István Horváth

Interaction of a synthetic polyanion with rat liver mitochondria.

Abstract The effect of a polyanion (a copolymer of methacrylate, malaete and styrene in a 1:2:3 proportion with an average molecular weight of 10 000) on respiration, ATPase activity and ADP/ATP exchange activity of rat liver mitochondria and submitochondrial particles has been studied. The polyanion (at 17–150 μg/ml concentration, 100 μg polyanion corresponding to 0.83 μequiv. of carboxylic groups) inhibits the oxidation of succinate and NAD-linked substrates in state 3 in a concentration-dependent manner. The extent of this inhibition can be decreased by elevating the concentration of ADP. State 4 respiration is not affected by the polyanion. It has also a slight inhibitory effect on the oxidation of the above mentioned substrates in the uncoupled state (a maximum inhibition of 37% at 166 μg/ml polyanion concentration), which is unaffected by ADP. The strong inhibition of state 3 respiration can be relieved by 2,4-dinitrophenol to the low level observed in the uncoupled state. Ascorbate+TMPD oxidation is slightly inhibited in state 3, while it is not inhibited at all in the uncoupled state. The polyanion, depending on its concentration, strongly inhibits also the DNP-activated ATPase activity of mitochondria (50% inhibition at 40 μg/ml polyanion concentration). The ATPase activity of sonic submitochondrial particles is also inhibited. However, this inhibition is incomplete (reaching a maximum of 65%) and higher concentrations of the polyanion are required than to inhibit the ATPase activity of intact mitochondria. The polyanion inhibits the ADP/ATP translocator activity of mitochondria, measured by the “back exchange” of [2- 3 H]ADP. After a short preincubation of the mitochondria with the polyanion, the concentration dependence of the inhibition by the polyanion corresponds to that of the DNP-activated ATPase activity of intact mitochondria. It is concluded that, in intact mitochondria, the polyanion has at least a dual effect, i.e. it partially inhibits the respiratory chain between cytochrome b and cytochrome c , and strongly oxidative phosphorylation by blocking the ADP/ATP translocator.

Reduction of Fe(III)ADP complex by liver microsomes

An NADPH-driven enzymatic reduction of an Fe(III)ADP complex by rat liver microsomes has been demonstrated directly for the first time during the initial phase of lipid peroxidation by using two different analytical methods. The reduction rate increased upon increasing the ratio of ADP to ferric iron. Fe(III)ADP reducing activity of both detergent-solubilized microsomes and purified NADPH:cytochrome-P-450 (cytochrome-c) reductase decreased to about 20% compared to that of the native microsomes. Superoxide dismutase and KCN did not inhibit the reduction.

Prostaglandin and thromboxane synthesizing activity in isolated murine hepatocytes and nonparenchymal liver cells.

Prostanoid synthesis from 3H-arachidonic acid was compared in isolated parenchymal and nonparenchymal murine liver cells. The cells incorporated arachidonic acid into phospholipids but no prostanoid synthesis could be measured during 30 min incubation. Conditions necessary for prostanoid synthesis were different in parenchymal and nonparenchymal cells and the products were also different. Prostanoid synthesis could be induced by in vitro partial hepatectomy: parenchymal cells synthesized thromboxane A2 whereas nonparenchymal cells produced prostaglandin E2 and F2 alpha. Prostaglandin E2 and F2 alpha synthesis could be provoked also by homogenization of the nonparenchymal cells prepared from normal liver, while the homogenates of parenchymal cells prepared from normal liver did not synthesize thromboxane. Imidazole and indomethacin inhibited the production of thromboxane and prostaglandins, respectively. Our results suggest that the various cell types of the liver respond by the synthesis of different and specific prostanoids after the same injury.

Mitochondrial substrate oxidation-dependent protection against lipid peroxidation

The effect of mitochondrial substrate oxidation on the NADPH-dependent lipid peroxidation of intact mitochondria, microsomes and of homogenate from rat liver was studied. It was found that addition of either succinate or beta-OH-butyrate decreased the rate of malondialdehyde production of mitochondria. The effect of succinate was found to be marked (80-90% inhibition). Addition of succinate strongly inhibited the lipid peroxidation of a reconstituted system containing both mitochondria and microsomes. Increasing the amount of mitochondria in this system resulted in enhancement of the inhibition. The same succinate effect on malondialdehyde formation in liver homogenate was also observed. These findings suggest that mitochondria supplied with respiratory substrates may play a role in the protection against the lipid peroxidation in the liver cell.

Specific binding of thrombin-antithrombin III complex to hepatocytes.

Thrombin-antithrombin III complex binds selectively to isolated hepatocytes, whereas antithrombin III alone does not. The binding is time and concentration dependent at 37 degrees C: the apparent Km value is 0.8/microM. The rate of binding is approximately 1.6 X 10(5) molecules h-1 cell-1 at this concentration. At 4 degrees C there is no measurable interaction between the complex and the hepatocytes. The binding is also prevented by pretreatment of cells with trypsin. On the other hand, about 80% of the thrombin-antithrombin III complex bound to hepatocytes is releasable by trypsin digestion. NaF or carboxyatractyloside does not inhibit the process. The interaction of thrombin-antithrombin III complex with hepatocytes seems to be specific, since the complexes of antithrombin III with other proteinases, like trypsin or plasmin, are not bound at the concentrations used. Based on these data, a mechanism for the binding of the inactive complexed form of thrombin to hepatocytes is suggested.

Ionophores and intact cells II. Oleficin acts on mitochondria and induces disintegration of the mitochondrial genome in yeast Saccharomyces cerevisiae

The non-macrolid polyene antibiotic oleficin, which has been shown to function as an ionophore of Mg2+ in isolated rat liver mitochondria, preferentially inhibited growth of the yeast Saccharomyces cerevisiae on non-fermentable substrates. It uncoupled and inhibited respiration of intact cells and converted both growing and resting cells into respiration-deficient mutants. The mutants arose as a result of fragmentation of the mitochondrial genome. Another antibiotic known to be an ionophore of divalent cations, A23187, also selectively inhibited growth of the yeast on non-fermentable substrates, but did not produce the respiration-deficient mutants, neither antibiotic inhibited the energy-dependent uptake of divalent cations by yeast cells nor opened the plasma membrane for these cations. The results indicate that in Saccharomyces cerevisiae both oleficin and A23187 preferentially affected the mitochondrial membrane without acting as ionophores in the plasma membrane.

Lipidg Peroxidation in Liver and Ehrlich Ascites Cell Mitochondria

Ehrlich ascites cell mitochondria are highly resistant to lipid peroxidation as compared to liver mitochondria from host animals. Succinate protects mitochondria from peroxidative damage, proteins from cross-links, enzymes from inactivation of the enzymes and membranes from permeability changes. The sensitivity of Ehrlich ascites cell mitochondrial membranes to lipid peroxidation is significantly increased in submitochondrial particles. Lipid peroxidation in tumour mitochondrial membranes can not be diminished by succinate as effectively as in liver mitochondria. Ascites cell mitochondria seems to be protected very efficiently from peroxidative damage by a glutathione-dependent mechanism.

The effect of EDTA-Fe(III) complexes with different chemical structure on the lipid peroxidation in brain microsomes

Unlike EDTA-Fe(III) (1:1), the oxidized form of EDTA-Fe(II) complex enhanced lipid peroxidation in brain microsomes. Mossbauer spectroscopy and electron paramagnetic resonance analysis of oxidized EDTA-Fe(II), capable of inducing lipid peroxidation, showed the presence of EDTA-Fe(III) complex, which was different from the separately prepared (not oxidized) EDTA-Fe(III). Lipid peroxidation initiated by the oxidized EDTA-Fe(II) complex was dependent on the presence of NAD(P)H and functionally intact microsomes. No inhibitory effect was found by generally used free radical scavengers and catalase. Our results clearly indicate that the chemically different EDTA-Fe(III) complexes differ in their capability of initiating the NAD(P)H-dependent lipid peroxidation in brain microsomes.

Inhibition of lipid peroxidation by heme-nonapeptide derived from cytochrome c

Heme-nonapeptide, derived from cytochrome c, inhibited both the NADPH- and NADH-dependent lipid peroxidation of brain microsomes but, in the case of liver microsomes, this inhibitory effect manifested itself in the presence of SKF-525A (a specific blocker of cytochrome P-450) only. Heme-nonapeptide prevented the transient accumulation of lipid peroxides in microsomes during lipid peroxidation. The oxygen consumption of microsomes in the presence of NADPH or NADH was stimulated by heme-nonapeptide. From these results we concluded that, in vitro, there are two independent mechanisms of lipid peroxidation in liver microsomes. It is suggested that, in vivo, the heme-peptide-sensitive mechanism, observed in brain microsomes, is more important.

The mode of action of primycin.

Primycin, an antibiotic active against Gram-positive microorganisms increased the permeability ofBacillus subtilis cell membranes when used in bacteriostatic concentrations. On addition of the antibiotic to the washed cell suspension, a dose-dependent increase in the conductivity was observed. Furthermore, an enhanced leakage of the nucleotides (measured by the32P-ATP release from the32P-labelled culture) could be detected.To get more information about the mechanism of the primycin-membrane interaction, the effect of the antibiotic on the ATPase activity of membrane vesicles prepared from bothBacillus subtilis andEscherichia coli B was studied. Activation was found at about 0.5 nmol antibiotic/μg protein and its extent was approximately the same as with sonicated membranes used as controls. Stimulation of ATPase activity was also achieved with vesicles prewashed with 3 mM Tris-HCl buffer.Purified membrane ATPase fromBacillus subtilis could not be activated by primycin at all; above 0.3 nmol/μg protein concentration the enzyme was inhibited. When acting on membrane vesicles isolated fromEscherichia coli B, inhibition without previous activation was observed, although sonication caused a substantial activation on the ATPase of these membranes.These observations confirmed our suggestion that the primary target of primycin action is the cell membrane in Gram-positive microorganisms.