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Biomedical subjects

M Ingelman-Sundberg

Publications and source records attributed to M Ingelman-Sundberg.

At least 235 records · Page 13Linked to original sources
PubMed1980

Incorporation of purified components of the rabbit liver microsomal hydroxylase system into phospholipid vesicles.

Methods are described for incorporation of purified forms of rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450LM2, P-450LM3 and P-450LM4 (LM, liver microsomes) into phospholipid vesicles. It was found that each cytochrome could individually be incorporated into preformed phospholipid vesicles in the absence of cholate. However, NADPH-cytochrome P-450 reductase prevented incorporation of P-450 by this method, a phenomenon possibly inherent in the formation of complexes between P-450 and the reductase in solution. Using the cholate-gel filtration technique it was possible to prepare monolamellar phosphatidylcholine vesicles containing any of the cytochromes and P-450 reductase in good yields. It was found that P-450LM3-containing vesicles had a mean diameter of 47 nm, whereas vesicles formed under the same conditions but containing P-450LM4 were much smaller (mean diameter 33 nm). Vesicles formed with P-450LM2 were homogeneous in density (1.04 g/cm3) according to isopycnic centrifugation in Ficoll but not in size (44-72 nm). These findings, taken together with results obtained from treatment of the cytochromes in soluble form and in reconstituted vesicles with the non-penetrating reagent, p-diazobenzene sulphonate, indicate a unidirectional, relatively peripheral orientation of P-450LM4 with the major part localized on the outside of the vesicles. Experiments with trypsin and cytochrome c-reduction demonstrated a unidirectional orientation of P-450 reductase towards the outside of the vesicles.

Animals↗
PubMed1980

Correlation between changes in enzymatic activities and induction of different forms of rat liver microsomal cytochrome P-450 after phenobarbital-, 3-methylcholanthrene- and 16 alpha-cyanopregnenolone treatment.

Multiple forms of liver microsomal cytochrome P-450 in rats were identified on SDS-polyacrylamide gels stained for protein and peroxidase activity after induction with phenobarbital, 3-methylcholanthrene, and 16 alpha-cyanopregnenolone. The induced forms were correlated to the in vitro metabolism of biphenyl, benzo(a)pyrene and the steroids 4-androstene-3,17-dione and 5 alpha-androstane-3 alpha,17 beta-diol. Induction of two forms with apparent molecular weights of 54,000 (RLvMc P-450 54) AND 50,000 (RLvMc P-450 50) was obtained with phenobarbital, induction of RLvMc P-450 55 and RLvMc P-450 58 with 3-methylcholanthrene and induction of FLvMc P-450 54 with 16 alpha-cyanopregnenolone. The RLvMc P-450 50 was mainly associated with the formation of benzo(a)pyrene-4,5-dihydrodiol, and 7 alpha-hydroxy-4-androstene-3,17-dione. The RLvMc P-450 55 and/or the RLvMc P-450 58 was mainly associated with the formation of 2- and 3-hydroxybiphenyl and benzo(a)pyrene-7,8-dihydrodiol and FLvMc P-450 54 was to some extent associated with the formation of benzo(a)pyrene-4,5-dihydrodiol and several metabolites of 5 alpha-androstane-3 alpha, 17 beta-diol. It is suggested that SDS-polyacrylamide gel electrophoresis may be a valuable complement to enzyme assays in evaluating effects of drugs and environmental chemicals on the liver microsomal hydroxylase system.

Androstane-3,17-diol↗
PubMed1979

Isolation and incorporation of rabbit liver epoxide hydrase into phospholipid vesicles.

Methods are described for the incorporation into phospholipid vesicles of epoxide hydrase isolated from liver microsomes of phenobarbital-treated rabbits. Chromatography on a Sephadex G-50 column of epoxide hydrase and egg yolk phosphatidylcholine treated with sodium cholate yielded homogeneous vesicles with a diameter of about 25 nm and containing 80 to 85% of the protein applied. At high substrate concentrations, the vesicles catalyzed the hydration of benzo(a)pyrene-4,5-oxide and styrene-7,8-epoxide at a rate similar to that obtained with the enzyme in a soluble form. However, the kinetics of styrene glycol formation catalyzed by the vesicular or microsomal preparations were complex. Convex Lineweaver-Burk plots and concave Hill plots were obtained, whereas normal Michaelis-Menten kinetics characterized the hydration catalyzed by the enzyme in a soluble form. The results could be explained if reconstitution of the enzyme into the vesicles gives rise to low affinity high capacity sites for the substrate on the enzyme, or alternatively facilitates the interaction of the substrate with such sites already present. It is suggested that reconstituted liposomes containing both the liver microsomal hydroxylase system and epoxide hydrase may prove to be a good model system for evaluating substrate specificity and factors of importance in the formation of toxic and carcinogenic metabolites by these enzymes.

Animals↗
PubMed1979

Protein amino acid analysis by an isotope ratio gas chromatography mass spectrometry computer technique.

A method for quantitative analysis of protein amino acids by a gas chromatography mass spectrometry computer system is described. Amino acids were analysed as their N-trifluoroacetyl n-butyl ester derivatives. Isotope ratio determination was used as the quantitating technique via multiple internal standards. The exact composition of a deuterated amino acid mixture was determined against a standard amino acid calibration mixture and in turn the protein amino acid composition was determined against the deuterated amino acid mixture. The amount of protein taken for analysis was 100 micrograms and the procedure, excluding hydrolysis, could be performed with 2 1/2 hours. The introduction of the internal standards prior to protein hydrolysis provides a method with good precision (mean coefficient of variation less than 5%). The method, tested on insulin, gave results which agreed well with the known composition of the protein and with simultaneous analysis on ion exchangers.

Amino Acids↗
PubMed1979

Catalytic properties of the liver microsomal hydroxylase system in reconstituted phospholipid vesicles.

Two types of cytochrome P-450, P-450LM2 and P-450LM3, have been purified from rabbit liver microsomes and incorporated into phospholipid vesicles by a cholate gel filtration technique together with purified preparations of NADPH-cytochrome P-450 reductase. The catalytic properties of the vesicles have been compared with a system reconstituted with small amounts of dilauroylphosphatidylcholine (DLPC). 6 beta-Hydroxylation of androstenedione proceeded at a rate 10 times higher in the vesicles compared to the DLPC-system. The kinetics for the reaction were the same in the vesicles as in intact microsomes i.e. sigmoidal substrate curves were obtained and Hill-coefficients of about 1.4 were calculated in these systems. In contrast, Michaelis-Menten kinetics were obtained for 6 beta-hydroxylation in the DLPC-system. The results could indicate cooperativity between different P-450 molecules in the intact membrane but not in the DLPC-system. P-450LM2-catalyzed 16-hydroxylation of androstenedione was in contrast to the situation with P-450LM3 inhibited in the vesicles as compared to the DLPC system. It is suggested that for evaluation of substrate specificity and other properties of different types of liver microsomal P-450, phospholipid vesicles may be a more relevant integration level than the DLPC-system.

Androstenedione↗
PubMed1979

Isolation and characterization of cytochrome P-450meg.

The cytochrome P-450-dependent steroid 15 beta-hydroxylase system from Bacillus megaterium has been resolved into three components, 1) a NADPH-specific, FMN-containing flavoprotein reductase, molecular weight 55-60 000; 2) an iron-sulfur protein, molecular weight 13,000 and 3) cytochrome P-450meg, molecular weight 52,000. The cytochrome component has been purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel, and its amino acid composition has been determined. Cytochrome P-450meg has a pI of 4.9, a Stokes radius of 27 A and a sedimentation constant of 3.3 S. Electron paramagnetic resonance and optical spectra are typical of a low-spin cytochrome P-450. The fluorescence spectrum is indicative of a tryptophane residue in a relatively non-polar environment. In recombination experiments, the electron flow was shown to proceed from the reductase via the iron-sulfur protein to the cytochrome. It is also possible to exchange the different components of the mitochondrial 11 beta-hydroxylase system from bovine adrenals for corresponding components in B. megaterium. Substrate specificity studies indicate that only steroids with a 3-oxo-delta 4-configuration are hydroxylated by the B. megaterium hydroxylase system. When oxidizing agents were used, hydroxylation occurred both in positions 15 alpha and 15 beta. Further substrate specificity studies have shown that aniline and imipramine can function as substrates for the bacterial system.

Bacillus megaterium↗
PubMed1979

Molecular properties of cytochrome P-45011 beta from adrenal cortex mitochondria.

Cytochrome P-45011 beta has been solubilized and partially purified from bovine adrenal cortex mitochondria using chromatography on Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450 preparations were about 90% pure as judged by SDS-polyacrylamide gel electrophoresis. In the presence of purified preparations of adrenodoxin reductase and adrenodoxin, the partially purified P-450 preparations catalyzed NADPH-supported 11 beta-hydroxylation of unconjugated and sulphoconjugated deoxycorticosterone. In presence of Triton X-100 the partially purified cytochrome P-45011 beta had a Stoke's radius of 4.5 nm, a sedimentation coefficient of 3.1 S and a partial specific volume of about 0.85 cm3/g. These results indicate that the cytochrome P-45011 beta-Triton X-100 complex has a molecular weight of about 100 000 and that P-45011 beta bound about 1.1 g of Triton X-100 per g of protein. The P-45011 beta-Triton X-100 complex was catalytically active in hydroxylation reactions supported by NADPH or the hydroxylating agent ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome P-45011 beta is an active form of the protein.

Adrenal Cortex↗
PubMed1979

Gas chromatography-mass spectrometry in analysis of protein amino acid composition.

A method for quantitative analysis of protein amino acids using isotope ratio gas chromatography-mass spectrometry computer techniques is described. The amino acids are analysed as their N-trifluoroacetyl-n-butyl esters and the quantitation is accomplished by the inclusion of deuterated amino acid mixtures of known composition. The inclusion of internal standards prior to protein hydrolysis provides a method with high precision (coefficient of variation less than 5%). The method, which requires 100 microgram and can be performed in just over 2 h, when tested on insulin gave results which agreed well with the known composition of the protein and with simultaneous analysis on ion exchangers. Thus, the technique offers an alternative to automatic amino acid analysers (utilizing ion exchangers) for quantitative amino acid analysis.

Amino Acids↗
PubMed1978

The active form of cytochrome P-45011beta from adrenal cortex mitochondria.

Cytochrome P-45011beta has been solubilized and partially purified from bovine adrenal cortex mitochondria by means of chromatography on Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450 preparations were about 90% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but had a low specific content of P-450 (between 1 and 2 nmol of P-450 per mg of protein). In the presence of purified preparations of adrenodoxin reductase and adrenodoxin, the partially purified P-450 preparations catalyzed NADPH-supported 11beta-hydroxylation of unconjugated and sulfoconjugated deoxycorticosterone. In the reconstituted system the hydroxylation of deoxycorticosterone sulfate proceeded at a much higher rate than in intact mitochondria, indicating that in the former case interactions between the hydrophilic substrate and P-450 were facilitated. In the presence of Triton X-100 the partially purified cytochrome P-45011beta had a Stokes radius of 4.5 nm, a sedimentation coefficient of 3.1 S, and a partial specific volume of about 0.85 cm3/g. These results indicate that the cytochrome P-45011beta . Triton X-100 complex had a molecular weight of about 100,000 and that P-45011beta bound about 1.1 g of Triton X-100 per g of protein. The P-45011beta . Triton X-100 complex was catalytically active in hydroxylation reactions supported by NADPH or the hydroxylating agent ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome P-45011beta is the active form of the protein.

Adrenal Cortex↗
PubMed1978

Qualitative alterations of cytochrome P-450 in mouse liver microsomes after administration of acrylamide and methylmethacrylate.

Cytochrome P-450 in mouse liver microsomes was characterized by SDS-polyacrylamide gel electrophoresis after intraperitoneal injection of 80 mg phenobarbital, 4.5 and 45 mg acrylamide and 60 and 600 mg methylmethacrylate per kg body weight each day for four days. Four different forms of cytochrome P-450 with molecular weights of 47,000, 50,000, 54,000 and 56,000 were identified by staining for peroxidase activity and protein. The amount of cytochrome P-450 with a molecular weight of 47,000 (MLvMcP-450(47) decreased in the phenobarbital group and in both acrylamide groups. After methylmethacrylate treatment, this form increased at the low dose but was totally repressed at the high dose. The cytochrome P-450 form with a molecular weight of 50,000 (MLvMcP-450(50) was significantly increased only in the phenobarbital group. An increase in the total amount of cytochrome P-450 was only observed following treatment with phenobarbital.

Acrylamides↗
PubMed1976

Specific reductive metabolism of steroid sulphates in rat liver.

The reductive metabolism of steroid sulphates and corresponding unconjugated steroids has been investigated in male and female rat liver. Enzyme activities studied were 5alpha-reductase, 5beta-reductase and 3alpha-, 3beta- and 20beta-hydroxy-steroid oxidoreductases in microsomal and soluble cellular fractions. With the exception of microsomal 3alpha-reductase all enzymes were equally or more active using sulphoconjugated substrates than using corresponding unconjugated substrates. 20beta-reduction of corticosterone and deoxycorticosterone occurred only with the 21-sulphurylated derivatives. This sulphate-specific 20beta-reductase, which is only present in male rat liver, probably participates in the formation of the sex-specific 20beta-reduced corticosterone metabolites present in bile from male but not from female rats. Sulphoconjugated deoxycorticosterone and corticosterone were also better substrates for microsomal 5alpha-reductase and sulphoconjugated 5alpha-dihydrotestosterone for microsomal 3beta-hydroxysteroid oxidoreductase than the corresponding unconjugated analogues. In conclusion, these results indicate that steroid sulphates generally are good substrates for hepatic reducing enzymes; in many cases even better substrates than the unconjugated analogues.

Animals↗