Carlo Gregolin

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.

[47] Phospholipid hydroperoxide glutathione peroxidase

In acute inflammation the activated leukocytes generate cytotoxic oxygen free radicals. The role of these radical species in the cellular damage following an acute inflammatory reaction is well known. On the other hand the extent of the cellular damage must be dependent on both the rate of the free-radical generation and the scavenging capacity of the tissues. Among the enzymes acting in the inhibition of this damage, a key role seems to be played by the new selenoenzyme phospholipid hydroperoxide glutathione peroxidase. Indeed the reduction of membrane hydroperoxides constitutes a secondary line of defence against lipid peroxidation, preventing the decomposition of hydroperoxides leading to the formation of new radicals. This enzyme inhibits lipid peroxidation and is as active as glutathione peroxidase on phospholipid hydroperoxides, on which no previously known peroxidase is active. Its protective activity for biomembranes, and the kinetic analysis in the presence of detergents, suggest its interfacial character. The inhibition of lipid peroxidation in the membranes apparently requires this enzyme, along with glutathione and vitamin E, in order to reduce the rate of the initiation reactions. This synergism bears out the role of this enzyme in the multilevel defence system against free-radical damage in tissues.

Formation of α-tocopherol radical and recycling of α-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes: An electron paramagnetic resonance study

The events accompanying the inhibitory effect of alpha-tocopherol and/or ascorbate on the peroxidation of soybean L-alpha-phosphatidylcholine liposomes, which are an accepted model of biological membranes, were investigated by electron paramagnetic resonance, optical and polarographic methods. The presence of alpha-tocopherol radical in the concentration range 10(-8)-10(-7) M was detected from its EPR spectrum during the peroxidation of liposomes, catalysed by the Fe3+-triethylenetatramine complex. The alpha-tocopherol radical, generated in the phosphatidylcholine bilayer, is accessible to ascorbic acid, present in the aqueous phase at physiological concentrations. Ascorbic acid regenerates from it the alpha-tocopherol itself. A kinetic rate constant of about 2 X 10(5) M-1 X s-1 was estimated from the reaction as it occurs under the adopted experimental conditions. The scavenging effect of alpha-tocopherol on lipid peroxidation is maintained as long a ascorbic acid is present.

High-performance liquid chromatography of hydroperoxy derivatives of stearoyllinoleoylphosphatidyl-choline and of their enzymatic reduction products

Abstract Two high-performance liquid chromatographic methods are presented to identify hydroperoxy and hydroxy derivatives of stearoyllinoleoylphosphatidylcholine (SLPC). When SLPC was peroxidized by Fe3+ -ascorbate, the peroxidation products were mainly 9- and 13-hydroperoxylinoleic acid derivatives of SLPC. The corresponding 9- and 13-hydroxy derivatives were formed if glutathione and a recently discovered phospholipid hydroperoxide glutathione peroxidase were also present. Analyses of the β-position fatty acids released by phospholipase A2 were carried out on an aminic column and of the whole phospholipid following acetic anhydride treatment on a reversed-phase column.

Hydrogen peroxide and hematin in microsomal lipid peroxidation

Lipids of rat liver microsomes underwent peroxidation with production of malondialdehyde in the presence of H2O2 and hematin. Rates of peroxidation of 27-33 nmol of MDA formed/mg of microsomal protein/30 min were measured with 5 mM H2O2 and 10 microM hematin at 22 degrees C. Histidine (0.01 M) caused a 55% inhibition. Hematin could be added to the reaction mixtures either simultaneously with H2O2 or afterwards, when all H2O2 had been destroyed by catalase present in the microsomal preparation. Catalase was necessary for formation of MDA. Indeed, when heat-denatured microsomes were employed, incubation with H2O2 and the iron complex led to formation of lipid hydroperoxides; however, no production of MDA was observed, unless exogenous catalase was added together with H2O2 and hematin to the reaction mixture. The role of H2O2 in microsomal lipid peroxidation is that of promoting the formation of fatty acid hydroperoxides. These are decomposed in the presence of hematin, with formation of free radicals, bicyclic endoperoxides and MDA. Catalase is necessary to remove H2O2, which, after starting the peroxidation process, blocks the decomposition of lipid hydroperoxides, apparently by binding to the iron complex.

Inactivation of soluble 3-hydroxy-3-methylglutaryl CoA reductase by ATP.

Endoplasmic reticulum-bound HMG CoA reductase catalyzes the rate-limiting reaction of cholesterol synthesis in hepatocytes and other cells [I]. The activity of the enzyme in isolated rat liver microsomes is profoundly reduced by incubation with ATPMg prior to assay [2]. Inactivation is prevented or blocked by addition of EDTA [3]. Washing of the microsomes by repeated centrifugation prevents [2], and a protein fraction separated from the 100 000 X g supernatant of rat liver [2] or human fibroblasts [4] homogenates restores, the ATP-Mg-dependent inactivation. Beg et al. [2] indicated the possibility that HMG CoA reductase activity is subject to modulation through enzyme phosphorylation. However, Chow et al. [5] could not find phosphorylation or adenylation of the enzyme protein under conditions leading to inactivation. According to Brown et al. [4] the inactivation factor in the 100 000 X g supernatant would promote the conversion of the microsomal enzyme from an active to an inactive form. Both ATP and ADP are equally effective. Preparations of HMG CoA reductase solubilized by freezing and glycerol extraction are not susceptible to inactivation. The present paper reports evidence that inactivation by ATP is a characteristic property of HMG COG reductase protein both in the membrane-bound and in the soluble form. The presence of the inactivation