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Journal of Lipid Research Apr 1986Biosynthesis of wax esters, one of the two major products of the meibomian gland, was found to be catalyzed mainly by the microsomes of the bovine meibomian gland. The...
Biosynthesis of wax esters, one of the two major products of the meibomian gland, was found to be catalyzed mainly by the microsomes of the bovine meibomian gland. The microsomal preparation catalyzed hexadecanoyl-CoA reduction to hexadecanol without any accumulation of the aldehyde intermediate. Maximal rates of reduction occurred at pH 6.5 and required both NADH and NADPH; the latter alone gave considerable rates whereas NADH alone was ineffective. Exogenous hexadecanal reduction catalyzed by the same preparation showed a preference for NADH. The hexadecanoyl-CoA saturation pattern was slightly sigmoidal and concentrations higher than 125 microM inhibited reduction. The fatty alcohol generated from hexadecanoyl-CoA was found as free alcohol and as wax esters. Esterification of hexadecanol to wax esters catalyzed by the meibomian gland microsomal preparation required exogenous acyl-CoA or ATP and CoA and was not affected by exogenous cholesterol. Maximal rates of esterification were observed at neutral pH. Hexadecanoyl-CoA concentrations higher than 125 microM inhibited esterification. Hexadecanol showed a typical substrate saturation pattern with an apparent Km of 125 microM. Radio gas-liquid chromatography showed that, in the presence of exogenous hexadecanoyl-CoA, hexadecanol gave hexadecyl hexadecanoate whereas in the presence of ATP and CoA both C16 and C18 endogenous acids were used to esterify the alcohol. Consistent with the composition of the meibomian gland secretion, exogenous acyl-CoA longer than C14 and shorter than C20 gave maximal rates of esterification of hexadecanol.
Topics: Aldehyde Oxidoreductases; Animals; Cattle; Coenzyme A-Transferases; Eyelids; Hydrogen-Ion Concentration; Meibomian Glands; Microsomes; NAD; NADP; Subcellular Fractions; Substrate Specificity; Sulfurtransferases; Time Factors
PubMed: 3459788
DOI: No ID Found -
Chemical & Pharmaceutical Bulletin Mar 2010An automated synthesis system using a solid-phase extraction (SPE) system and column packed with octadecylsilica (ODS), which was coated with phospholipid and loaded...
An automated synthesis system using a solid-phase extraction (SPE) system and column packed with octadecylsilica (ODS), which was coated with phospholipid and loaded with dog liver microsomes, was developed for synthesis of glucuronides. Preparation of the microsome-immobilized SPE column, glucuronidation of drugs to synthesize the glucuronides and elution of the products were performed by an automated synthesis system. The phospholipid-coated SPE column and then the microsome-immobilized SPE column were readily prepared by allowing a solution containing L-alpha-dipalmitoylphosphatidylcholine to flow through the SPE column, and then by recycling a buffer solution containing dog liver microsomes through the resulting phospholipid-coated SPE column. The microsome-immobilized SPE column exhibiting the uridine diphosphate (UDP)-glucuronosyltransferase activity catalyzed the glucuronidation of mefenamic acid and estradiol to the corresponding glucuronides in the presence of UDP-glucuronic acid, and three glucuronides of mefenamic acid and estradiol were synthesized using the automated synthesis system, by simply recycling a buffer solution containing UDP-glucuronic acid through the microsome-immobilized SPE column loaded with the substrate. We used beta-cyclodextrin as a solubilizing agent for the synthesis of the glucuronides of estradiol that is practically insoluble in aqueous solutions. The productivity of these glucuronides using the microsome-immobilized SPE column was higher than that using the free microsomes (batch method). Furthermore, we developed a fully automated synthesis-isolation system by coupling the automated synthesis system to an automated preparative HPLC system. The automated synthesis system as well as the fully automated synthesis-isolation system should be very useful for synthesizing glucuronides for drug development.
Topics: Animals; Dogs; Female; Glucuronides; Liver; Microsomes; Molecular Conformation; Phospholipids; Silicon Dioxide; Solid Phase Extraction; Stereoisomerism; Surface Properties
PubMed: 20190440
DOI: 10.1248/cpb.58.354 -
Molecules (Basel, Switzerland) Mar 2020Novel purine and purine isosteres containing a ferrocene motif and 4,1-disubstituted (-, -, -, -, -, , -, -, -) and 1,4-disubstituted (- and -) 1,2,3-triazole rings were...
Novel purine and purine isosteres containing a ferrocene motif and 4,1-disubstituted (-, -, -, -, -, , -, -, -) and 1,4-disubstituted (- and -) 1,2,3-triazole rings were synthesized. The most potent cytotoxic effect on colorectal adenocarcinoma (SW620) was exerted by the 6-chloro-7-deazapurine (IC = 9.07 µM), 6-chloropurine (IC = 14.38 µM) and (IC = 15.50 µM) ferrocenylalkyl derivatives. The -9 isomer of 6-chloropurine containing ferrocenylmethylene unit showed a favourable in vitro physicochemical and ADME properties including high solubility, moderate permeability and good metabolic stability in human liver microsomes.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cytotoxins; Epithelial Cells; Ferrous Compounds; Humans; Inhibitory Concentration 50; Liver; Metallocenes; Microsomes; Permeability; Purines; Solubility; Stereoisomerism; Structure-Activity Relationship; Triazoles
PubMed: 32235404
DOI: 10.3390/molecules25071570 -
The Journal of Biological Chemistry Jun 1995A gastric serine protease(s) was found in porcine gastric antral mucosa and was shown to be distributed in the endoplasmic reticulum (ER)-microsome fraction and also in...
A gastric serine protease(s) was found in porcine gastric antral mucosa and was shown to be distributed in the endoplasmic reticulum (ER)-microsome fraction and also in the vesicle fraction. Two forms of the protease were purified over 6,000-fold from the ER-microsome fraction. Analyses of various molecular and enzymatic characteristics including the N-terminal and partial internal amino acid sequences of both forms revealed that they share the same properties and are indistinguishable from porcine pancreatic trypsin. This is the first time that trypsin or a protease almost identical with trypsin has been found to be present intracellularly in normal tissues. The gastric trypsin activities from the ER-microsome and the vesicle fractions were located in distinct density regions upon density gradient centrifugation, which indicates association of the protease with different organelle membranes. Taken together, these results suggest that there may be a novel function of trypsin in the gastric mucosa; it might function as a specific degrading or processing enzyme as an intracellular protease.
Topics: Amino Acid Sequence; Animals; Centrifugation; Chromatography, DEAE-Cellulose; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Gastric Mucosa; Microsomes; Molecular Sequence Data; Swine; Trypsin
PubMed: 7782340
DOI: 10.1074/jbc.270.24.14748 -
The Journal of Biological Chemistry Oct 1981Since there exists some controversy in the literature as to whether paraquat augments microsomal lipid peroxidation via superoxide anion (O2-), the role of paraquat and...
Since there exists some controversy in the literature as to whether paraquat augments microsomal lipid peroxidation via superoxide anion (O2-), the role of paraquat and active oxygen species in NADPH-dependent lung microsomal lipid peroxidation was investigated. Incubation of buffered aerobic mixture of bovine lung microsome and NADPH, in the presence or absence of exogenously added iron, resulted in a progressive formation of lipid peroxides whose accumulation could be followed at 535 nm as malondialdehyde. Paraquat strongly inhibited this lipid peroxidation. Thus, malondialdehyde formation was 50% inhibited by 4 X 10(-5) M paraquat in the reaction mixture. The malondialdehyde color development by lipid peroxides was not affected by this concentration of paraquat. Lipid peroxidation was also strongly inhibited by singlet oxygen scavengers, e.g. dimethylfuran and diphenylfuran, and by catalase. Hydroxyl radical scavengers, e.g. mannitol, benzoate, and ethanol, had little effect in malondialdehyde production. Superoxide dismutase, which removes O2- efficiently, did not inhibit malondialdehyde production by lung microsomes and rather enhanced its formation. A scheme in which paraquat and active O2 species may be involved with microsomal lipid peroxidation is presented.
Topics: Animals; Catalase; Cattle; Erythrocytes; Kinetics; Lipid Peroxides; Lung; Microsomes; NADP; Oxidation-Reduction; Paraquat; Superoxide Dismutase
PubMed: 7275991
DOI: No ID Found -
The Journal of Biological Chemistry Jun 1975MgATP-dependent calcium sequestering activity of rat liver microsomes has been characterized. This activity is linear over a 90-min period and specifically requires...
MgATP-dependent calcium sequestering activity of rat liver microsomes has been characterized. This activity is linear over a 90-min period and specifically requires MgATP. Substitution of CTP, UTP, GTP, or ADP will not support calcium uptake. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle microsomes, is required to maintain net accumulation of calcium. The reaction is temperature-dependent and has an apparent Vmax of 11.2 nmol/mg of protein/min. The apparent Km for calcium is 23.2 muM calculated from total calcium concentration, and approximately 4.6 muM based on free calcium concentration, and apparent Km for ATP is 1.8 mM. Calcium uptake activity normally measured in presence of 100 mM KCl is only slightly depressed if 100 mM NaCl is substituted and is considerably depressed when 200 mM sucrose replaces KCl. An appropriate hydrolysis of ATP is associated with the calcium uptake. Separation of smooth and rough endoplasmic reticulum on sucrose gradients indicates a considerably lower specific activity per mg of protein in the fraction enriched with rough endoplasmic reticulum. Azide, at a level which completely inhibits liver mitochondrial calcium sequestration, has no effect on the liver microsomal system. Oligomycin, which inhibits ATP-dependent calcium uptake of liver mitochondria, has a considerably lesser effect on calcium uptake of liver microsomes. p-Chloromercuribenzoate and mersalyl inhibit the liver microsomal calcium pump at levels as low as 10- minus 7 M. Calcium uptake activity is considerably reduced in adult female rats. Weanling rats, both male and female, have calcium uptake activities like that of the adult males. Because of the higher activity in the male rat, the fatty acid composition of the liver microsomal phospholipids was analyzed. The male rat had a higher percentage of linoleic and palmitic acids in the microsomal phospholipids. Endoplasmic reticulum and plasma membrane are postulated to play a role in regulation of the levels of free cytoplasmic calcium in the mammalian liver.
Topics: Adenosine Triphosphate; Animals; Antimycin A; Azides; Biological Transport, Active; Calcium; Edetic Acid; Endoplasmic Reticulum; Female; Kinetics; Male; Microsomes, Liver; Oligomycins; Oxalates; Phospholipids; Rats; Sex Factors; Time Factors
PubMed: 806589
DOI: No ID Found -
Biopharmaceutics & Drug Disposition Apr 2017The metabolic capacity of the intestine and its importance as the initial barrier to systemic exposure can lead to underestimation of first-pass, and thus overestimation...
Optimization of intestinal microsomal preparation in the rat: A systematic approach to assess the influence of various methodologies on metabolic activity and scaling factors.
The metabolic capacity of the intestine and its importance as the initial barrier to systemic exposure can lead to underestimation of first-pass, and thus overestimation of oral bioavailability. However, the in vitro tools informing estimates of in vivo intestinal metabolism are limited by the complexity of the in vitro matrix preparation and uncertainty with the scaling factors for in vitro to in vivo extrapolation. A number of methods currently exist in the literature for the preparation of intestinal microsomes; however, the impact of key steps in the preparation procedure has not been critically assessed. In the current study, changes in enterocyte isolation, the impact of buffer constituents heparin and glycerol, as well as sonication as a direct method of homogenization were assessed systematically. Furthermore, fresh vs. frozen tissue samples and the impact of microsome freeze thawing was assessed. The rat intestinal microsomes were characterized for CYP content as well as metabolic activity using testosterone and 4-nitropheonol as probes for CYP and UGT activity, respectively. Comparisons in metabolic activity and scaled unbound intestinal intrinsic clearance (CL ) were made to commercially available microsomes using 25 drugs with a diverse range of metabolic pathways and intestinal metabolic stabilities. An optimal, robust and reproducible microsomal preparation method for investigation of intestinal metabolism is proposed. The importance of characterization of the in vitro matrix and the potential impact of intestinal scaling factors on the in vitro-in vivo extrapolation of F needs to be investigated further. © 2017 The Authors Biopharmaceutics & Drug Disposition Published by John Wiley & Sons Ltd.
Topics: Animals; Cytochrome P-450 Enzyme System; Glucuronosyltransferase; In Vitro Techniques; Intestinal Mucosa; Intestines; Male; Microsomes; Rats
PubMed: 28207929
DOI: 10.1002/bdd.2070 -
Drug Discoveries & Therapeutics May 2017Microsomal aromatase enzymes of humans and rats have been used in antiaromatase assays, but enzyme activity is species-specific. The current study extracted hepatic...
Microsomal aromatase enzymes of humans and rats have been used in antiaromatase assays, but enzyme activity is species-specific. The current study extracted hepatic microsomes of Nile tilapia (Oreochromis niloticus) to investigate and compare the antiaromatase activity of chrysin, quercetin, and quercitrin. This activity was evaluated using a dibenzylfluorescein (DBF) assay. Results revealed that the age and body weight of Nile tilapia affected the yield of extracted microsomes. Extraction of hepatic microsomes of Nile tilapia was most effective when using a reaction medium with a pH of 8.0. A DBF assay using Nile tilapia microsomes revealed significant differences in levels of antiaromatase activity for chrysin, quercetin, and quercitrin. Chrysin was the most potent aromatase inhibitor, with an IC50 of 0.25 mg/mL. In addition, chrysin is an aromatase inhibitor that also inhibits the proliferation of cancer cells. Hepatic microsomes of Nile tilapia can be used to investigate and compare the antiaromatase activity of different compounds.
Topics: Animals; Antioxidants; Aromatase Inhibitors; Cell Proliferation; Cichlids; Flavonoids; Hep G2 Cells; Humans; Hydrogen-Ion Concentration; MCF-7 Cells; Microscopy, Electron, Scanning; Microsomes, Liver; Quercetin
PubMed: 28320984
DOI: 10.5582/ddt.2017.01006 -
Stroke Jul 2012Cyclooxygenase-2 (COX-2) and Microsomal Prostaglandin E2 Synthase-1 (mPGES-1) catalyze isomerization of the cyclooxygenase product PGH2 into PGE2. Deletion of...
BACKGROUND AND PURPOSE
Cyclooxygenase-2 (COX-2) and Microsomal Prostaglandin E2 Synthase-1 (mPGES-1) catalyze isomerization of the cyclooxygenase product PGH2 into PGE2. Deletion of COX-2/mPGES-1 suppresses carotid artery atherogenesis and angiotensin II-induced aortic aneurysms formation, and attenuates neointimal hyperplasia after vascular injury in mice. The upregulation of COX-2/mPGES-1 in the wall of ruptured human cerebral aneurysms is not known.
METHODS
Ten patients with intracranial aneurysms (5 ruptured and 5 nonruptured) underwent microsurgical clipping. During the procedure, a segment of the aneurysm dome was resected and immunostained with monoclonal antibodies for COX-1, COX-2, and mPGES-1. A segment of the superficial temporal artery was also removed and immunostained with monoclonal antibodies for COX-1, COX-2, and mPGES-1.
RESULTS
All 10 aneurysm tissues stained positive for mPGES-1 monoclonal antibody. Expression of mPGES-1 was more abundant in ruptured aneurysm tissue than in nonruptured aneurysms, based on a semiquantitative grading. None of the superficial temporal artery specimens expressed mPGES-1. COX-2 was upregulated in the same distribution as was mPGES-1. COX-1 was present constitutively in all tissues.
CONCLUSIONS
COX-2/mPGES-1 are expressed in the wall of human cerebral aneurysms and more abundantly so in ruptured aneurysms than in nonruptured. We speculate that the protective effect of aspirin against rupture of cerebral aneurysms may be mediated in part by inhibition of COX-2/mPGES-1.
Topics: Adult; Aged; Aneurysm, Ruptured; Cyclooxygenase 2; Female; Humans; Intracranial Aneurysm; Intramolecular Oxidoreductases; Male; Microsomes; Middle Aged; Prostaglandin-E Synthases; Up-Regulation
PubMed: 22588264
DOI: 10.1161/STROKEAHA.112.655829 -
European Journal of Biochemistry Oct 1976A general assay for epoxide hydratase using epoxides derived from polycyclic aromatic hydrocarbons as substrates is described. Addition of dimethylsulphoxide to the...
A general assay for epoxide hydratase using epoxides derived from polycyclic aromatic hydrocarbons as substrates is described. Addition of dimethylsulphoxide to the incubation mixture after incubation allowed unreacted epoxide and its phenolic by-product to be extracted into light petroleum whilst the product dihydrodiol remained in the aqueous phase. The product was then extracted into ethyl acetate and estimated radiochemically. This assay gave low extraction blanks (0.8-3.8%) when six K-region epoxides of polycyclic hydrocarbons were used, with high recoveries of the corresponding dihydrodiol in the ethyl acetate phase (65-89%). Radiochromatograms demonstrated that all the radioactivity in the ethyl acetate extracts of active incubations above that of boiled enzyme blanks was confined to a single band that always cochromatographed with the authentic trans-dihydrodiol. Using this assay, the kinetic parameters of six K-region epoxides were estimated. In all cases the apparent Km was low (2-5.9 muM). This is about 100-fold lower than the known apparent Km of epoxide hydratase for styrene oxide, an alkene oxide that is widely used as a substrate for epoxide hydratase. The rate of hydration varied with the substrate. Thus the maximum velocity for hydration of phenanthrene 9,10-oxide greater than 7-methylbenz[a]anthracene 5,6-oxide approximately benz[a]anthracene 5,6-oxide approximately benzo[a]pyrene 4,5-oxide greater than 3-methylcholanthrene 11,12-oxide greater than dibenz[a,h] anthracene 5,6-oxide. This relationship between the individual epoxides was found in microsomal fractions from both rat skin and rat liver, although the activity was always much lower in skin microsomes. All six arene oxides derived from polycyclic hydrocarbons were substrates for the homogeneous epoxide hydratase that was isolated from rat liver microsomal fractions using styrene oxide, an alkene oxide, as substrate to follow the purification.
Topics: Animals; Epoxide Hydrolases; Epoxy Compounds; Hydro-Lyases; Kinetics; Microsomes, Liver; Polycyclic Compounds; Rats; Structure-Activity Relationship
PubMed: 991864
DOI: 10.1111/j.1432-1033.1976.tb10862.x