-
The Journal of Biological Chemistry Jun 1993When reconstituted with cytochrome b5 and NADPH cytochrome P450 oxidoreductase, cytochrome P450 2E1 metabolized lauric, stearic, oleic, linoleic, linolenic, and...
When reconstituted with cytochrome b5 and NADPH cytochrome P450 oxidoreductase, cytochrome P450 2E1 metabolized lauric, stearic, oleic, linoleic, linolenic, and arachidonic acid to multiple metabolites. Two major metabolites, accounting for 78% of the total metabolism, were produced with arachidonic acid. The Vmax for total metabolite formation from arachidonic acid was 5 nmol/min/nmol P450 with an apparent Km of 62 microM. Gas chromatography-mass spectrometry analysis identified the two major metabolites as monohydroxylated eicosatetraenoic acids (HETEs). The major HETE was 19-hydroxyeicosatetraenoic acid (19-HETE) and comprised 46% of the total metabolite produced. The second metabolite was the omega-2 hydroxylated metabolite (18-HETE) and comprised 32% of the total product formed. Chiral analysis demonstrated that 19-HETE was 70% 19(S)-HETE and 30% 19(R)-HETE. In contrast, 18-HETE was essentially 100% R isomer. Approximately 18% of the total metabolite produced from arachidonic acid coeluted with epoxyeicosatrienoic acid (EET) standards. The EET metabolites were 56.4% 14,15-EET and 43.6% as a mixture of 11,12-EET and 8,9-EET. 5,6-EET was not detected. Anti-P450 2E1 IgG inhibited arachidonic acid metabolism by renal and hepatic microsomes prepared from acetone-treated rabbits. With renal cortex microsomes, the formation of 18-HETE and 19-HETE was inhibited 67 and 25%, respectively, by the antibody. Liver microsomal formation of 18-HETE was inhibited by 87% and 19-HETE by 70%. Thus, under conditions where cytochrome P450 2E1 is induced, the enzyme could contribute significantly to the formation of the omega-1 and omega-2 hydroxylated metabolites of arachidonic acid.
Topics: Acetone; Animals; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2E1; Cytochrome P-450 Enzyme System; Enzyme Induction; Ethanol; Fatty Acids; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Kidney Cortex; Male; Mass Spectrometry; Microsomes; Microsomes, Liver; Organ Specificity; Oxidoreductases, N-Demethylating; Rabbits; Stereoisomerism; Substrate Specificity
PubMed: 8509425
DOI: No ID Found -
European Journal of Biochemistry Oct 1995Immunolocalization studies indicated that, in contrast to other enzyme markers of human pancreatic secretion, bile-salt-dependent lipase (BSDL) was partly but...
Immunolocalization studies indicated that, in contrast to other enzyme markers of human pancreatic secretion, bile-salt-dependent lipase (BSDL) was partly but specifically associated with endoplasmic reticulum membranes. In microsomes, temperature-induced phase separation using Triton X-114 elucidated the partition of BSDL between the aqueous phase and the detergent-rich phase containing hydrophilic and membrane proteins, respectively. The size of the membrane-associated BSDL (approx. 100 kDa) is compatible with that of the fully processed enzyme. Fucosylated O- and N-linked oligosaccharide structures were detected by means of specific lectins. The membrane-associated BSDL might therefore be released from membranes between the trans-Golgi compartment (where terminal fucose residues were added) and the zymogen granules where BSDL was mainly found in the soluble fraction. Even though BSDL associated with membranes was enzymically active, it appeared less efficient than the soluble form. The association of BSDL with membranes was pH-dependent and optimal association occurred between pH 5-6. The membrane-associated BSDL was released by KBr which suggests that the association of BSDL with microsomal membranes involves ionic interactions. Lipid-protein interactions are probably not involved in this association as BSDL did not associate with liver microsome membranes. We attempted to characterize the putative ligand and showed that BSDL and a 94-kDa protein, immunologically related to a glucose-regulated protein of 94 kDa (Grp94), were co-immunoprecipitated by specific antibodies directed against each individual species. It is suggested that the biogenesis of the human pancreatic BSDL involves an association with intracellular membranes and that its folding may be assisted by molecular chaperones.
Topics: Adult; Female; Glucose; Glycosylation; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Membranes; Lipase; Male; Membrane Proteins; Microscopy, Immunoelectron; Microsomes; Middle Aged; Pancreas; Sterol Esterase
PubMed: 7588748
DOI: 10.1111/j.1432-1033.1995.209_1.x -
Biochimica Et Biophysica Acta Jan 1997Tropical and sub-tropical higher plant species show marked growth inhibition when exposed to chilling temperatures. In root tip segments of coffee seedlings which were...
Tropical and sub-tropical higher plant species show marked growth inhibition when exposed to chilling temperatures. In root tip segments of coffee seedlings which were subjected for 6 days to temperatures of 10, 15, 20 and 25 degrees C, in darkness, we have detected an increased amount of malondialdehyde formed in the 10 degrees C treatment, accompanied by higher electrolyte leakage. The electron paramagnetic resonance (EPR) technique and the fatty acid spin probes 5-, 12- and 16-doxylstearic acid were used to assess cellular membrane fluidity. At the depth of the 5th and 16th carbon atom of the alkyl chains the nitroxide radical detected more rigid membranes in seedlings subjected to 10 degrees C compared with 15 and 25 degrees C. At the C-12 position of the chains the probe showed very restricted motion and was insensitive to chilling induced membrane alterations. EPR parameters for intact tissues and microsome preparations from root tips showed that the fluidity was essentially the same when evaluated at C-5 and C-16 positions of the chains, and was considerably more fluid for microsomal membranes in the region of the C-12 position of the bilayers. The rotational motion of the nitroxide at C-16 position of the chains experienced a phase transition at about 15 degrees C. The calculated energy barriers for reorientational motion of the probe 16-doxylstearic acid were higher at temperatures of 5-15 degrees C than in the interval of 15-25 degrees C, suggesting that below the phase transition the membrane lipids assume a more ordered and compacted array. Membrane rigidity induced by chilling was interpreted as due to lipid peroxidation that could have been facilitated by higher density of peroxidizable chains below the membrane phase transition.
Topics: Coffee; Cold Temperature; Electron Spin Resonance Spectroscopy; Lipid Peroxidation; Malondialdehyde; Membrane Fluidity; Microsomes; Plant Roots; Spin Labels; Thermodynamics
PubMed: 9030214
DOI: 10.1016/s0005-2736(96)00177-0 -
The Journal of Investigative Dermatology Jun 1994The phospholipase A2 (PLA2) activities that are localized in the keratinocyte cytosolic and microsomal fractions were biochemically and pharmacologically characterized....
The phospholipase A2 (PLA2) activities that are localized in the keratinocyte cytosolic and microsomal fractions were biochemically and pharmacologically characterized. The cytosol and to a lesser extent the microsome were sensitive to heat treatment and stable in the presence of sulfhydryl reducing agents. Both fractions were almost totally inactivated by reduction of pH to 2. The cytosolic activity demonstrated a sevenfold preference for arachidonic acid over oleic acid in the sn-2 position of substrate phospholipid and the microsome exhibited a fourfold preference. Neither the cytosol nor the microsome was inactivated by a neutralizing mouse monoclonal antibody 3F10 generated against recombinant human (rh) type II 14-kDa PLA2. Western immunoblot analysis of both fractions identified a high-molecular-mass protein in keratinocyte cytosol but not the microsome that migrated with rh 85-kDa PLA2. Neither the cytosol nor the microsome possessed immunoreactive bands that migrated with rh type II 14-kDa PLA2 when probed with monoclonal antibody 3F10. Further analysis of the cytosolic activity showed that it was activated by submicromolar concentrations of Ca2+, reduced by arachidonyl trifloromethylketone, a selective 85-kDa PLA2 inhibitor, but was unaffected by C-7 phosphonate phospholipid, a selective 14-kDa PLA2 transition state inhibitor. Taken together, the data supports the existence of a PLA2 activity in the cytosol that displays characteristics that are indistinguishable from those exhibited by the 85-kDa PLA2. Alternatively, both the cytosol and microsome were devoid of type II 14-kDa-like PLA2 activity. The failure of 12-epi scalaradial, a 14-kDa PLA2 inhibitor, to modify A23187-stimulated keratinocyte prostaglandin E2 release, was consistent with the biochemistry and suggests that the 85-kDa PLA2 may play an important role in keratinocyte prostaglandin E2 formation.
Topics: Antibodies, Monoclonal; Calcimycin; Cells, Cultured; Cytosol; Dinoprostone; Humans; Infant, Newborn; Keratinocytes; Male; Microsomes; Molecular Weight; Phospholipases A; Phospholipases A2; Substrate Specificity; Tumor Cells, Cultured
PubMed: 8006465
DOI: 10.1111/1523-1747.ep12384234 -
The Journal of Biological Chemistry Mar 1990Studies reported from this laboratory have demonstrated that O-glycosidic glycoproteins of salivary, pulmonary, and gastrointestinal origin are acylated by fatty...
Studies reported from this laboratory have demonstrated that O-glycosidic glycoproteins of salivary, pulmonary, and gastrointestinal origin are acylated by fatty acyltransferase residing in Golgi and microsome-enriched fraction (Slomiany, A., Liau, Y.H., Takagi, A., Laszewicz, W., and Slomiany, B.L. (1984) J. Biol. Chem. 259, 13304-13308). Here we report on the successful purification of this enzyme from rough microsomal membranes of rat gastric mucosa and its identification in a number of diverse tissues and organs, such as heart, liver, pancreas, lung, kidney, salivary glands, and lymphoblasts. The enzymatic activity has been released from the stripped and salt-extracted microsomes with 0.5% Triton X-100 and recovered from 100,000 x g supernatant by affinity chromatography on Cibacron blue F3GA column. The retained fatty acyltransferase protein was selectively displaced from the column with 50 microM palmitoyl-CoA. On nonreducing polyacrylamide gel electrophoresis, the enzymatic activity was associated with a 234-kDa complex, and on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the complex afforded 65- and 67-kDa protein bands. Incubation of microsomes with trypsin prior to enzyme extraction resulted in a 50% inactivation of the fatty acyltransferase and generation of 53- and 55-kDa protein bands, which also had affinity to Cibacron blue F3GA and were displaced from the column together with the active (intact) enzyme. We suggest that the fatty acyltransferase is an integral rough microsomal protein partially exposed to cytosol, which catalyzes the fatty acyl-CoA-protein reaction on the cytosolic site of the rough endoplasmic reticulum and that this enzyme is responsible for processing of the group of protein which are entering rough endoplasmic reticulum-Golgi secretory pathway.
Topics: Acyl Coenzyme A; Acyltransferases; Animals; Detergents; Electrophoresis, Polyacrylamide Gel; Gastric Mucosa; Intracellular Membranes; Kinetics; Male; Microsomes; Molecular Weight; Octoxynol; Peptide Mapping; Polyethylene Glycols; Rats; Rats, Inbred Strains; Solubility; Trypsin
PubMed: 2318887
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1993The alkenyl and aromatic glucosinolates in oilseed rape (Brassica napus) are biosynthesized from chain-extended homologues of protein amino acids, including methionine...
The alkenyl and aromatic glucosinolates in oilseed rape (Brassica napus) are biosynthesized from chain-extended homologues of protein amino acids, including methionine and phenylalanine. Homologues of these two amino acids, homophenylalanine (2-amino-4-phenylbutyric acid) and dihomomethionine (2-amino-6-methylthiohexanoic acid) were synthesized both with and without a 1-14C label. Microsomal preparations from oilseed rape leaves were shown to contain enzyme systems which metabolize these compounds, with loss of 14CO2, and produce the aldoxime intermediates possible in the biosynthetic pathway utilizing homophenylalanine. These were characterized by comparison with authenticated synthetic compounds. Potential intermediates on the pathway between homophenylalanine and its corresponding aldoxime, the N-hydroxyamino- and the oximino acids, were synthesized and their possible role in the pathway investigated.
Topics: Fatty Acids, Monounsaturated; Glucosinolates; Mass Spectrometry; Microsomes; Plant Oils; Rapeseed Oil
PubMed: 8262954
DOI: No ID Found -
The Biochemical Journal Aug 1976Obesity in obese-hyperglycaemic mouse is associated with an increase in number and size of adipocytes. Adipocytes from the obese mouse showed increased incorporation of...
Obesity in obese-hyperglycaemic mouse is associated with an increase in number and size of adipocytes. Adipocytes from the obese mouse showed increased incorporation of [14C]acetate and[14C]glucose into triacylglycerol. This increased capacity of triacylglycerol formation was correlated with increased activities of various triacylglycerol-forming enzymes measured in the microsomal fraction of adipose tissue from obese mice. Microsomal fractions from lean and obese mice contained sn-glycerol 3-phosphate acyltransferase, phosphatidate phosphohydrolase and diacylglycerol acyltransferase. Phosphatidate phosphohydrolase was also detected in the soluble fraction. In the presence of Mg2+, the phosphatidate phsophohydrolase from the soluble and the microsomal fractions was active towards membrane-bound phosphatidate. Among the three enzymes studied here, the increase in Mg2+-dependent phosphatidate phosphohydrolase was most prominent in adipose tissue of obese mice.
Topics: Acetates; Acyltransferases; Adenosine Triphosphate; Adipose Tissue; Animals; Cell Fractionation; Coenzyme A; Epididymis; Glucose; Glycerol-3-Phosphate O-Acyltransferase; Magnesium; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Microsomes; Palmitates; Phosphoric Monoester Hydrolases; Triglycerides
PubMed: 186034
DOI: 10.1042/bj1580327 -
Arthritis Research & Therapy 2005Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme that catalyzes the conversion of prostaglandin (PG)H2 to PGE2. Proinflammatory stimuli markedly... (Review)
Review
Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme that catalyzes the conversion of prostaglandin (PG)H2 to PGE2. Proinflammatory stimuli markedly increase levels of mPGES-1 expression both in vivo and in vitro. mPGES-1 knockout studies and animal models of inflammatory arthritis also provide a strong basis for the contribution of mPGES-1 in the increased local production of PGE2 observed in inflammatory arthritis. The focus of this article is to review some recent advances in our understanding of mechanisms specific to the regulation of inducible mPGES-1 in inflammatory arthritis.
Topics: Animals; Dinoprostone; Enzyme Induction; Humans; Intramolecular Oxidoreductases; Microsomes; Prostaglandin-E Synthases
PubMed: 15899061
DOI: 10.1186/ar1748 -
The Journal of Cell Biology Jan 1963The effect of trypsin on the morphology of the rat liver microsomal fraction isolated by differential centrifugation has been investigated. The microsomes were incubated...
The effect of trypsin on the morphology of the rat liver microsomal fraction isolated by differential centrifugation has been investigated. The microsomes were incubated at 37 degrees C and centrifuged thereafter under the conditions of their initial isolation. The trypsin-treated microsomes and the untreated controls were fixed in unbuffered osmium tetroxide and embedded first in gelose and then in methacrylate. In the trypsin-treated microsomes, there was a removal of the ribosomes from the rough vesicles. Parallel chemical determinations showed that the total nitrogen and total phosphorus of the pellet were lowered. Particles, densely stained with phosphotungstic acid (PTA) and homogeneous in appearance, were found within microsome smooth vesicles in a fluffy layer which collects on the top of the microsome pellet. The morphology of these PTA-stained particles remained unchanged after incubation with trypsin.
Topics: Animals; Liver; Microsomes; Microsomes, Liver; Rats; Ribosomes; Trypsin
PubMed: 13931790
DOI: 10.1083/jcb.16.1.81 -
European Journal of Biochemistry Dec 1996In many tissues from different species, pregnenolone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7 alpha position by a cytochrome P450...
In many tissues from different species, pregnenolone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7 alpha position by a cytochrome P450 (P450)-containing microsomal enzyme complex. In addition, 7-hydroxysteroids have been shown to activate immune processes in mice. The reported production of 7 beta-hydroxypregnenolone and 7 beta-hydroxy-DHEA was not supported by formal identification, and the P450 responsible for 7 alpha-hydroxylation and 7 beta-hydroxylation of pregnenolone and DHEA have not been identified. Based on results of analyses by crystallization to constant specific activity and gas chromatography/mass spectrometry, we report that mouse-liver and mouse-brain microsomes carried out 7 beta-hydroxylation of pregnenolone and DHEA, and that yeast-expressed mouse cytochrome P450-1A1 (P450 1A1) transformed pregnenolone into 7 beta-hydroxypregnenolone (Km = 25.1 +/- 0.4 microM, turnover number = 979 +/- 30 pmol.min-1.nmol-1 mouse P450 1A1). Neither 7-hydroxy derivatives of DHEA nor 7 alpha-hydroxypregnenolone was produced by P450 1A1. The presence of P450 1A1 in liver and brain microsomes was shown by Western blot analysis, and induction of mouse P450 1A1 by beta-naphthoflavone resulted in increased 7 beta-hydroxylation of pregnenolone in liver microsomes. Studies of the brain-microsome 7 beta-hydroxylating enzyme with pregnenolone or DHEA gave Km of 5.0 microM and 4.9 microM, respectively, and Vmax of 4.5 pmol.min-1.mg-1 and 6.1 pmol.min-1.mg-1, respectively, and showed the absence of cross-inhibitions between the two steroids. These findings indicate that, in addition to unidentified P450, P450 1A1 is involved in 7 beta-hydroxylation of pregnenolone and may contribute in part to the production of the 7-hydroxylated steroids necessary for activation of immune defenses.
Topics: Animals; Brain; Cytochrome P-450 CYP1A1; Dehydroepiandrosterone; Gas Chromatography-Mass Spectrometry; Kinetics; Mice; Mice, Inbred C57BL; Microsomes; Microsomes, Liver; Pregnenolone; Saccharomyces cerevisiae; Substrate Specificity
PubMed: 9022692
DOI: 10.1111/j.1432-1033.1996.0641r.x