chemical nature of the sixth ligand. The measured half-reduction potentials for cytochromes a₃ and b_T are dependent on the concentrations of added ATP, adenosine diphosphate (ADP), and orthophosphate. The relationship is consistent with a ligand exchange mechanism in which the ligand on the cytochrome is dependent on the phosphate potential ($\text{ATP}\text{ADP}$ × P_i). The equilibrium constants obtained by the ligand exchange treatment of the E_m values for cytochrome a₃ are consistent with those obtained by direct measurement of the equilibrium constants for the spectrally measured changes.     

Rat liver glutaminase. Regulation by reversible interaction with the mitochondrial membrane

Partially purified rat liver mitochondrial glutaminase shows a sigmoidal dependence on glutamine concentration, and an absolute requirement for inorganic phosphate as activator. Reconstitution with a mitochondrial membrane fraction changes the kinetic properties of the enzyme making the glutamine dependence more hyperbolic and reducing the concentration of phosphate required for half-maximum activation. Glutaminase activity in isolated mitochondria is known to be increased as a result of mitochondrial swelling. In mitochondria suspended in isotonic medium, the properties of glutaminase resemble of the isolated enzyme while in swollen mitochondria the kinetic properties revert to those exhibited by the enzyme in association with the mitochondrial membrane. It is postulated that mitochondrial glutaminase is regulated in situ by reversible association with the inner mitochondrial membrane which is mediated by mitochondrial swelling. This mechanism may explain the short-term hormonally induced activation of the enzyme observed in isolated hepatocytes.     

Effects of SH group reagents on creatine kinase interaction with the mitochondrial membrane

Solubilization of the specific mitochondrial isoenzyme of creatine kinase (CKm) from rabbit heart mitochondria by treatment with SH group reagents has been studied. From the various compounds tested only the negatively charged organomercurials are able to induce an extensive solubilization of the enzyme. This effect is fully reversible since the solubilized enzyme readily reassociates with the membrane when the bound organomercurial is removed by treatment of the homogenate by an excess of dithiothreitol. Solubilization by negatively charged organomercurials can be partly prevented by pretreatment of mitochondria with either disulfide or uncharged organomercurials. No clear-cut relationship has been pointed out when the amount of SH titrated by various reagents has been compared with the extent of CKm solubilization. More detailed studies with para-chloromercuribenzoate (pCMB) show that extensive CKm solubilization (about 75%) occurs for pCMB concentration as low as 25 microM, whereas pronounced inhibition of the enzyme is observed only for concentrations greater than 200 microM. By cross-reassociation of enzyme solubilized either by para-hydroxymercuribenzoate (pHMB) or by 20 mM sodium phosphate (NaPi) with mitochondria depleted of CKm by pHMB or by NaPi treatment, SH groups whose titration impedes CKm reassociation with the mitochondrial membrane have been tentatively located on the enzyme. Thus, negatively charged organomercurials, could induce a reversible conformational modification of the enzyme which is no longer able to bind on the inner mitochondrial membrane. Furthermore, our results show that the binding of an excess of mitochondrial CK, which has been previously reported, could reflect unspecific binding since it occurs only on mitoplasts incubated in very hypotonic medium, but not in isotonic medium.     

On the mechanism of oligomycin inhibition of Ca-induced mitochondrial respiration

The addition of oligomycin in the presence of Ca²⁺ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca²⁺-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5–20 μM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca²⁺-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca²⁺ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.     

Cytochrome c interaction with membranes. II. Comparative study of the interaction of c cytochromes with the mitochondrial membrane

A comparative study of the interaction of various cytochromes c with phospholipid vesicles and with mitochondrial membranes was undertaken. Both mammalian and yeast types of cytochrome c bind preferentially in the oxidized form as evidenced by the midpoint redox potential (E_m 7.0) becoming more negative upon binding. Cytochrome c which is reincorporated into cytochrome c-depleted mitochondria is kinetically comparable with the native cytochrome c component; rate of cytochrome b oxidation is maximally restored at ratios of c₁:c:a of 1:1:1. Comparison between the electron paramagnetic spectrum of cytochrome c labeled at methionine 65 or cysteine 103 reveals that upon binding to the mitochondrial membrane, the former is immobilized and not the latter. This result suggests that cytochrome c binds to the membrane at the side at which methionine 65 is located.     

Regulation of mitochondrial membrane permeability by a factor from mitochondrial interaction]

It was shown that a factor from the thyroxine-injured mitochondria caused inhibition of mitochondrial swelling in isotonic KNO3 and NH4NO3 solutions in the presence of rotenone. These data were interpreted as permeability inhibition for K+ and H+ ions.     

NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl-xL

Bcl-2 family proteins are essential regulators of cell death and exert their primary pro- or antiapoptotic roles at the mitochondrial outer membrane. Previously, pro- and antiapoptotic Bcl-2 proteins have been shown to interact with the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. VDAC is a 283-residue integral membrane protein that forms an aqueous pore in the outer mitochondrial membrane, through which metabolites and other small molecules pass between the cytosol and intermembrane space. The essential life-sustaining function of VDAC in metabolite trafficking is believed to be regulated by proteins of the Bcl-2 family. The protective role of antiapoptotic Bcl-xL may be through its interaction with VDAC. Here, VDAC has been expressed, purified, and refolded into a functional form amenable to NMR studies. Various biophysical experiments indicate that micelle-bound VDAC is in intermediate exchange between monomer and trimer. Using NMR spectroscopy, gel filtration, and chemical cross-linking, we obtained direct evidence for binding of Bcl-xL to VDAC in a detergent micelle system. The VDAC-interacting region of Bcl-xL was characterized by NMR with chemical shift perturbation and transferred cross-saturation. The interaction region was mapped to a putative helical hairpin motif of Bcl-xL that was found to insert into detergent micelles. Our results suggest that Bcl-xL can bind to one or two VDAC molecules forming heterodimers and heterotrimers. Our characterization of the VDAC/Bcl-xL complex offers initial structural insight into the role of antiapoptotic Bcl-xL in regulating apoptotic events in the mitochondrial outer membrane.     

Topography of oligomycin sensitivity conferring protein in the mitochondrial adenosinetriphosphatase-ATP synthase

The topographical organization of oligomycin sensitivity conferring protein (OSCP) in the mitochondrial adenosinetriphosphatase (ATPase)-ATP synthase complex has been studied. The accessibility of OSCP to monoclonal antibodies has been qualitatively visualized by using the protein A-gold electron microscopy immunocytochemistry or quantitatively estimated by immunotitration of OSCP in depolymerized or intact membranes. Besides, OSCP cannot be labeled by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which selectively labels the hydrophobic core of membrane proteins. These observations demonstrate an external location of OSCP on the inner face of the inner mitochondrial membrane. The position of OSCP relative to other peptides of the complex has been analyzed by cross-linking experiments using either zero length N-(ethoxycarbonyl)-2-ethoxydihydroquinoline or 11-A span dimethyl suberimidate cross-linkers in the ATPase-ATP synthase complex. The OSCP cross-linked products were identified either by immunocharacterization with anti-alpha, anti-beta, or anti-OSCP monoclonal antibodies or by their molecular weight. OSCP was cross-linked with either the alpha- or beta-subunits of F1 or to a subunit of Mr 24 000. Other types of cross-linking were obtained by the labeling of OSCP with [cysteamine-35S]-N-succinimidyl 3-[[2-((2-nitro-4-azidophenyl)amino)ethyl]dithio]propionate ([35S]SNAP) and reconstitution of SNAP-OSCP with F1 in urea-treated submitochondrial particles. Under these conditions, OSCP is found to be adjacent to two other peptides of molecular weight close to 30 000. A comparison is made between the topology and the organization of the b-subunit of Escherichia coli and OSCP, suggesting an analogy between OSCP and the hydrophilic part of the b-subunit.     

Cytochrome c interaction with the mitochondrial membrane: A spin label study

Horse-heart ferrocytochrome c has been labeled with N-(2,2,5,5-tetramethyl-3-pyrrolidinyl-1-oxyl) iodoacetamide at methionine-65. The paramagnetic resonance spectrum of labeled ferricytochrome c indicates a weak immobilization of the radical (τ_c = 9.3·10⁻¹⁰ sec) which becomes stronger upon binding of labeled cytochrome c to cytochrome c-depleted mitochondrial membranes (τ_c = 3.3·10⁻⁹ sec). The hyperfine coupling constant remains, however, unchanged (16.7 ± 0.1 gauss) indicating that the cytochrome c binding site is highly polar. The region where cytochrome c is bound to the membrane is insensitive to large variations of medium viscosity.     

Modifiers of the oligomycin sensitivity of the mitochondrial F1F0-ATPase

The mitochondrial F₁F₀ complex is highly sensitive to macrolide antibiotics and especially targeted by oligomycins. These compounds bind to the membrane-embedded sector F₀ and block proton conductance through the inner membrane, thus inhibiting both ATP synthesis and hydrolysis. Oligomycin sensitivity is universally recognized as a clue of the functional integrity and matching between F₀ and F₁. Since oligomycin binding implies multiple interactions with amino acid residues of F₀, amino acid substitutions often affect the inhibition efficiency. Moreover, variegated factors spanning from membrane properties to xenobiotic incorporation and detachment of the oligomycin-insensitive F₁ sector can alter the oligomycin sensitivity of the enzyme complex. The overview on the multiple factors involved strengthens the link between altered oligomycin sensitivity and physiopathological conditions associated with defective ATPases. An improved understanding of the mechanisms involved may also favor drug design to counteract oxidative damage, which stems from most mitochondrial dysfunctions.