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Bovine and dogfish (Squalus acanthias) heart submitochondrial particles and cytochrome c oxidase (EC 1.9.3.1) were prepared. Biphasic Eadie--Hofstee plots from steady-state polarographic assays were obtained for both species. Phospholipid analyses indicated that cardiolipin was absent from this active dogfish enzyme.
Interactions of adriamycin with ferritin-bound iron have been investigated. It is demonstrated (i) that adriamycin stimulates an iron-dependent lipid peroxidation in submitochondrial particles in the presence of ferritin, and (ii) that incubation of adriamycin with ferritin results in a slow transfer of iron to adriamycin with formation of an adriamycin-iron complex. The results are discussed in relation to the possible role for intracellular iron in adriamycin toxicity.
From the cell mass and culture supernatant of Myxococcus xanthus strain Mx X12 an antibiotic activity against yeasts, molds and some Gram-positive bacteria could be extracted. It consisted of 4 biologically active compounds which were named myxalamid A, B, C and D. The main component, myxalamid B, was shown to block in beef heart submitochondrial particles the respiratory chain at the site of complex I, i.e. NADH: ubiquinone oxidoreductase. The myxalamids are new antibiotics.
Specific antibody has been obtained against cytochrome b (pig heart mitochondria). It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome b site of the inner mitochondrial membrane. It has no effect on the isolated submitochondrial particles which are inside-out inner membrane vescicles free of any outer membrane or outside-out inner membrane. These findings indicate a probably not transmembranous topologic localization of cytochrome b; this component of the respiratory chain seems located near the outer side of the inner mitochondrial membrane.
ICRF-187 ((+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane) has shown promise (Speyer et al., N. Engl. J. Med. 319, 745 (1988] as a cardioprotective agent against what may be an iron-based adriamycin-induced cardiotoxicity. ICRF-187, which is membrane permeable, likely exerts its action through its rings-opened hydrolysis product which has a structure similar to EDTA and which, likewise, strongly binds metal ions. Both Fe3(+)-adriamycin and Cu2(+)-adriamycin reacted directly with ICRF-187, promoting a ring-opening hydrolysis of ICRF-187 that resulted in the displacement of the metal ion from its complex with adriamycin. Thus ICRF-187 can be considered to be acting as a "suicide protective agent" in its reaction with metal ion-adriamycin complexes. That this metal ion complex-promoted hydrolysis was preceded by mixed ligand complex formation is evidenced by the fact that the first-order rate constant for loss of metal ion from the adriamycin complex exhibits saturation behaviour at high ICRF-187 concentrations. Also direct spectroscopic evidence was obtained both for a Cu2(+)-adriamycin-ICRF-187 mixed ligand complex and a Cu2+ (ICRF-187)2 complex. The Fe3(+)-adriamycin complex inactivates the cytochrome c oxidase and NADH cytochrome c reductase activity on submitochondrial particles. The protection that ICRF-187 affords against this loss of activity may be explained both on the basis of simple Fe3+ removal from Fe3(+)-adriamycin and also on formation of a less active Fe3(+)-adriamycin-ICRF-187 mixed ligand complex.
It was found that mitochondrial oligomycin-sensitive ATPase (OS-ATPase) possesses the esterase activity with respect to some carboxylic acid esters with phenols and arylalcane alcohols. The substrate specificity of the esterase found was studied. The effects of some inhibitors and activators of ATPase on the enzyme activity were demonstrated. It was found that ADP inhibits the enzyme from submitochondrial particles containing factor F1 and does not inhibit the enzyme from the particles devoid of this factor. The data obtained suggest that esterase is localized in the hydrophobic part of the oligomycin-sensitive ATPase complex and are indicative of the functional interrelationship between the esterase and ATPase activities.
Almost complete slow activation of the deactivated purified Complex I was observed after the steady-state NADH: cytochrome c reductase reaction turnovers catalyzed by the endogenous (tightly-bound) ubiquinone and contaminant Complex III. The rotenone-sensitive NADH oxidase was reconstituted from bovine heart Complex I and Escherichia coli quinol-oxidase. The ratio between active and inactive Complex I during the steady-state NADH oxidase reaction catalyzed by the reconstituted system was shown to be proportional to the rate of quinol oxidation. Analogous result was obtained for the NADH oxidase reaction catalyzed by the submitochondrial particles. It is concluded that the ratio between active and inactive Complex I depends on the quinone redox state. One of the functions of ubiquinone-binding site(s) in Complex I is the control of its active/inactive state.
10 sera were studied from patients with primary biliary cirrhosis (PBC), that were anomalous in their reactivity against mitochondrial antigens as detected by Western blotting. They had low reactivity against the major, M2 reactive antigen (Mr for beef heart mitochondria, 74 Kd) but reacted against an antigen of Mr 52 Kd (species independent) which was apparently inaccessible in submitochondrial particles (SMP) on ELISA and which was not present in chloroform-released ATPase preparations. In all respects this differed from the characteristics of the M2 antigens and it is concluded that these sera are detecting predominantly the M4-reactive antigen.
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.
The purified, soluble F1-ATPase was modified by several covalently reacting inhibitors, either known or considered to bind to the active site bearing beta-subunit, to cause partial inhibition up to 99%. The modified enzyme was then reconstituted in the presence of OSCP (oligomycin sensitivity conferring protein) with submitochondrial particles (SMP) almost completely (greater than 99%) denuded of active F1-ATPase and was assayed for oligomycin-sensitive ATPase and oxidative phosphorylation activities. The inhibitors used were 1-fluoro-2,4-dinitrobenzene (FDNB), N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCD), quinacrine mustard (QM), 5-(dimethylamino)-naphthalene-1-sulfonyl chloride (dansyl-Cl), 5'-[p-(fluoro-sulfonyl)benzoyl]adenosine (FSBA), and N,N'-dicyclohexylcarbodiimide (DCCD). The SMP reconstituted with unmodified F1 exhibited oxidative phosphorylation and oligomycin-sensitive ATPase (in the presence of uncouplers) activities as high as 500 nmol min-1 mg-1 and 8 mumol min-1 mg-1, respectively. The systems reconstituted with F1 modified to cause various degrees of inhibition with FDNB, EEDQ, CMCD, QM, and dansyl-Cl exhibited the same degree of inhibition of oxidative phosphorylation and oligomycin-sensitive ATPase activities as the inhibition of the ATPase activity of the modified F1 before reconstitution. The systems reconstituted with FSBA-modified F1 showed the following relative degrees of inhibition: oxidative phosphorylation greater than oligomycin-sensitive ATPase of particles greater than ATPase of soluble F1. In contrast, the systems reconstituted with DCCD-modified F1 showed much greater inhibition of oligomycin-sensitive ATPase than of oxidative phosphorylation activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Cinnarizine and flunarizine are piperazine derivatives with calcium antagonist and anticonvulsant properties and are used widely in the treatment of vertigo and circulatory disorders. They have been implicated recently in the aggravation, or even the induction, of parkinsonism in elderly patients. Because the aetiology of parkinsonism has been suggested as having a mitochondrial component, we have investigated the effects of both compounds on mitochondrial respiration and on the activities of the individual respiratory chain complexes. In intact mitochondria from rat liver, both drugs inhibited respiration rates, with substrates entering at Complex I (glutamate/malate) and Complex II (succinate). These effects could be explained by potent inhibitions (Ki 3-10 microM) of both complexes. Complex I is inhibited at a site near the ubiquinone-binding site, which is not competitive with respect to ubiquinone, whereas the inhibition of Complex II is apparently caused by competition with ubiquinone. Furthermore, the inhibition of NADH oxidation by flunarizine in submitochondrial particles caused an NADH-dependent generation of superoxide. These inhibitory properties of both compounds could be significant factors in the aggravation or induction of parkinsonism in elderly patients, in whom mitochondrial function already may be impaired.
The hydrophobic subunit 8 of the yeast ATP synthase was modified using the non-penetrating amino reactive specific reagent: isethionylacetimidate. The polypeptide was modified when using the isolated ATP synthase and sodium bromide-treated submitochondrial particles. It is shown that the only lysine of the protein was modified by the reagent. It is concluded that the hydrophilic C terminal part of the protein containing lysine 47 is located on the inner side of the inner mitochondrial membrane.
A study of kinetic properties of mitochondrial ATPase in Morris hepatoma 3924A is reported. The results show that submitochondrial particles isolated from the tumor tissue exhibited a three-fold increase in both the Km for ATP hydrolysis and Ki for the competitive inhibitor [beta, gamma-imido]ATP with regard to normal rat liver. Eadie-Hofstee analysis of the kinetics of ATP hydrolysis show that both the high and the low affinity constants for ATP were enhanced in the hepatoma with respect to the rat liver enzyme. Kinetic analysis of passive proton conduction through the F0 sector of ATPase does not reveal any difference between Morris hepatoma and rat liver. In Morris hepatoma particles, 50% inhibition of the hydrolase activity required 10 times more oligomycin than in control particles. On the contrary, 50% inhibition of proton conduction occurred in both hepatoma and rat liver particles at the same concentration of oligomycin. It is concluded that in Morris hepatoma the catalytic process in F1 and the functional connection between F1 and F0 of the ATP synthase are altered with regard to control rat liver.
Submitochondrial particles prepared by treatment of mitochondria with ammonia and silicotungstic acid were found to be deficient in coupling factor 6 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting and had reduced ATP-Pi exchange activity. Requirement of coupling factor 6 for passive proton conductance through mitochondrial F0 was investigated by assaying the ability of depleted particles to sustain NADH-induced proton fluxes as measured by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence. The depleted particles themselves showed negligible quenching, but the quenching increased markedly after treating the particles with oligomycin. The data show for the first time that coupling factor 6-depleted complexes have an active proton channel that can be blocked by oligomycin. Therefore, coupling factor 6 is not essential for inhibitor-sensitive proton conductance through mitochondrial F0.
The capacity of mitochondria isolated from tumor cells to conserve the transmembrane electrochemical proton gradient set up by respiration has been studied. In a K+ medium, mitochondria from Ehrlich ascites tumor cells exhibit a capacity to conserve aerobic delta microH comparable to that displayed by normal rat liver mitochondria. Mitochondria from Morris hepatoma 3924A show a decreased capacity to store delta microH+, which is principally due to lowering of delta pH. In a Na+ medium, both species of tumor mitochondria show a significant decrease of aerobic delta pH, while delta psi is the same, with respect to rat liver mitochondria. Experiments on passive swelling show that mitochondria from ascites tumor cells have an enhanced permeability to chloride salts of monovalent cations and increased activity of the Na+ (K+)-H+ exchange system of the mitochondrial membrane with respect to normal mitochondria. The enhanced activity of this system in ascites cells is also shown by the characteristics of respiration-linked proton translocation in submitochondrial particles and subsequent anaerobic proton diffusion. It is concluded that the decreased capacity of mitochondria from tumor cells to conserve aerobic delta pH is due to enhanced cyclic flow of Na+ across the membrane.
Injection of large doses of ammonium salts lead to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We have tested the effect of injecting 7 mmol/kg of ammonium acetate on the production of superoxide and on the activities of some antioxidant enzymes in rat liver, brain, erythrocytes and plasma. Glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver and brain (both in cytosolic and mitochondrial fractions) and also in blood red cells, while glutathione reductase activity remained unchanged. Superoxide production in submitochondrial particles from liver and brain was increased by more than 100% in both tissues. Both diminished activity of antioxidant enzymes and increased superoxide radical production could lead to oxidative stress and cell damage, which could be involved in the mechanism of acute ammonia toxicity.
2-Hydroxy-5-nitrobenzyl bromide, a highly reactive reagent towards tryptophan residues in proteins, is shown to activate the passive proton flux through the inner mitochondrial membrane of bovine heart submitochondrial particles (ETPH). When added at low concentrations, the reagent increased both the ATPase activity of the particles and the passive proton transport rate through the membrane. The presence of oligomycin reduced the extent of the 2-Hydroxy-5-nitrobenzyl bromide action on the proton conductivity suggesting that it acted primarily on the H+-ATPase complex. Similar effects were observed on F1-depleted particles, whilst no effect was observed on the isolated F1-ATPase activity. The results suggest that polypeptides bearing tryptophan residues may be involved in the gating function of proton channels of the mitochondrial membrane and this is particularly evident for the F0F1-ATPase complex.
The mechanism of the hydroperoxide-induced release of calcium from rat liver mitochondria was investigated. In calcium-loaded mitochondria both the hydroperoxide-induced hydrolysis of pyridine nucleotides and the release of calcium is inhibited by intramitochondrial ATP. In submitochondrial particles, in the absence of ATP a hydrolytic product of NAD+, probably ADP-ribose, binds covalently to a protein of the inner mitochondrial membrane. It is proposed that a compound deriving from NAD(P)+ is an important factor in the calcium release mechanism in rat liver mitochondria.