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Biomedical subjects

M Ingelman-Sundberg

Publications and source records attributed to M Ingelman-Sundberg.

At least 217 records · Page 12Linked to original sources
PubMed1985

Phorbol ester-induced alteration of differentiation and proliferation in human hematopoietic tumor cell lines: relationship to the presence and subcellular distribution of protein kinase C.

The intracellular translocation of protein kinase C (PKC) from the soluble to the membranous fraction has been shown previously to correlate with biological activity of phorbol esters in several systems. In this paper, we describe that PKC translocation was a general phenomenon in all PKC containing cell types when five 12-O-tetradecanoylphorbol-13-acetate (TPA) responsive and nonresponsive hematopoietic tumor cell lines were investigated. The nonresponsive cell line U-266 contained undetectable levels of PKC. The dose of TPA required for translocation was similar to the TPA concentration necessary to suppress erythroid differentiation in K-562 cells and to induce macrophage differentiation in U-937 cells, but 100-fold higher than that required for suppression of proliferation in K-562 and U-937 cells. By contrast, PKC translocation and TPA induced proliferation inhibition exhibited a similar dose dependence in a subline of U-937 (U-937 RES) adapted to growth in the presence of 10(-9) M TPA. It is suggested that U-937 RES is deficient in a TPA dependent but PKC independent signal pathway.

Biological Transport↗
PubMed1984

Mechanism of rate control of the NADPH-dependent reduction of cytochrome P-450 by lipids in reconstituted phospholipid vesicles.

The NADPH-supported reduction of cytochrome P-450 LM2 (liver microsomal isozyme 2) in reconstituted phospholipid vesicles in general exhibits two-exponential kinetics. The physiologically relevant rapid partial reaction is favoured in amount with increasing reductase/P-450 ratio. A lipid specificity was observed in that negatively charged lipids favour that process, too. The rate constant increases concomitantly. The data are consistent with the formation of a reactive 1:1 complex the amount of which determines the rate constant. The dissociation constants amount to 0.048 microM for a microsomal lipid extract, 0.051 microM for a 3:1 (w/w) mixture of dioleoylglycerophosphoethanolamine and phosphatidylserine, and 0.47 microM for dioleoylglycerophosphocholine, respectively, in the respective reconstituted systems. At low reductase/P-450 ratio the amount of the rapidly reduced P-450 exceeds the equilibrium concentration of a 1:1 complex. Preformed 1:1 associates, therefore, cannot fit the derived mechanism. Instead, a cluster model based on P-450 association does correspond to the data.

Cytochrome P-450 Enzyme System↗
PubMed1984

Induction of the ethanol-inducible form of rabbit liver microsomal cytochrome P-450 by inhibitors of alcohol dehydrogenase.

Cytochrome P-450 LMeb was purified from liver microsomes obtained from rabbits treated with either benzene or imidazole and was shown to have identical N-terminal amino acid sequence as that of cytochrome P-450 LM3a. The amino acid compositions of the proteins were indistinguishable. Quantitation of P-450 LMeb in various types of microsomes using radial immunodiffusion, revealed that pyrazole- or imidazole-treatment of the animals caused a 2-3-fold induction of the enzyme, accompanied by 2-3-fold increases of the rates of ethanol and aniline oxidation.

Alcohol Dehydrogenase↗
PubMed1984

Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450.

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.

Animals↗
PubMed1983

Reduction of cytochrome P-450 LM2 by NADPH in reconstituted phospholipid vesicles is dependent on membrane charge.

The kinetics of the reduction of cytochrome P-450 LM2 mediated by NADPH-cytochrome P-450 reductase in reconstituted phospholipid vesicles was examined. An inefficient reduction of the hemoprotein in phosphatidylcholine vesicles was observed. However, by introducing negatively charged phospholipids into the membrane, the rate of reduction increased in a concomitant manner to the resulting net negative charge of the vesicles. In the presence of benzphetamine, the extent of cytochrome P-450 LM2 reduced 1 s after the addition of NADPH to the system was a linear function of the electrophoretic mobilities of the vesicles used. A similar relationship between the net negative charge of the vesicles, as measured electrophoretically, and the reduction rate was also attained in the absence of substrate. The enhanced reduction was mainly reflected in an altered phase distribution of the reduction; the extent of fast phase reduction in the absence or in the presence of added substrate was dependent upon the electrophoretic mobilities of the vesicles. A similar change in the distribution of the reduction phases was observed upon decreasing the phosphatidylcholine content of the vesicles; the fast phase reduction being more pronounced in membranes with higher relative amounts of the protein components. A decrease of the rate of O-demethylation of p-nitroanisole catalyzed by P-450 LM2 parallel to the extent of fast phase reduction was observed upon dilution of neutral phosphatidylcholine membranes with phospholipid. By contrast, no effect of lipid dilution was evident in negatively charged membranes. The results are consistent with the hypothesis that the extent of fast phase reduction is governed by the amount of complex formed between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membranes; negative membranes appear to favor the formation of such complexes, whereas similar complexes are less formed, or are not functional, in neutral membranes.

Electrochemistry↗
PubMed1983

Hydroxyl radical-mediated, cytochrome P-450-dependent metabolic activation of benzene in microsomes and reconstituted enzyme systems from rabbit liver.

The mechanism of benzene oxygenation in liver microsomes and in reconstituted enzyme systems from rabbit liver was investigated. It was found that the NADPH-dependent transformation of benzene to water-soluble metabolites and to phenol catalyzed by cytochrome P-450 LM2 in membrane vesicles was inhibited by catalase, horseradish peroxidase, superoxide dismutase, and hydroxyl radical scavengers such as mannitol, dimethyl sulfoxide, and catechol, indicating the participation of hydrogen peroxide, superoxide anions, and hydroxyl radicals in the process. The cytochrome P-450 LM2-dependent, hydroxyl radical-mediated destruction of deoxyribose was inhibited concomitantly to the benzene oxidation. Also the microsomal benzene metabolism, which did not exhibit Michaelis-Menten kinetics, was effectively inhibited by six different hydroxyl radical scavengers. Biphenyl was formed in the reconstituted system, indicating the cytochrome P-450-dependent production of a hydroxycyclohexadienyl radical as a consequence of interactions between hydroxyl radicals and benzene. The formation of benzene metabolites covalently bound to protein was efficiently inhibited by radical scavengers but not by epoxide hydrolase. The results indicate that the microsomal cytochrome P-450-dependent oxidation of benzene is mediated by hydroxyl radicals formed in a modified Haber-Weiss reaction between hydrogen peroxide and superoxide anions and suggest that any cellular superoxide-generating system may be sufficient for the metabolic activation of benzene and structurally related compounds.

Animals↗
PubMed1983

Oxidation-reduction potential of soluble and membrane-bound rabbit liver microsomal cytochrome P-450 LM2.

The redox midpoint potentials of rabbit liver microsomal cytochromes P-450 and of soluble and membrane-bound rabbit liver microsomal cytochrome P-450 LM2 were determined using EPR-spectroscopy and absorption difference spectrometry with NADPH or dithionite as reductants. Using EPR, a redox midpoint potential of -0.36 V was obtained both for the low spin and the high spin components of microsomal cytochrome P-450. Spectrophotometrical determinations yielded very similar values: -0.37 V and -0.34 V for the low and high spin signals, respectively. Soluble cytochrome P-450 LM2 had a midpoint potential of -0.32 V. This redox potential was not significantly affected by incorporation of the protein into an artificial membrane structure or, furthermore, by the presence of cytochrome b5 the same membrane.

Animals↗
PubMed1982

Interindividual variation in benzo(a)pyrene metabolism and composition of isoenzymes of cytochrome P-450 as revealed by SDS-gel electrophoresis of human liver microsomal fractions.

Human liver microsomal fractions from 13 different individuals were characterized with respect to SDS-polyacrylamide gel electrophoretic profiles and regiospecificity in the metabolism of the polyaromatic hydrocarbon benzo(a)pyrene. Pronounced interindividual differences in the composition of microsomal proteins in the molecular weight region of Mr=49,000-60,000 were found. Furthermore, gel electrophoresis combined with staining for peroxidase activity indicated most of the variations among the profiles of microsomal proteins being attributed to interindividual differences in the composition of isoenzymes of cytochrome P-450. One type of human liver microsomal fraction was selectively induced in specimens obtained from patients with known regular drug intake before death and correlated well in molecular weight to the phenobarbital-inducible form of cytochrome P-450 in the rabbit. Large variations among the human liver microsomal samples were also seen in the benzo(a)pyrene metabolism. The results indicate the presence of 7-8 different froms of cytochrome P-450 in human liver microsomes and that the interindividual variations seen in drug metabolism may at least in part be explained by interindividual variations in the distribution of these isoenzymes.

Adolescent↗
PubMed1982

On the significance of the cytochrome P-450-dependent hydroxyl radical-mediated oxygenation mechanism.

1. Reconstituted membrane vesicles containing purified preparations of cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase effectively destroyed 2-deoxy-D-ribose in an NADPH-dependent process. 2. The destruction was mediated by hydroxyl radicals formed in an iron-catalysed Haber-Weiss reaction between superoxide anions and hydrogen peroxide liberated from the haemoprotein. 3. Administration of ethanol or benzene to rabbits, compounds known to be oxygenated by the hydroxyl radical-dependent mechanism, resulted in induction of a species of cytochrome P-450 effective in the radical-dependent metabolism of both chemicals. 4. Benzene treatment of rabbits also resulted in an enhanced hydroxyl radical-dependent metabolism of ethanol and benzene in liver microsomes. 5. It is suggested that, for certain substrates, hydroxyl radical-mediated cytochrome P-450-dependent oxygenation reactions are of importance for the microsomal metabolism of these compounds. 6. It is speculated that radical-producing species of cytochrome P-450 may contribute to hydroxyl radical-mediated cell damage.

Animals↗
PubMed1981

Membrane charge as effector of cytochrome P-450LM2 catalyzed reactions in reconstituted liposomes.

The phospholipid specificity of rabbit liver microsomal cytochrome P-450LM2 catalyzed hydroxylation reactions was examined in reconstituted phospholipid vesicles. An apparent linear relationship between the negative charge of the vesicles and the rate of P-450LM2-catalyzed O-dealkylation of p-nitroanisole or 7-ethoxycoumarin was obtained. The membrane charge-mediated increase in hydroxylation activities was found not to be due to (i) an altered lipid/water partition coefficient of the substrate, (ii) a change in the apparent Michaelis constant of P-450LM2 for the substrate, (iii) a different activation energy of the O-demethylation of p-nitroanisole, (iv) different spin states of P-450LM2 or (v) an altered secondary structure of this enzyme as monitored by circular dichroism. However, when the formation of the ferrous carbonyl complex of P-450LM2 was followed under aerobic or anaerobic conditions after the addition of NADPH to the vesicles, an increased negative charge of the membrane was accompanied by an increased reducibility of P-450LM2. A similar linear relationship between the reducibility of cytochrome b5 and the negative charge of the liposomes was also evident in membranes containing NADPH-cytochrome P-450 reductase and cytochrome b5. It is proposed that the interaction of the reductase with P-450LM2 is inefficient in neutral vesicles and thus rate determining for the overall hydroxylation activities.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1981

The mechanism of cytochrome P-450-dependent oxidation of ethanol in reconstituted membrane vesicles.

The mechanism of cytochrome P-450-dependent oxidation of ethanol has been investigated using reconstituted phospholipid vesicles containing purified preparations of rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450 LM2. Incorporation of cytochrome b5 into the vesicles resulted in a 5-fold enhancement of cytochrome P-450-catalyzed O-dealkylation of 7-ethoxycoumarin, whereas the cytochrome P-450-dependent ethanol oxidation was slightly inhibited. Superoxide dismutase, added in increasing amounts to the vesicles, inhibited the formation of superoxide anions and, in a concomitant manner, also the production of acetaldehyde from ethanol in the system. Also horseradish peroxidase inhibited ethanol oxidation catalyzed by the vesicles; acetaldehyde formation and H2O2 formation decreased in a concomitant manner as the amount of the peroxidase was increased. Externally added hydrogen peroxide markedly stimulated cytochrome P-450-dependent ethanol oxidation, but not until the concentration of H2O2 reached 0.3 mM, whereas the hydroxyl radical scavenger mannitol completely inhibited the cytochrome P-450-dependent acetaldehyde production. Oxidation of ethanol was also accomplished using vesicles containing cytochrome b5 instead of cytochrome P-450 and in other systems regenerating superoxide anions, e.g. the xanthine-xanthine oxidase system and dihydroxyfumarate. The results are consistent with an iron-catalyzed Haber-Weiss mechanism for regeneration of hydroxyl radicals which subsequently react with ethanol, thereby giving the corresponding aldehyde.

Animals↗