Inhibitor effects on redox-linked protonations of the b haems of the mitochondrial bc1 complex.

The effects of pH and inhibitors on the spectra and redox properties of the haems b of the bc1 complex of beef heart submitochondrial particles were investigated. The major findings were: (1) both haems have a weakly redox-linked protonatable group with pKox and pKred of around 6 and 8; (2) at pH values above 7, haem bH becomes heterogeneous in its redox behaviour. This heterogeneity is removed by the Qi site inhibitors antimycin A, funiculosin and HQNO, but not by the Qo site inhibitors myxothiazol or stigmatellin; (3) of all inhibitors tested only funiculosin had a large effect on the Em/pH profile of either haem b. In all cases where definite effects were found, the haem most affected was that thought to be closest to the site of inhibitor binding; (4) spectral shifts of haem groups caused by inhibitor binding were usually, but not always, of the haem group closest to the binding site; (5) titrations with succinate/fumarate were in reasonable agreement with redox-mediated data provided that strict anaerobiosis was maintained. Apparent large shifts of haem midpoint potentials with antimycin A and myxothiazol could be produced in aerobic succinate/fumarate titrations in the presence of cyanide, as already reported in the literature, but these were artefactual; (6) the heterogeneous haem bH titration behaviour can be simulated with a model similar to that proposed by Salerno et al. (J. Biol. Chem. (1989) 264, 15398-15403) in which there is redox interaction between haem bH and ubiquinone species bound at the Qi site. Simulations closely fit both the haem bH data and known semiquinone data only if it is assumed that semiquinone bound to oxidised haem bH is EPR-silent.

Mitochondrial precursor protein. Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence.

A hybrid precursor protein constructed by fusing the mitochondrial matrix-targeting signal of rat preornithine carbamyl transferase to murine cytosolic dihydrofolate reductase (designated pO-DHFR) was expressed in Escherichia coli. Following purification under denaturing conditions, pO-DHFR was capable of membrane translocation when diluted directly into import medium containing purified mitochondria but lacking cytosolic extracts. This import competence was lost with time, however, when the precursor was diluted and preincubated in medium lacking mitochondria, unless cytosolic proteins (provided by rabbit reticulocyte lysate) were present. Identical results were obtained for purified precursor made by in vitro translation. The ability of the cytosolic proteins to maintain the purified precursor in an import-competent state was sensitive to protease, N-ethylmaleimide (NEM), and was heat labile. Further, this activity appeared to be signal sequence dependent. ATP was not required for the maintenance of pO-DHFR competence, nor did purified 70-kDa heat shock protein (the constitutive form of Hsp70) substitute for this activity. Interestingly, however, purified Hsp70 prevented aggregation of the precursor in an ATP-dependent manner and, as well, retarded the apparent rate and extent of pO-DHFR folding. Partial purification of reticulocyte lysate proteins indicated that competence activity resides within a large mass protein fraction (200-250 kDa) that contains Hsp70. Sucrose density gradient analysis revealed that pO-DHFR reversibly interacts with components of this fraction. Pretreatment of the fraction with NEM, however, significantly stabilized the subsequent formation of a complex with the precursor. The results indicate that Hsp70 can retard precursor polypeptide folding and prevent precursor aggregation; however, by itself, Hsp70 cannot confer import competence to pO-DHFR. Maintenance of import competence correlates with interactions between the precursor and an NEM-sensitive cytosolic protein fraction. Efficient dissociation of the precursor from this complex appears to require a reactive thiol moiety on the cytosolic protein(s).

Cardiolipin molecular species in rat heart mitochondria are sensitive to essential fatty acid-deficient dietary lipids.

Feeding various dietary lipids did not alter the mass of phospholipids in rat heart mitochondria, but the phospholipids' fatty acid compositions changed. Although the compositional changes in mitochondrial phosphatidylcholine and phosphatidylethanolamine were negligible, linoleic acid [cis 18:2(n-6)] of cardiolipin was replaced by other fatty acids from dietary lipids. An analysis of the molecular species showed an even greater extent of change than was observed by fatty acid composition analysis. The quantity of 18:2(n-6)/18:2(n-6) molecular species [18:2(n-6) in C-1 position/18:2(n-6) in C-2 position] in cardiolipin decreased when rats were fed lipids that were depleted of an essential fatty acid but not deficient in essential fatty aids (eicosatrienoic acid did not appear). The decrease in 18:2(n-6)/18:2(n-6) species in cardiolipin was most pronounced in rats fed sardine oil, in which the ratio of (n-3) to (n-6) polyunsaturated fatty acids was 0.2. The O2 consumption rate of rat heart mitochondria decreased as the quantity of 18:2 (n-6)/18:2(n-6) cardiolipin species decreased.

[The effect of mitochondrial Ca 2+-transporting Ca 2+-binding glycoprotein on development of Ehrlich ascites carcinoma]. / Vliianie mitokhondrial'nogo Ca 2+-transportiruiushchego Ca 2+-sviazyvaiushchego glikoproteina na razvitie astsitnoi kartsinomy Erlikha.

Single administration of Ca2(+)-transporting-Ca2(+)-binding glycoprotein simultaneously with intraperitoneal inoculation of Ehrlich ascites tumor cells into mice led to a decrease in the animals weight growth by approximately 34% as compared with the animals which were not treated with the glycoprotein. The effect observed was clearly pronounced, especially within the first days after inoculation of Ehrlich ascites tumor. Lifetime of individual experimental animals was increased whereas that of the whole group remained unchanged.

[The temperature dependence of the respiration and phospholipid content of the mitochondria in the organs of heat-adapted rats]. / Temperaturnaia zavisimost' dykhaniia i fosfolipidnyi sostav mitokhondrii organov u adaptirovannykh k teplu krys.

A shift of breaking point to higher temperatures was illustrated on Arrhenius plots in kidney, liver and heart mitochondria of heat-adapted rats on 4.1, 2.7, 2.4 degrees C respectively. Q10 mitochondrion respiration fall in adapted animals in a range of 26-36 degrees C was indicative of temperature compensation. It was shown the elevation of phospholipid plasmalogen form content in heart and kidney mitochondria; as well as saturated fatty acids content in liver mitochondria. Structure and function changes were suggested to be the basic point of elevation of mitochondrion functional ability at high ambient temperature.

Isolation and amino acid sequence of a novel 6.8-kDa mitochondrial proteolipid from beef heart. Use of FAB-MS for molecular mass determination.

We have isolated a 6.8 kDa proteolipid from an acidic chloroform/methanol extract of bovine cardiac muscle. The molecular mass of the polypeptide was measured by fast atom bombardment-mass spectrometry (FAB-MS) (m/z6834.1). Its amino acid sequence was partly determined by direct sequencing and completed by characterization of cyanogen bromide and tryptic fragments (sequencing, FAB-MS and amino acid analysis). The polypeptide consists of 60 amino acid residues. Polyclonal antibodies raised in rabbit allowed its localization by electroimmunoblotting in mitochondria.

Evolution of cardiac involvement in progressive ophthalmoplegia with deleted mitochondrial DNA.

A 43-year-old woman with progressive external ophthalmoplegia developed a bifascicular block and dilatation of the right ventricle during 4 years of follow-up. Histochemical and electron microscopy studies detected mitochondrial abnormalities in ocular, skeletal muscle and cardiac biopsies. This case registers disease progression from the external ocular to the skeletal and cardiac muscles. Mitochondrial DNA was deleted in relation to the morphological abnormality.

Detection of hydroxyl radical in the mitochondria of ischemic-reperfused myocardium by trapping with salicylate.

Although the presence of free radicals has been indicated in ischemic-reperfused heart, the exact nature and source of these free radicals are not known. The present study utilized a chemical trap, salicylic acid, to trap hydroxyl radical which could be detected as hydroxylated benzoic acid using high pressure liquid chromatography. Since the hydroxylated product is extremely stable, heart was subjected to subcellular fractionation after ischemia and reperfusion, and each fraction was separately examined for the presence of hydroxyl radical. The results indicated for the first time the presence of hydroxyl radical in the mitochondrial fraction during early reperfusion, which decreased in intensity as the reperfusion progressed.

Purification and properties of the voltage-dependent anion channel of the outer mitochondrial membrane.

The methods for the purification of functionally active mitochondrial porin or voltage-dependent anion channel of the outer mitochondrial membrane are critically evaluated. Two rapid and efficient methods are now available. Both make use of a hydroxyapatite/celite column as a single chromatographic step. However, in one method with long polar head-group detergents, porin passes through the column, whereas in the other method, with shorter polar head-group detergents, porin is first bound to the column and then eluted by the addition of salts. On the basis of these results, a model for the arrangement of porin in the detergent-protein micelles is proposed.

Levels of dihomo-gamma-linolenate are depressed in heart phosphatidylcholine and phosphatidylethanolamine in the biotin-deficient chick.

Male broiler chicks were fed a purified diet with 0 or 500 micrograms/kg of added biotin for 21 days posthatch. Fatty acid composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were determined in erythrocytes and in heart mitochondria and microsomes of chicks at 12 and 21 days of age. The biotin-deficient (BD, 0 microgram biotin) chicks had lower body weights, lower feed efficiencies, and higher scores for dermatitis and twisted leg than the biotin-adequate (BA, 500 micrograms biotin) chicks. The erythrocyte arachidonate level in PC was higher in BD chicks compared to the BA controls, and the level increased with age in PC and PE in the erythrocytes. The level of dihomo-gamma-linolenate (20:3 omega 6) in PC and PE in heart mitochondria and microsomes was consistently decreased in BD chicks compared with that in BA chicks. In heart subcellular organelles, the level of arachidonate in PC increased, but the level of dihomo-gamma-linolenate in PE decreased with age. These data demonstrate that biotin deficiency results in dramatic changes in the fatty acid composition of specific phospholipids in chick erythrocytes and heart subcellular organelles.

Subcellular calcium content in cardiomyopathic hamster hearts in vivo: an electron probe study.

In the Syrian cardiomyopathic hamster heart, abnormal cellular calcium regulation, resulting in cellular calcium overload, is believed to play a role in the pathogenesis of cardiac hypertrophy and failure. Alternatively, the primary abnormality may be coronary vasospasm, resulting in reperfusion-induced necrosis. According to the latter hypothesis, only those cells that suffer an ischemic insult would contain elevated calcium levels. To determine whether a generalized elevation in myocytic calcium exists in myopathic hamster hearts, we measured cellular and subcellular calcium concentrations by electron probe microanalysis in cryosections of 50-day and 96-day myopathic and control hearts, rapidly frozen in vivo. Total calcium content of ventricular homogenates from each group was also measured by atomic absorption spectrophotometry. No significant differences in subcellular calcium were found by electron probe microanalysis among 50-day and 96-day myopathics and their age-matched controls. In 50-day myopathic and control hearts, mitochondrial calcium was 0.7 +/- 0.2 and 0.9 +/- 0.2, respectively, and A-band calcium was 3.0 +/- 0.4 and 2.6 +/- 0.4 mmol calcium/kg dry wt(+/- SEM). Results from 96-day animals were similar. Localized regions of elevated calcium were found only at sites of necrotic foci: in Na+-loaded cells (mitochondria: 4.7 +/- 1.3 (SEM) mmol/kg dry wt), in dying cells (mitochondria: 72 +/- 22 (SEM) mmol/kg dry wt) or as extracellular deposits (7-10 mol/kg dry wt). Total calcium content of hearts from myopathic hamsters, as determined by atomic absorption spectrophotometry, was also 13 times (50-day) and 50 times (96-day) higher than controls. These results demonstrate that there is a marked heterogeneity in cellular calcium content in myopathic hamster hearts, but the data do not support the hypothesis of a generalized cellular calcium overload.

Molecular basis of complement activation in ischemic myocardium: identification of specific molecules of mitochondrial origin that bind human C1q and fix complement.

Mitochondria may be a source of molecules that activate complement during ischemic injury to myocardium, providing therewith a stimulus for infiltration of polymorphonuclear leukocytes. To identify specific molecules that activate the classical complement pathway, detergent lysates of canine cardiac mitochondria were fractionated by polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose paper (NCP). The NCP replicas of the gels were incubated with isolated C1q and fresh sera as a source of complement, washed briefly, and overlaid with sensitized sheep erythrocytes (RBC) in agarose. A cluster of four to six molecules between 45 and 53 kDa as well as four others, 34, 30, 26, and 23 kDa, consumed complement thereby preventing complement-mediated lysis of sensitized sheep RBC in the agarose overlay. Additional molecules reactive with C1 were identified by their ability to bind isolated human C1q and to serve as assembly sites for later acting complement components. Sites of localization of complement were demonstrated by incubating NCP replicas of fractionated mitochondria with antisera specific for C1q, C3, C5, and C9, followed by peroxidase-conjugated anti-immunoglobulin and substrate. A total of 12 C1q binding molecules ranging in size from 67 kDa to 23 kDa, which can fix later acting complement components, were identified. At least two of these reacted with antisera prepared against canine cardiac lymph collected in the first 3-4 hours after a 45-minute coronary artery occlusion. These studies present direct evidence that specific molecules, released from subcellular fractions of myocardial cells rich in mitochondria, can activate the complement cascade.

Subcellular distribution of cardiac fatty acid-binding protein in bovine heart muscle and quantitation with an enzyme-linked immunosorbent assay.

Several types of the 14-15 kDa fatty acid-binding proteins (FABPs) are known to occur in the cytosol of mammalian cells. With antibodies raised against the cardiac-type protein from bovine heart, immunoblots indicated a more widespread distribution of the cardiac FABP in subcellular fractions, such as mitochondria and nuclei. A detailed view was obtained when the post-embedding protein A-gold labeling method was applied to cross-sections of heart cells and isolated subcellular fractions. Cardiac FABP in myocytes was associated with myofibrils and localized within mitochondria and nuclei. After subfractionation of mitochondria, the binding protein was recovered with matrix proteins only. A non-competitive enzyme-linked immunosorbent assay (ELISA) of the direct type was developed specifically for bovine cardiac FABP. This assay was sensitive in the range of 0.05 to 1 ng, and concentrations of cardiac FABP per mg protein were found for cytosol, matrix and nuclei to be around 3.18, 0.18 and 0.03 micrograms, respectively. The newly found compartmentation of cardiac FABP in the heart cell must be considered when the true functions of the protein, yet to be defined, are studied.

Phosphate transport protein of rat heart mitochondria: location of its SH-groups and exploration of their environment.

(1) The properties of the SH groups of the phosphate transport protein of rat heart mitochondria were investigated on the basis of inhibition caused by SH reagents under different conditions. (2) The essential thiol groups are located near the external surface, as they are accessible to impermeable reagents from the external space. (3) The environment of the sulfhydryl groups influences their reactivity, as alteration of the external pH affects adversely their reactions with ionizable and non-ionizable SH reagents. (4) Intramitochondrial pH exerts a transmembrane effect: alkalinization augments and acidification diminishes the reaction rate of the sulfhydryl groups on the opposite surface of the membrane. (5) Changes of the concentration of the transported substrate occurring exclusively in the extramitochondrial space do not influence the reactivity of the essential SH groups. (6) It is concluded that in transport studies the phosphate transport protein of heart and liver mitochondria show basic similarity. It is suggested that the amino-acid sequence around the NEM-reactive cysteine (i.e., Lys-41 - Cys-42 - Arg-43) does not participate in substrate binding.

Mitoskelin: a mitochondrial protein found in cytoskeletal preparations.

A 70 kD protein, which we have named mitoskelin, is highly enriched in cytoskeletal preparations from bovine cardiac muscle. Mitoskelin has three main variants with isoelectric points between 5.6 and 5.8. Immunoblotting with polyclonal antibodies directed against mitoskelin shows that, like intermediate filament proteins, the majority of mitoskelin resists solubilization from a myocardial homogenate by a series of extraction solutions ranging from very low salt to 0.6 M KI buffers and by 0.1-1% Nonidet P-40 detergent. By double-label immunofluorescence on cells and tissues, mitoskelin is colocalized with the mitochondrial marker cytochrome c oxidase. Mitoskelin is associated with the inner membranes of mitochondria as shown by immunoelectron microscopy and immunoblotting. Immunological cross-reactivity and similarities of molecular weight, pI, distribution, and chromatographic properties indicate that mitoskelin is the 70 kD component of complex I (NADH: ubiquinone oxidoreductase), a portion of the mitochondrial oxidative phosphorylation system. No function or activity has yet been demonstrated for the 70 kD component of the 25-polypeptide complex I. Dialysis against physiological buffers allows purified, urea-solubilized mitoskelin to form 10 nm wide filamentous structures that do not closely resemble intermediate filaments. These results suggest the exciting possibility that mitochondria may contain a membrane-associated filamentous skeleton.

Cardiac mitochondrial calcium content during fatal doxorubicin toxicity.

The purpose of this study was to determine whether abnormalities of mitochondrial divalent cation metabolism are early, causative events in doxorubicin (DXR, Adriamycin) cardiotoxicity. We used electron probe microanalysis (EPMA) to examine the calcium (Ca) and magensium (Mg) content of in situ mitochondria in cryosections of rat hearts, rapidly frozen at 6 hr and 1, 3, and 5 days after a single iv injection of 20 mg/kg DXR. This dose produced 100% mortality in 7 days, with a mean survival of 5.8 days. Mean control mitochondrial Ca and Mg was 0.7 and 28 mmol/kg dry wt, respectively (+/- SEM), and did not change in the DXR-injected animals, even in severely symptomatic rats 5 days after DXR. This suggests that an alteration in mitochondrial divalent cation metabolism is unlikely to be a primary event in the pathogenesis of DXR-induced cardiotoxicity, and that the mitochondrial Ca accumulation demonstrated in previous studies represents a secondary event in cells damaged by another mechanism.

Protective effect of a prostacyclin-mimetic on the ischaemic-reperfused rabbit myocardium.

To assess whether the administration of the stable prostacyclin-mimetic ZK 36374 (iloprost) protects the myocardium in a dose-dependent manner against ischaemia and reperfusion, isolated rabbit hearts were infused with three different concentrations of iloprost: 2.7, 27 and 270 nM. Diastolic and developed pressures were monitored; coronary effluent was collected and assayed for creatine phosphokinase (CPK) activity and for noradrenaline concentration; mitochondria were harvested and assayed for respiratory activity; ATP production and calcium content and tissue concentration of adenosine triphosphate (ATP) and creatine phosphate (CP) were determined. Treatment with iloprost altered neither developed pressure under normoxic conditions nor the rate and extent of depletion of ATP and CP during ischaemia. The ischaemic-induced deterioration of mitochondrial function, however, was attenuated. On reperfusion, hearts treated with iloprost recovered better than the untreated hearts with respect to left ventricular performance, replenishment of ATP and CP stores and mitochondrial function. The reperfusion-induced mitochondrial calcium overload and release of CPK and of noradrenaline were also significantly reduced. The effect of iloprost was dose-dependent. The lower concentration (2.7 nM) failed to modify ischaemic and reperfusion damage. The best protective effect was found at 27 nM. An increase of the dose to 270 nM did not result in further protection. It is concluded that iloprost infusion provides a dose-dependent protection of the heart against some of the deleterious effects of ischaemia and reperfusion and, in particular, prevents mitochondrial calcium overload and maintains mitochondrial function. Because this protection occurred in the absence of negative inotropic effect during normoxia or of a coronary dilatory effect during ischaemia, it cannot be attributed to an energy sparing effect or to improvement of oxygen delivery. Therefore, alternative mechanisms of action are to be considered.

Age-related changes in respiration coupled to phosphorylation. II. Cardiac mitochondria.

Age-related changes in mitochondrial adenine nucleotide metabolism may underlie the progressive decline in cardiac function. Oxidase activity coupled with phosphorylation, adenine nucleotide translocase (AdNT) activity, adenine nucleotide pool size and membrane lipid composition were determined using cardiac mitochondria from young (3 months), mature (12 months) and aged (24 months) Fischer 344 male rats which had been fed NIH-31 diet. While an age-associated 15% decrease in respiratory activity was not significant, AdNT activity of the aged rat was 20% lower (P less than 0.05) than that of the young rat. The exchangeable matrix adenine nucleotide pool (ATP + ADP) tended to decrease with age. In comparison to the young, membrane lipids of cardiac mitochondria from aged rat had a 43% higher (P less than 0.01) cholesterol/phospholipid-Pi ratio and a significantly lower (P less than 0.01) phosphatidyl ethanolamine/phosphatidyl choline ratio. The overall change in the fatty acid pattern of mitochondrial membrane lipids resulted in a significant (P less than 0.01) decrease in the n-6/n-3 fatty acid ratio. All values obtained for the mature rat fell between those of the young and aged rats. These data suggest that the reduced cardiac AdNT activity in the aged rat is a consequence of both a diminished pool of exchangeable adenine nucleotides and a lower AdNT velocity. Age-related changes in the lipid components of the membrane matrix in which the AdNT is embedded may underlie the decrease in respiratory activity.

Use of immunoblotting to characterize the mitochondrial antigens recognized by anti-mitochondrial autoantibodies.

Sera with anti-mitochondrial autoantibodies detected by indirect immunofluorescence and/or enzyme-linked immunosorbent assay (ELISA) were examined by immunoblotting against pig heart mitochondria. Seven types of reactions were defined, according to the pattern of the labelled bands. Type I sera reacted with 12 bands located within four zones. The most intensively labelled bands were located at 70, 67, 58, 63 and 43 kDa. Other types gave decreasing band numbers. When beef heart mitochondria were used, sera belonging to each of the above types had a profile of labelled bands which sometimes differed from those obtained with pig heart mitochondria. When the chloroform extracted F1-ATPase from beef heart mitochondria was used to prepare the immunoblots, primary biliary cirrhosis (PBC) sera with anti-mitochondria antibodies reacted with all the bands although zone A bands were less labelled. Rat liver mitochondria gave seven bands with type I sera among which the 57 and 35 kDa bands were specific for rat liver mitochondria, as shown by absorption tests. Sera of PBC patients were also tested in immunoblotting against rat liver subcellular fractions including mitoplasts, submitochondrial particles, inner membrane, outer membrane, matrix proteins and inter-membrane proteins. Antigenic bands of A and B zones were localized in the inner membrane and/or in the matrix proteins and the 35 kDa band in inter-membrane proteins. The outer membrane gave no reaction. The most frequent anti-mitochondrial autoantibody types in PBC were type II, then I, whilst for chronic active hepatitis type III was the most common. Type V was only seen in a patient suffering from a typical PBC. Some sera from patients with syphilis, collagenous colitis or progressive systemic sclerosis labelled one or two bands distinct from those labelled by the PBC sera. Sera from patients with drug-induced hepatitis with endoplasmic reticulum antibodies and with systemic lupus erythematosus were generally found negative by immunoblotting.