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Biomedical subjects

L Ernster

Publications and source records attributed to L Ernster.

At least 37 records · Page 2Linked to original sources
PubMed1994

Activity and immunohistochemistry of DT-diaphorase in hamster and human kidney tumours.

We have studied the biochemical and immunohistochemical changes of DT-diaphorase in diethylstilbestrol (DES)-induced hamster kidney tumours and human biopsies from normal kidneys and renal clear cell carcinoma. The activities of primary and secondary antioxidants in these hamster and human tissues are also reported. DT-diaphorase is decreased in the different subcellular fractions of hamster and human tissues. In hamster kidney the activities of the one-electron quinone reductases show a nearly two-fold increase. Immunohistochemical findings confirm the decrease in DT-diaphorase in hamster and human tissues. This image is of special interest in the case of nephroblastoma (Wilms' tumour), since it has been proposed that the DES-induced tumour is a 'nephroblastoma-like' one. Primary anti oxidant enzymatic activities, i.e. superoxide dismutase and glutathione peroxidase, are increased in hamster kidney bearing DES-induced tumours and decreased in human renal clear cell carcinoma. Glutathione disulphide reductase is decreased in hamster and human tumours. The role of these enzymatic activities in the carcinogenic process is also discussed.

Animals↗
PubMed1993

Occurrence of prenylated proteins in plant cells.

In this paper evidence is presented for the occurrence of prenylated proteins in plants. When spinach leaves were incubated in the presence of [3H]mevalonate non-extractable lipids were found in the protein fraction after extraction with organic solvents. Alkaline hydrolysis liberated phytol, polyprenyl phosphates-11-15 and also, in contrast to animal cells, polyprenols-11-15. Complete removal of farnesol and geranylgeraniol required the cleavage of thioether linkages by iodomethane. The results indicate that several polyisoprenoid lipids in plant cells are covalently bound to proteins. So far a protein fraction dominated by one or more proteins in the 23 kDa region has been identified.

Chromatography, High Pressure Liquid↗
PubMed1993

Biosynthesis of ubiquinone and plastoquinone in the endoplasmic reticulum-Golgi membranes of spinach leaves.

The localization of ubiquinone (UQ) and plastoquinone (PQ) biosynthesis in subfractions isolated from spinach leaves has been studied. UQ-9 and UQ-10 were found mainly in mitochondria, whereas PQ was enriched in chloroplasts, but also found in Golgi membranes. alpha-Unsaturated polyprenol-11 was also present at a low concentration in chloroplasts. Autoradiography revealed the presence of nonaprenyl-4-hydroxybenzoate (NPHB) and nonaprenyl-2-methylquinol (NPMQ) transferase activities involved in quinone biosynthesis in all subfractions, but the specific activities involved in quinone biosynthesis in the total microsomal fraction were 20 times higher than those in mitochondria and chloroplasts. The isolated Golgi vesicles were particularly enriched in both activities. When the incubation medium containing total microsomes or Golgi membranes was supplemented with NADH, NADPH, S-adenosylmethionine, and an ATP-generating system, NPHB and NPMQ were transferred to UQ-9 and PQ, respectively. trans-Prenyltransferase, which synthesizes the side chain of UQ and PQ, was present in the total microsomal fraction. With farnesyl-PP as substrate, no product was formed, but with geranyl-PP, solanesyl-PP was synthesized and transferred to 4-hydroxybenzoate present in the total microsomal fraction. The results show that these membranes from spinach contain farnesyl-PP synthetase. It is concluded that the plant leaf Golgi membranes contain the enzymes for both UQ and PQ biosynthesis and that a specific transport and targeting system is required for selective transfer of UQ to the mitochondria and of PQ to the chloroplast.

Cell Fractionation↗
PubMed1993

Ubiquinol: an endogenous antioxidant in aerobic organisms.

Ubiquinone (coenzyme Q), in addition to its function as an electron and proton carrier in mitochondrial and bacterial electron transport linked to ATP synthesis, acts in its reduced form (ubiquinol) as an antioxidant, preventing the initiation and/or propagation of lipid peroxidation in biological membranes and in serum low-density lipoprotein. The antioxidant activity of ubiquinol is independent of the effect of vitamin E, which acts as a chain-breaking antioxidant inhibiting the propagation of lipid peroxidation. In addition, ubiquinol can efficiently sustain the effect of vitamin E by regenerating the vitamin from the tocopheroxyl radical, which otherwise must rely on water-soluble agents such as ascorbate (vitamin C). Ubiquinol is the only known lipid-soluble antioxidant that animal cells can synthesize de novo, and for which there exist enzymic mechanisms that can regenerate the antioxidant from its oxidized form resulting from its inhibitory effect of lipid peroxidation. These features, together with its high degree of hydrophobicity and its widespread occurrence in biological membranes and in low-density lipoprotein, suggest an important role of ubiquinol in cellular defense against oxidative damage. Degenerative diseases and aging may be manifestations of a decreased capacity to maintain adequate ubiquinol levels.

Aerobiosis↗
PubMed1992

Distribution and redox state of ubiquinones in rat and human tissues.

The distribution and redox state of ubiquinone in rat and human tissues have been investigated. A rapid extraction procedure and direct injection onto HPLC were employed. It was found in model experiments that in postmortem tissue neither oxidation nor reduction of ubiquinone occurs. In rat the highest concentrations of ubiquinone-9 were found in the heart, kidney, and liver (130-200 micrograms/g). In brain, spleen, and intestine one-third and in other tissues 10-20% of the total ubiquinone contained 10 isoprene units. In human tissues ubiquinone-10 was also present at highest concentrations in heart, kidney, and liver (60-110 micrograms/g), and in all tissues 2-5% of the total ubiquinone contained 9 isoprene units. High levels of reduction, 70-100%, could be observed in human tissues, with the exception of brain and lung. The extent of reduction displayed a similar pattern in rat, but was generally lower.

Animals↗
PubMed1992

The mode of action of lipid-soluble antioxidants in biological membranes. Relationship between the effects of ubiquinol and vitamin E as inhibitors of lipid peroxidation in submitochondrial particles.

The effects of ubiquinol and vitamin E on ascorbate- and ADP-Fe(3+)-induced lipid peroxidation were investigated in beef heart submitochondrial particles before and after extraction of ubiquinone and vitamin E, and after reincorporation of either or both of these components. It is concluded that ubiquinol is capable of inhibiting lipid peroxidation without the mediation of vitamin E, but may also amplify the antioxidant effect of the latter. It is pointed out that ubiquinol is the only known lipid-soluble antioxidant that can be synthesized de novo in animal cells, and for which there exists an enzymic mechanism--the mitochondrial electron-transport system--that can regenerate the antioxidant from its oxidized form resulting from its inhibitory effect on lipid peroxidation. These features, together with its high degree of hydrophobicity and its general occurrence in biological membrane and in low-density lipoprotein, suggest a highly important role of ubiquinol in cellular defense against oxidative damage.

Animals↗
PubMed1992

The mode of action of lipid-soluble antioxidants in biological membranes: relationship between the effects of ubiquinol and vitamin E as inhibitors of lipid peroxidation in submitochondrial particles.

The effects of ubiquinol and vitamin E on ascorbate- and ADP-Fe3+-induced lipid peroxidation were investigated by measuring oxygen consumption and malondialdehyde formation in beef heart submitochondrial particles. In the native particles, lipid peroxidation showed an initial lag phase, which was prolonged by increasing concentrations of ascorbate. Lipid peroxidation in these particles was almost completely inhibited by conditions leading to a reduction of endogenous ubiquinone, such as the addition of succinate or NADH in the presence of antimycin. Lyophilization of the particles followed by three or four consecutive extractions with pentane resulted in a complete removal of vitamin E and a virtually complete removal of ubiquinone, as revealed by reversed-phase high pressure liquid chromatography. In these particles, lipid peroxidation showed no significant lag phase and was not inhibited by either increasing concentrations of ascorbate or conditions leading to ubiquinone reduction. Treatment of the particles with a pentane solution of vitamin E (alpha-tocopherol) restored the lag phase and its prolongation by increasing ascorbate concentrations. Treatment of the extracted particles with pentane containing ubiquinone-10 resulted in a restoration of the inhibition of lipid peroxidation by succinate or NADH in the presence of antimycin, but not the initial lag phase or its prolongation by increasing concentrations of ascorbate. Malonate and rotenone, which prevent the reduction of ubiquinone by succinate and NADH, respectively, abolished, as expected, the inhibition of the initiation of lipid peroxidation in both native and ubiquinone-10-supplemented particles. Reincorporation of both vitamin E and ubiquinone-10 restored both effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Distribution of glutathione peroxidases and glutathione reductase in rat brain mitochondria.

The distribution of glutathione reductase (GR), glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat brain mitochondria was investigated, using a fractionation procedure for the separation of inner and outer membranes, contact sites between the two membranes and a soluble fraction mainly originating from the mitochondrial matrix. The data indicate that GR and GPx are concentrated in the soluble fraction, with a minor portion of the two enzymes being associated with the contact sites. PHGPx is localized largely in the inner membrane. The possible functional significance of these findings is discussed.

Animals↗
PubMed1991

Inhibition of lipid peroxidation by ubiquinol in submitochondrial particles in the absence of vitamin E.

The relationship between the antioxidant effects of reduced coenzyme Q10 (ubiquinol, UQH2) and vitamin E (alpha-tocopherol) was investigated in beef heart submitochondrial particles in which lipid peroxidation was initiated by incubation with ascorbate + ADP-Fe3+. These effects were examined after extraction of coenzyme Q10 (UQ-10) and vitamin E from the particles and reincorporation of the same components alone or in combination. The results show that UQH2 efficiently inhibits lipid peroxidation even when vitamin E is absent. It is concluded that UQH2 can inhibit lipid peroxidation directly, without the mediation of vitamin E.

Adenosine Diphosphate↗
PubMed1991

Initiation of lipid peroxidation in submitochondrial particles: effect of respiratory inhibitors.

Initiation of lipid peroxidation in the inner mitochondrial membrane was investigated using respiratory substrates and inhibitors and various iron chelates. An iron chelate was required for initiation of lipid peroxidation in the presence of either NADH or NADPH. The two nicotinamide nucleotides exhibited different activities in initiating lipid peroxidation with regard to concentration and to the effects of rotenone and rhein. Succinate and both nicotinamide nucleotides supported lipid peroxidation in the presence of thenoyl trifluoroacetone (TTFA), without a requirement for exogenously added iron. ADP stimulated lipid peroxidation in the case of NAD(P)H and TTFA, but inhibited it in the case of succinate and TTFA. Lipid peroxidation is thought to be enzymatically induced in both the NADH and the succinate dehydrogenase regions of the respiratory chain, and evidence is presented for a novel pathway of NADPH oxidation that may also be involved. Possible initiation mechanisms are discussed.

Adenosine Diphosphate↗
PubMed1990

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)

Adenosine Triphosphatases↗
PubMed1990

Hydrogen peroxide production by monoamine oxidase in isolated rat-brain mitochondria: its effect on glutathione levels and Ca2+ efflux.

H2O2 production and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H2O2 which was inhibited by malate + pyruvate or isocitrate, consistent with a need for mitochondrial NADPH to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H2O2 generated by monoamine oxidase. The H2O2 accumulation was also inhibited by externally added reduced glutathione or NADPH but not NADH. External NADPH was oxidized by added oxidized glutathione but not alpha-ketoglutarate + NH4+. These results suggest that the removal of H2O2 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and NADPH, possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H2O2 generated by monoamine oxidase enhances Ca2+ release from mitochondria and may thus function as a regulator of mitochondrial Ca2+ efflux.

Animals↗
PubMed1990

Effect of superoxide dismutase on the autoxidation of substituted hydro- and semi-naphthoquinones.

The effect of superoxide dismutase on the autoxidation of hydro- and semi-1,4-naphthoquinones with different substitution pattern and covering a one-electron reduction potential range from -95 to -415 mV was examined. The naphthoquinone derivatives were reduced via one or two electrons by purified NADPH-cytochrome P-450 reductase or DT-diaphorase, respectively. Superoxide dismutase did not alter or slightly enhance the initial rates of enzymic reduction, whereas it affected in a different manner the following autoxidation of the semi- and hydroquinones formed. Autoxidation was assessed as NADPH oxidation in excess to the amounts required to reduce the quinone present, H2O2 formation, and the redox state of the quinones. Superoxide dismutase enhanced 2--8-fold the autoxidation of 1,4-naphthosemiquinones, following the reduction of the oxidized counterpart by NADPH-cytochrome P-450 reductase, except for the glutathionyl-substituted naphthosemiquinones, whose autoxidation was not affected by superoxide dismutase. Superoxide dismutase exerted two distinct effects on the autoxidation of naphthohydroquinones formed during DT-diaphorase catalysis: on the one hand, it enhanced slightly the autoxidation of 1,4-naphthohydroquinones with a hydroxyl substituent in the benzene ring: 5-hydroxy-1,4-naphthoquinone and the corresponding derivatives with methyl- and/or glutathionyl substituents at C2 and C3, respectively. On the other hand, superoxide dismutase inhibited the autoxidation of naphthohydroquinones that were either unsubstituted or with glutathionyl-, methyl-, methoxyl-, hydroxyl substituents (the latter in the quinoid ring). The inhibition of hydroquinone autoxidation was reflected as a decrease of NADPH oxidation, suppression of H2O2 production, and accumulation of the reduced form of the quinone. The enhancement of autoxidation of 1,4-naphthosemiquinones by superoxide dismutase has been previously rationalized in terms of the rapid removal of O2-. by the enzyme from the equilibrium of the autoxidation reaction (Q2-. + O2----Q + O2-.), thus displacing it towards the right. The superoxide dismutase-dependent inhibition of H2O2 formation as well as NADPH oxidation during the autoxidation of naphthohydroquinones--except those with a hydroxyl substituent in the benzene ring--seems to apply to those organic substrates which can break down with simultaneous formation of a semiquinone and O2-.. Inhibition of hydroquinone autoxidation by superoxide dismutase can be interpreted in terms of suppression by the enzyme of O2-.- dependent chain reactions or a direct catalytic interaction with the enzyme that might involve reduction of the semiquinone at expense of O2(-.).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗