Dynamic organization of mitochondrial membranes: A crosslinking study with dimethylsuberimidate

Rat liver mitochondria were treated with dimethylsuberimidate, a bifunctional alkylating agent, and the effects were evaluated kinetically. Concurrently with the modification of amino groups, mitochondrial proteins were crosslinked and the organelles lost their osmotic response. When the dimethylsuberimidate reaction was performed in the presence of succinate, more primary amino groups were available when compared with a sucrose medium. Concomitantly, osmotic stabilization and crosslinking of mitochondrial proteins were accelerated. The activity of aspartate aminotransferase was also studied in crosslinked mitochondria. The enzyme activity was only slightly modified when mitochondria were amidinated in a sucrose medium and solubilized thereafter with Triton X-100 or cetyltrimethylammonium bromide. In contrast, in the presence of succinate, 60% of activity was lost after solubilization with Triton X-100, but not after solubilization with cetyltrimethylammonium bromide. This finding was correlated with the changes in intramitochondrial localization of the enzyme (A. Waksman and A. Rendon, 1974,Biochimie54, 907–924). When carbonylcyanide-p-trifluoromethoxyphenylhydrazone was added in both cases (sucrose or sucrose plus succinate), the rates of osmotic stabilization, amidination reaction, crosslinking of proteins, and aspartate aminotransferase activity were similar to those observed in a sucrose medium alone. The present results suggest that organizational changes of the mitochondrial membranes induced by succinate, including intramitochondrial protein movement, are prevented by carbonylcyanide-p-trifluoromethoxyphenylhydrazone.     

The oxidative inactivation of mitochondrial electron transport chain components and ATPase

Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (.OH) alone, superoxide anion radical (O2-) alone, or mixtures of .OH and O2-, by gamma radiolysis in the presence of 100% N2O (.OH exposure), 100% O2 + formate (O2- exposure), or 100% O2 alone (.OH + O2- exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by .OH (10% inactivation at 15-40 nmol of .OH/mg of SMP protein, 50-90% inactivation at 600 nmol of .OH/mg of SMP protein) and by .OH + O2- (10% inactivation at 20-80 nmol of .OH + O2-/mg of SMP protein, 45-75% inactivation at 600 nmol of .OH + O2-/mg of SMP protein). Importantly, O2- was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O2-/mg of SMP protein, 40% inactivation at 600 nmol of O2-/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O2-/mg of SMP protein, 30% inactivation at 600 nmol of O2-/mg of SMP protein), and a poor inactivator of succinate oxidase (less than 10% inactivation at 600 nmol of O2-/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at .OH, O2-, OH + O2-, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of .OH/mg of SMP protein. The .OH-dependent inactivations reported above were largely inhibitable by the .OH scavenger mannitol. In contrast, the O2(-)-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with .OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of chlorambucil, a bifunctional alkylating agent, on the histone variant biosynthesis of HEp-2 cells

The effect of chlorambucil on the synthesis of histone variants of a cancer cell line HEp-2 is analysed and compared to that of nontreated and hydroxyurea treated cells. Cell proteins were labelled with [14C]lysine and [14C]arginine and histone variants resolved by one- or two-dimensional electrophoresis. Chlorambucil shows no significant decrease in total protein synthesis but shows a significant decrease in histone biosynthesis. It does not selectively inhibit the synthesis of the S-phase variants, i.e., H2A.1, H2A.2, H3.2 or the G1/G2 phase (basal) histone variants, i.e., H2A.Z, H2A.X and H3.3. On the contrary, hydroxyurea treated cells, which also show no significant decrease in amino acid incorporation into total cellular protein but do exhibit a significant inhibition of histone biosynthesis, show a selective inhibition of the synthesis of S-phase variants, but have no effect on the synthesis of basal histone variants. On the basis of histone variants being synthesized in the presence of chlorambucil, it is shown that although chlorambucil shows a specificity for histone synthesis inhibition it has a general action over the whole variant complement and is not coupled to S-phase synthesis in a way typical for DNA synthesis inhibiting drugs.     

Isolation of yeast mutants sensitive to the bifunctional alkylating agent nitrogen mustard

Summary Mutants of Saccharomyces cerevisiae with enhanced sensitivity to the DNA cross-linking agent nitrogen mustard (HN2) have been isolated and partially characterized with respect to their phenotypic and genetic properties. The screening technique, based on HN2-sensitivity as sole criterion, yields approximately 1 sensitive isolate in 200 clones when applied to an intensively mutagenized population of a resistant parent strain. Mutants characterized so far are all due to recessive nuclear genes and represent at least seven complementation groups. They exhibit different degrees as well as different patterns of sensitivity towards monofunctional and bifunctional alkylating agents, and ultraviolet light.     

Bizelesin, a bifunctional cyclopropylpyrroloindole alkylating agent, inhibits simian virus 40 replication in trans by induction of an inhibitor.

Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA replication was analyzed in SV40-infected cells, using two-dimensional (2D) neutral agarose gel electrophoresis, and in a cell-free SV40 DNA replication assay. Within 1 h of bizelesin addition to infected cells, a similar rapid decrease in both the level of SV40 replication intermediates and replication activity was observed, indicating inhibition of initiation of SV40 DNA replication. However, prolonged bizelesin treatment (>/=2 h) was associated with a reduced extent of elongation of SV40 replicons, as well as the appearance on 2D gels of intense spots, suggestive of replication pause sites. Inhibition of elongation and induction of replication pause sites may result from the formation of bizelesin covalent bonds on replicating SV40 molecules. The level of in vitro replication of SV40 DNA also was reduced when extracts from bizelesin-treated HeLa cells were used. This effect was not dependent upon the formation of bizelesin covalent bonds with the template DNA. Mixing experiments, using extracts from control and bizelesin-treated cells, indicated that reduced DNA replication competence was due to the presence of a trans-acting DNA replication inhibitor, rather than to decreased levels or inactivation of essential replication factor(s).     

The effect of Zn2+ ions on mitochondrial electron transport

Zn²⁺ ions linearly inhibit the electron transport in uncoupled mitochondria, Mg²⁺/ATP submitochondrial particles, and electron transport complex III between ubiquinone and the b cytochromes. A second effect is observed in coupled mitochondria only, where less than 4 μm Zn²⁺ causes a respiratory stimulation and a reduction of the b cytochromes; higher concentrations reverse these effects. The inhibition is completely reversed by chelating agents. The binding site has a unique affinity for Zn²⁺ with a dissociation constant of 1·10^?6.     

Antioxidative effect of ubiquinones on mitochondrial membranes.

Peroxidation of mitochondria occurs extensively in ubiquinone-depleted membranes. Reincorporation into the membranes of either the physiological ubiquinone or a short-chain homologue protects mitochondria against peroxidation. The ability to prevent this phenomenon is more evident in mitochondria that have incorporated ubiquinone-3 and might be ascribed to an ordering structural effect on the lipid bilayer.     

Induction of the mitochondrial permeability transition by the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Sorting cause and consequence of mitochondrial dysfunction

The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) alters DNA and stimulates the activity of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme involved in DNA repair. The consumption of cellular NAD(+) by PARP-1 is accompanied by ATP depletion, mitochondrial depolarization and release of proapoptotic proteins, but whether a causal relationship exists among these events remains an open question. Most of cellular NAD(+) is stored in the mitochondrial matrix and becomes available for cytosolic and nuclear processes only after its release through the permeability transition pore (PTP), a voltage-gated inner membrane channel. Here we have explored whether MNNG affects mitochondrial function upstream of PARP-1 activation. We show that MNNG has a dual effect on isolated mitochondria. At relatively low concentrations (up to 0.1 mM), it selectively sensitizes the PTP to opening, while at higher concentrations (above 0.5 mM) it inhibits carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)-stimulated respiration. MNNG caused PTP opening and activation of the mitochondrial proapoptotic pathway in intact HeLa cells, which resulted in cell death that could be prevented by the PTP inhibitor CsA. We conclude that a key event in MNNG-dependent cell death is induction of PTP opening that occurs independently of PARP-1 activation.     

Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport

The respiratory pathways of glycolysis, the tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain are ubiquitous throughout nature. They are essential for both energy provision in heterotrophic cells and a wide range of other physiological functions. Although the series of enzymes and proteins that participate in these pathways have long been known, their regulation and control are much less well understood. Further complexity arises due to the extensive interaction among these pathways in particular, and also between cytosolic and mitochondrial metabolism in general. These interactions include those between mitochondrial function in the photosynthetic and photorespiratory processes, amino-acid biosynthesis and the regulation of cellular redox. Recently, a wide range of molecular and biochemical strategies have been adopted to elucidate the functional significance of these interactions.     

Effect of disseminated myocardial necrosis on ATPase activity, Ca2+ transport, and lipid peroxidation in cardiac mitochondrial and microsomal membranes

Isoproterenol-induced (5 mg/kg) disseminated necrosis of the rabbit myocardium led to a decrease in the efficiency of calcium pump of sarcoplasmic reticulum fragments. This was shown by the reduced Ca/ATP ratio, as well as by Ca2+ and Ca2+ ATPase accumulation rate. In these conditions, calcium transport to mitochondria increased. Lipid peroxidation plays a definite role in the impairment of membrane permeability since the concentration of malonic dialdehyde rises in microsomal and mitochondrial fractions.U