Marina Valente

Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides

The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and biomembranes from peroxidative degradation in the presence of glutathione. The activity of this protein has been assayed by measuring the inhibition of aged phosphatidylcholine liposome peroxidation induced by the Fe3+-triethylenetetramine complex. The peroxidation-inhibiting protein from pig liver has been purified 585-fold to homogeneity with overall recovery of activity of 12%. (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-Sepharose CL-6B and CM23-cellulose, affinity chromatography on glutathione-bromosulfophthalein-Sepharose and gel filtration on Sephadex G-50 were used. Gel filtration and SDS- polyacrylamide gel electrophoresis indicated a molecular weight of approximately 20 000. The protein inhibited peroxidation by Fe3+-triethylenetetramine following a 15 min preincubation of phosphatidylcholine liposomes in the presence of 5mM glutathione or 2-mercapthoethanol. The pure protein exhibited glutathione peroxidase activity on hydroperoxide groups of phosphatidylcholine and on cumene and t-butyl hydroperoxides, with specific activities of 2.2, 3.8 and 0.9 mumol/min per mg protein, respectively. The protein appears to be distinct from the selenoenzyme glutathione peroxidase and from any known glutathione S-transferase. The peroxidation was studied also with fresh phosphatidylcholine liposomes and was induced in this case by Fe-ascorbate. To obtain protection by the peroxidation-inhibiting protein and glutathione, preincubation was not necessary, but alpha-tocopherol, incorporated in the liposomes in the molar ratio 1:250 to phosphatidylcholine, was required. Lipid peroxidation of rat liver mitoplasts and microsomes was blocked when these preparations were incubated in the peroxidizing mixture in the presence of peroxidation-inhibiting protein and glutathione. The protection from Fe3+-triethylenetetramine-induced peroxidation is related apparently to reduction of hydroperoxide groups in polyunsaturated fatty acid residues of phospholipids and to inhibition of free radicals formation by chain branching. Protection from the Fe-ascorbate-induced peroxidation is apparently attributable to the same mechanism. However, the requirement of alpha-tocopherol for protection in the Fe-ascorbate-induced peroxidation suggests that the cooperation of a free-radical scavenger is necessary. It is probable that the glutathione peroxidase activity is involved also in the glutathione-dependent protection exhibited by the protein on lipid peroxidation of biomembranes.

Inhibitory action of quercetin on xanthine oxidase and xanthine dehydrogenase activity

Quercetin is an equally good inhibitor of xanthine oxidase (type O, oxygen-reducing enzyme) and xanthine dehydrogenase (type D, NAD+-reducing enzyme) activity of a preparation of the xanthine-oxidizing enzyme partially purified from rat liver. The inhibition seems competitive with the oxidase form and non-competitive (mixed-type) with the dehydrogenase form of the enzyme. These inhibitory properties should be referred to the flavonoid structure of quercetin rather than to its antioxidant power. The antioxidant properties of quercetin and its inhibitory effect on the xanthine-oxidizing enzyme are discussed with reference to hyperuricemic and ischemic states.

The protective action of pyruvate on recovery of ischemic rat heart: Comparison with other oxidizable substrates

The recovery of both contractile performance and metabolic response of rat heart following 1 h of ischemia after equilibration with glucose + insulin (glucose-ischemia) or with pyruvate (pyruvate-ischemia), was tested in normoxic reperfusion in the presence of glucose + insulin, pyruvate, lactate or acetate. In glucose-ischemia only the reperfusion with pyruvate results in a complete recovery of the contractile force (left ventricular pressure, LVP) (170%) and good recovery of high energy phosphate compounds. Lower LVP and tissue energy charge were found in glucose reperfusion and even less in lactate and acetate reperfusion. Disappearance of the IMP accumulated during ischemia is evident only in the pyruvate reperfusion indicating a higher metabolic recovery. On the contrary in pyruvate-ischemia all types of reperfusion tested were effective in reactivating the contractile force (although acetate to a lesser extent); the contractile activity was accompanied by a good recovery of phosphocreatine, ATP, energy charge and by the decrease of IMP. Large decreases of adenine nucleotides and NADP and lower decreases of NAD are observed during ischemia/reperfusion in both systems. Pyruvate-ischemia is quite similar to, if not worse than glucose-ischemia, for all the metabolic parameters considered, but not worse for the possibility of recovery. Some specific effect of pyruvate should be exerted during the ischemic phase. The mechanism of pyruvate protection is discussed in relationship to: (i) the possible activation of pyruvate dehydrogenase, (ii) the activation of NADPH-dependent peroxide scavenging systems, (iii) the direct scavenging action of pyruvate on H2O2.

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H2O2 on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H2O2 (50–150 µM) for a few minutes results in a long‐lasting depression of the Ca2+‐dependent exocytosis of glutamate, induced by KCl or by the K+‐channel inhibitor 4‐aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H2O2, although a transient decrease was observed after the treatment. H2O2 did not promote peroxidation, as judged by the formation of malondialdehyde. In indo‐1‐loaded synaptosomes, the treatment with H2O2 did not modify significantly the KCl‐induced increase of [Ca2+]i. H2O2 inhibited exocytosis also when the latter was induced by increasing [Ca2+]i with the Ca2+ ionophore ionomycin. The effects of H2O2 were unchanged in the presence of superoxide dismutase and the presence of the Fe3+ chelator deferoxamine. These results appear to indicate that H2O2, apparently without damaging the synaptosomes, induces a long‐lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.

NADH and NADPH inhibit lipid peroxidation promoted by hydroperoxides in rat liver microsomes

Lipid peroxidation induced through cytochrome P-450 activation of cumene hydroperoxide, linolenic acid hydroperoxide and peroxidized phosphatidylcholine in rat liver microsomes is markedly inhibited by either NADH or NADPH. This inhibition is not due to an antioxidant effect. Conversely, cumene hydroperoxide decomposition is stimulated by the reduced pyridine nucleotides but not by some modifiers of cytochrome P-450 (SKF-525A, metyrapon and aniline). The mechanism by which NADH and NADPH prevent lipid peroxidation may involve a reduction of the hydroperoxides mediated by cytochrome P-450 and occurring without formation of free radical forms that are usual sparkers of lipid peroxidation.

On the mechanism of inhibition of lipid peroxidation by manganese

Abstract The antioxidant action of Mn(II) on different peroxidizing systems was studied. Mn(II) inhibits lipid peroxidation induced by free radical producing systems but not the one induced by singlet oxygen. A close relationship between the action of Mn(II) and that of the classical antioxidant BHA was found. This indicates that Mn(II), likewise BHA, may act by scavenging the free radicals formed during initiation and propagation of lipid peroxidation.

Effects of palmitoyl coenzyme a on rat skeletal muscle sarcoplasmic reticulum

Palmitoyl coenzyme A (PCoA) inhibits Ca2+ uptake and stimulates Ca2+-activated ATPase in sarcoplasmic reticulum vesicles. The inhibitory effect on Ca2+-uptake is referable to a stimulation of Ca2+ release which is directly correlated to the concentration of PCoA added. The comparison of the Ca2+-releasing effect of PCoA in different experimental conditions indicates that concentrations of PCoA higher than 10 microM may be disruptive for the vesicles while concentrations of PCoA lower than this value can activate a Ca2+-releasing channel or more generally can increase the membrane permeability for Ca2+.

A procedure allowing measurement of cytosolic CA2+ in rat platelets. Inhibition of a plasma lipoprotein on fura 2-AM loading

Loading of the fluorescent Ca2+ probe fura 2 in rat platelets is highly inhibited by a plasmatic factor that is removed by gel filtration through a Sepharose C-2B column. Rat plasma also inhibits fura 2 loading in human platelets. The inhibitory effect is abolished by perchloric acid-deproteinization or heat denaturation of plasma suggesting a proteic nature of the inhibitory compound. Indeed the 10,000 x g supernatant of the heat denaturated plasma shows a positive effect on fura 2 accumulation, most likely by partially inhibiting its cellular effux. These effects are only negligibly shown by the corresponding fractions of human plasma. Results obtained by fractionation of rat plasma proteins by means of ion exchange DEAE Sepharose C-6B chromatography and ultracentrifugation through high density saline solutions indicate that the inhibition of cellular fura 2 loading is due to the HDL fraction of rat plasma.

Hydrolysis of ITP generates a membrane potential in submitochondrial particles

ITP hydrolysis catalysed by the ATPase of submitochondrial particles from both bovine heart and rat liver is shown to be linked to the generation of a membrane potential, and therefore also to proton translocation. The magnitude of the membrane potential is similar to that observed during ATP hydrolysis at equivalent concentrations of phosphate and nucleoside tri- and diphosphates. An explanation is suggested for why in other reports ITP was found to be a poor substrate for supporting energy-linked reactions that are driven by the membrane potential.

Inhibition of lipid peroxidation by manganese

The peroxidation of unsaturated lipids of biological membranes is a degenerative process usually sparked by free radicals or by singlet oxygen. MnCl2 is an inhibitor of lipid peroxidation [1] but its mechanism of action is still unknown. This report summarizes some results on the inhibitory effect of MnCl2 on peroxidizing systems such as ascorbate/FeSO4, NADPH/FeCl3/ADP, cumene hydroperoxide (CHP), hematin and rose bengal/light. 1 mM MnCl2 efficiently inhibited all the systems tested, with the exception of rose bengal/visible light where lipid peroxidation is not initiated by a free radical mechanism but by singlet oxygen which directly forms a hydroperoxide through an ‘ene’ reaction [2]. The lipid peroxidation induced by CHP was independent of iron ions since it occurred to the same extent even in the presence of 1 mM EDTA. Since MnCl2 also strongly inhibited the CHP-induced lipid peroxidation, a competition of MnCl2 for the binding of iron ions, which are well known promotors of lipid peroxidation, can be excluded. Figure 1 illustrates the formation of lipid hydroperoxides (LOOH) after photoactivation in the presence of rose bengal. In this case the formation of new hydroperoxides occurred also when the photoactivation was ended because of the branching reactions initiated by decomposition of the lipid hydroperoxides previously formed by rose bengal. The chelating agent EDTA lowered the rate of lipid hydroperoxide formation due to branching reactions, but did not decrease the amount of preformed lipid hydroperoxides. On the contrary, Mn2+, like the antioxidant agent butylated hydroxyanisole (BHA), decreased them. On this ground we suggest that Mn2+ could directly interact with the oxy and peroxy radicals (RO•, RO2•) originating from lipid hydroperoxide decomposition. Consequently these free radicals cannot spark new radical chains and lipoperoxidation is blocked. Recently Mn2+ was shown to act as a scavenger of Obb2 [4] and bbOH [5] and this supports the hypothesis of a direct antioxidant action of this ion. The inhibitory action of Mn-superoxide dismutase (SOD) on lipoperoxidation was also studied. The enzyme inhibits by 25–30% the lipid peroxidation sparked by CHP in experiments carried out in the presence of reduced glutathione.