Microsomal Material Research Articles

Modulation of trichloroethylene in vitro metabolism by different drugs in human

Toxicological interactions with drugs have the potential to modulate the toxicity of trichloroethylene (TCE). Our objective is to identify metabolic interactions between TCE and 14 widely used drugs in human suspended hepatocytes and characterize the strongest using microsomal assays. Changes in concentrations of TCE and its metabolites were measured by headspace GC–MS. Results with hepatocytes show that amoxicillin, cimetidine, ibuprofen, mefenamic acid and ranitidine caused no significant interactions. Naproxen and salicylic acid showed to increase both TCE metabolites levels, whereas acetaminophen, carbamazepine and erythromycin rather decreased them. Finally, diclofenac, gliclazide, sulphasalazine and valproic acid had an impact on the levels of only one metabolite. Among the 14 tested drugs, 5 presented the most potent interactions and were selected for confirmation with microsomes, namely naproxen, salicylic acid, acetaminophen, carbamazepine and valproic acid. Characterization in human microsomes confirmed interaction with naproxen by competitively inhibiting trichloroethanol (TCOH) glucuronidation (Ki=2.329mM). Inhibition of TCOH formation was also confirmed for carbamazepine (partial non-competitive with Ki=70μM). Interactions with human microsomes were not observed with salicylic acid and acetaminophen, similar to prior results in rat material. For valproic acid, interactions with microsomes were observed in rat but not in human. Inhibition patterns were shown to be similar in human and rat hepatocytes, but some differences in mechanisms were noted in microsomal material between species. Next research efforts will focus on determining the adequacy between in vitro observations and the in vivo situation.

Intracellular localization of photosensitizers.

The intracellular localization of photosensitizers can be studied by different methods. One method involves homogenization of the cells followed by differential ultracentrifugation which leads to fractions enriched in nuclear, mitochondrial, and microsomal material as well as a supernatant fraction. More detailed information can be obtained by electron microscopy of cells exposed to light in the presence of photosensitizers. This method is based on the assumption that damage is primarily induced at intracellular sites where the concentration of photosensitizer is high. By irradiating the cells at 6 degrees C, where biochemical reactions are slow, and then incubating them for different times at 37 degrees C, it is possible to follow the development of damage. The amount of photosensitized damage to enzymes or cell functions whose localization in the cells is known gives information about the intracellular localization of the sensitizer. Fluorescence microscopy is the most direct method and is widely applicable because most photosensitizers fluoresce. Lipophilic dyes generally localize in membrane structures. In future more attention should be paid to the localization of dyes in lysosomes, as suggested by early reports. Mitochondria, the endoplasmic reticulum and nuclear membrane are other important loci for intracellular localization of sensitizers.

Organ-related distribution of phospholemman in the spiny dogfish Squalus acanthias

The distribution of phospholemman among nine different organs of the spiny dogfish ( Squalus acanthias) has been determined on the basis of Western blotting of microsomal material. Only rectal gland (100%), brain (43%), heart (18%), and kidney (19%) (abundancies as percent of the concentration in rectal gland) contained the protein, but not gill and colon. The relative abundance in the brain makes this organ a preferential test system for phospholemman in fishes that lack a rectal gland like teleosts.

Addressing the metabolic activation potential of new leads in drug discovery: a case study using ion trap mass spectrometry and tritium labeling techniques.

Metabolic activation of drug candidates to electrophilic reactive metabolites that can covalently modify cellular macromolecules may result in acute and/or idiosyncratic immune system-mediated toxicities in humans. This presents a significant potential liability for the future development of these compounds as safe therapeutic agents. We present here an example of an approach where sites of metabolic activation within a new drug candidate series were rapidly identified using online liquid chromatography/multi-stage mass spectrometry on an ion trap mass spectrometer. This was accomplished by trapping the reactive intermediates formed upon incubation of compounds with rat and human liver microsomes as their corresponding glutathione conjugates and mass spectral characterization of these thiol adducts. Based on the structures of the GSH adducts identified, potential sites and mechanisms of bioactivation within the chemical structure were proposed. These metabolism studies were interfaced with iterative structural modifications of the chemical series in order to block these bioactivation sites within the molecule. This strategy led to a significant reduction in the propensity of the compounds to undergo metabolic activation as evidenced by reductions in the irreversible binding of radioactivity to liver microsomal material upon incubation of tritium-labeled compounds with this in vitro system. With the efficiency and throughput achievable with such an approach, it appears feasible to identify and address the metabolic activation potential of new drug leads during routine metabolite identification studies in an early drug discovery setting.

V‐ AND P‐TYPE Ca2+‐STIMULATED ATPases IN A CALCIFYING STRAIN OF PLEUROCHRYSIS SP. (HAPTOPHYCEAE)

Coccolithophorids are marine unicellular algae characterized by their ability to carry out controlled, subcellular calcification. The biochemical and kinetic features of membrane‐bound Ca2+‐stimulated ATPases have been examined. Membranes and organelles from axenic cultures of Pleurochrysis sp. (CCMP299) were isolated by means of sucrose density centrifugation. High levels of Ca2+‐stimulated ATPase were detected in chloroplasts, Golgi apparatus, plasma membrane, and coccolith vesicles. The sensitivity of the enzyme activity in the organelles and membranes was assessed with pharmacologic agents that are known to be specific for the several isoforms of Ca2+‐stimulated ATPase. The Ca2+‐stimulated ATPase activity in the Golgi and coccolith vesicle preparations was sensitive to nitrate, thiocyanate, and sodium azide and insensitive to vanadate, cyclopiazonic acid, and thapsigargin. ATP‐dependent H+ movement, but not 45Ca2+ transport, across the coccolith vesicle was demonstrated. The Ca2+‐stimulated ATPase in the plasma membrane preparation was sensitive to vanadate. ATP‐dependent, vanadate‐sensitive efflux of 45Ca2+ was demonstrated for microsomal material derived from gradient‐isolated plasma membrane. Polypeptides from isolated Golgi and coccolith vesicle preparations cross‐reacted to an antibody raised against a subunit of the oat root proton pump, whereas polypeptides from the chloroplast preparations did not cross‐react. These findings show that a V‐type Ca2+‐stimulated ATPase is located on the coccolith vesicle membrane and a P‐type Ca2+‐stimulated ATPase is located on the plasma membrane.

A STUDY OF MEMBRANE‐ASSOCIATED PHYTOCHROME: HYDROPHOBICITY TEST AND NATIVE SIZE DETERMINATION

Abstract—We confirmed that after extraction in the absence of added Mg2+, a small fraction of phytochrome was associated with pelletable, hydrophobic membranes. When microsomal material of several plant species was subjected to Triton X‐114 phase partitioning, a part of phytochrome migrated into the hydrophobic Triton phase in contrast to soluble phytochrome. The amount of bound phytochrome partitioning into the Triton phase varied from 6% for oats to 30% for zucchini and 50% for mustard and maize (0.5–10% of total phytochrome). Membrane‐associated phytochrome could be solubilized by the zwitterionic detergent CHAPS and with the nonionic detergent dodecylmaltoside. Subjected to gel filtration on Superose‐6/FPLC, oat phytochrome of the CHAPS solubilized sample was eluted in three different molecular weight ranges. There was a main fraction with the molecular weight of purified phytochrome, another fraction (approximately 20%) with a higher molecular weight, and a third small fraction appearing immediately after the void volume. Gel filtration after solubilization by dodecylmaltoside resulted in two distinct fractions: the one eluted at the position of the phytochrome dimer, and the other (approximately 15%) with an apparent molecular weight of 800 kDa. Phytochrome was detected, separated and quantified by SDS‐PAGE, and western blotting with the monoclonal antibody Z‐3B1. We assume that the distinct, phytochrome positive, high molecular weight fractions contain phytochrome associated with hydrophobic protein.

Expression of recombinant soluble and membrane-bound catechol O-methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain.

The rat and human recombinant soluble and membrane-bound catechol O-methyltransferase (S- and MB-COMT, respectively) were expressed using mammalian and baculovirus vectors. Low levels of rat and human S-COMT polypeptides were detected by immunoprecipitation in K-562 cell lines transfected with the S-COMT vectors. From K-562 cells transfected with the rat MB-COMT construct, both S- and MB-COMT recombinant proteins were detected by a rat COMT-specific anti-serum. Infection of lepidopteran Spodoptera frugiperda cells with recombinant S- or MB-COMT baculovirus constructs yielded high amounts of enzymically active and immunoreactive S- or MB-COMT proteins, respectively. Pulse/chase experiments with [35S]methionine-labelled insect cells infected with the MB-COMT baculovirus showed that the 30-kDa recombinant human MB-COMT polypeptide was not processed into the 25-kDa S-COMT form. Subcellular fractionations of insect cells, followed by immunoblotting with COMT antiserum, showed that recombinant S-COMT was found only in the soluble, cytoplasmic fraction, whereas MB-COMT resided both in soluble and membrane fractions. The recombinant MB-COMT sedimented in Percoll gradients at the density of 1.042 g/ml cosedimenting with the plasma-membrane marker. Fractionation and immunoblotting experiments on homogenized total rat brains indicated that the rat S-COMT (24 kDa) and some of the rat MB-COMT (28 kDa) was recovered in soluble fractions, whereas the microsomal material having COMT activity contained the MB-COMT polypeptide. The rat brain microsomal MB-COMT had a density of 1.042 g/ml in Percoll gradients, cosedimenting with the plasma-membrane and rough-endoplasmic-reticulum marker enzymes. The meta/para methylation ratio of dihydroxybenzoic-acid substrate by different recombinant and rat brain COMT-containing subcellular fractions was analysed.

Preparation of highly purified plasma membrane vesicles of controlled orientation from Penicillium cyclopium

A procedure for the production of plasma membrane vesicles, consisting of enzyme-assisted physical homogenization, differential centrifugation and partitioning in an aqueous polymer two phase system resulted in a fraction containing highly purified plasma membranes, as determined by vanadate-inhibited H + ATPase and near absence of intracellular membrane marker activities. H + ATPase latency tests indicated that at least 90% of the vesicles were oriented with the apoplastic surface facing outward (right-side-out orientation) in the upper phase, while inside out vesicles and other microsomal membrane material collected in the lower phase.

The effect of colchicine treatment on the buoyant density of noradrenaline storage particles in the rabbit superior cervical ganglion

The subcellular distribution of noradrenaline (NA) was studied in the rabbit superior cervical ganglion before and after colchicine treatment. One-third of the total NA could be sedimented with the microsomal material in control conditions. Upon colchicine treatment the percentage of the NA which was particle-bound increased only slightly. The absolute NA content of the superior cervical ganglion from colchicine-treated rabbits, however, showed a 3.5-fold increase compared to control animals. Density gradient centrifugation of the microsomal fraction revealed that the distribution of the NA-containing particles changed significantly. Before colchicine treatment the NA was found in a broad band covering the gradient fractions where ‘light’ and ‘heavy’ NA vesicles were expectted to equilibrate. After colchicine treatment the NA was concentrated in the ‘heavy’ NA vesicles part of the gradient. The dopamine β-hydroxylase activity and the NA content in the gradient are increased 3 times after colchicine treatment. These experimental results can be explained by the axoplasmic transport impairing action of colchicine. The neuronal cell body accumulates ‘heavy’ NA vesicles formed there, unable to transport them towards the periphery.

Isopycnic centrifugation of rat-liver microsomes in isoosmotic gradients of Percoll® and release of microsomal material by low concentrations of sodium deoxycholate

Rat-liver microsomes were subjected to isopycnic centrifugation under isoosmotic conditions in gradients of Percoll® containing 0%, 0.05%, 0.1% and 0.2% sodium deoxycholate, respectively. The buoyant density of the microsomes was in the range 1.043 to 1.046 g/ml, independent of the detergent concentration. Absorbance measurements and polyacrylamide gel electrophoresis revealed that the detergent causes an increase in the release of material from the microsomes. Electron microscopy studies showed that membrane disassembly was avoided if the microsomes were isolated at concentrations below 0.1% sodium deoxycholate.

The incorporation in vitro of sulphate ions into synaptic-membrane glycoproteins.

Synaptosomes from sheep brain cortex were incubated with carrier-free Na235SO4 and the synaptic plasma membranes were isolated. The membranes were free of contamination from cytosol, mitochondria and microsomal material and accounted for 30% of the radioactivity present in the synaptosomal particulate fraction. Control experiments demonstrated that the radioactivity present in the preparation was not due to non-specific binding of sulphate ions. The synaptic membranes contained at least six 35S-containing protein bands with molecular weights between 160 000 and 16 000. Analysis showed that the radioactivity was located in the carbohydrate moiety of a glycopeptide.

Purification and characterization of α1-proteinase inhibitor from microsomal fraction of normal human liver

A plasma α 1-proteinase inhibitor was purified, for the first time, from the microsomal fraction of normal human liver by combined procedures of deoxycholate extraction, (NH 4) 2SO 4 precipitation, gel filtration and immunoadsorbent column chromatographies. The final yield of the material was approx. 15 mg/300 g liver. The preparation was shown to be homogeneous according to polyacrylamide gel electrophoretic and immunological criteria. The estimated molecular weight of the glycoprotein as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was 53 000, a value comparable to that reported for plasma α 1-proteinase inhibitor. The liver material was, however, devoid of trypsin-, chymotrypsin- or elastase-inhibitory activity. The loss of activity could be due to deoxycholate treatment during the extraction procedure. SDS-polyacrylamide gel electrophoresis of reduced (β-mercaptoethanol) and alkylated (iodoacetamide) preparation revealed no change in mobility suggesting the presence of only polypeptide chain. The amino acid and carbohydrate compositions of the microsomal material were in close agreement with values reported in the literatures for the corresponding glycoprotein purified from the plasma. The liver microsomal α 1-proteinase inhibitor has a single amino terminal residue, namely, glutamic acid, as determined by the dansyl procedure, and a single carboxyl terminal residue, lysine as shown by the carboxypeptidase digestion method. These terminal amino acids are the same as those found in α 1-proteinase inhibitor purified from normal human plasma.

Teratogenic bioactivation of cyclophosphamide [formula omitted

Day 10 rat embryos grown as cultured explants in vitro developed characteristic defects when culture media contained cyclophosphamide (6.25 micrograms per milliliter or more), an hepatic microsomal fraction, and cofactors for a monooxygenase system. Cyclophosphamide concentrations as high as 250 micrograms per milliliter were innocuous when either the microsomal material or cofactors were omitted from the medium. These experiments represent the first direct demonstration of bioactivation of a proteratogen.

Preparation of glial plasma membrane from a cell fraction enriched in astrocytes

A technique for obtaining glial plasma membrane has been developed, starting with a bulk-prepared glial cell-enriched fraction from rabbit cerebral cortex. The astrocytic-enriched fraction was hand-homogenized in isotonic sucrose media, and the crude membrane fraction sedimented at 3,000g. The isolation of a membrane-enriched fraction was accomplished with sucrose density gradient centrifugation. The plasma membrane fraction was collected at the interphase between 31.5% and 25.5% sucrose. Enzymatic and electron-microscopical analyses indicated a 4-7-fold enrichment in plasma membrane, and a 15-20% contamination with microsomal and mitochondrial material. Some multilaminar membrane structures were also seen in the fraction.

Localization of enzymes in specialized regions of the microsomal membrane

Microsomal vesicle were centrifuged through sucrose density gradients containing deoxycholate. With 0.15% detergent electron transport enzymes and phosphatases could be separated. Increasing the deoxycholate concentration to 0.19% resulted in separation of the microsomal material into five bands containing (in order from the top of the gradient) adenosine monophosphatase, inosine diphosphatase and some glucose-6-phosphatase (band 1); NADH-linked (band 2) and NADPH-linked (band 3) electron transport enzymes; and glucose-6-phosphatase (bands 4 and 5). It appears that enzymes are arranged in specialized patchers in the microsomal membrane.

Inducing activity of fractionated microsomal material from theXenopus laevis gastrula stage.

The postmitochondrial supernatant fromXenopus gastrulae has been fractionated on sucrose gradients. Part of the microsomal material was treated with EDTA, which dissociates most of the polysomal and monosomal material into ribosomal subunits. In addition, a series of pooled fractions from the EDTA treated gradients has been applied to discontinuous gradients in more concentrated sucrose to separate membranous material from the remaining microsomal components.Pooled fractions from all gradients have been tested for inductive activity on amphibian gastrula ectoderm. The spinocaudal (trunk and tail) inducing activity was to some extent eneriched in the membrane fractions.

Subcellular structure of bovine thyroid gland. A study on bovine thyroid membranes by buoyant-density-gradient centrifugation in a B-XIV zonal rotor.

A combined mitochondrial and light mitochondrial fraction and a microsomal fraction were isolated from bovine thyroid gland and fractionated further in a B-XIV zonal rotor. A density gradient ranging from 20 to 50% (w/w) sucrose was used. The rotor was operated for 3 h at 45 000 rev./min. All manipulations were performed at 4 degrees C and at pH 7.4. 2. Membranous material was recovered in two zones: zone I, containing microsomal material derived from both smooth endoplasmic reticulum and plasma membranes and probably also from other smooth membranes; zone II, containing material from rough endoplasmic reticulum. 3. Increasing the pH of the medium up to 8.6, or the addition of Mg2+ to the medium resulted in the formation of a single zone at intermediate densities (aggregation of membranes?). An analogous effect was obtained after treatment with Pb (NO3) 2. 4. In the presence of heparin (50 i.u./ml) the bulk of the membranes was found in zone I. This was due to the release of ribosomes from the rough endoplasmic reticulum.

The presence of N-terminal methionine on nascent protein of rat liver and rabbit reticulocytes and its cleavage during polypeptide-chain elongation.

1. The size of nascent globin peptides from which the N-terminal methionine residue is cleaved has been investigated by comparing the proportion of N-terminal methionine and valine in short and long chains. Nascent chains were labelled in rabbit reticulocyte lysates, fractionated according to length by chromatography on Sephadex G-50, and analysed by the Edman degradation of selected pooled fractions. It was found that different peptide fractions contained either methionine or valine, but not both, as the N-terminal residue. Methionine was present at the N-terminus of globin chains containing up to approx. 50 amino acids whereas valine was found to be the N-terminal amino acid of longer peptides. 2. In similar experiments with nascent proteins of rat liver, labelled either in vivo or in a cell-free system containing microsomal material and cell sap, evidence was obtained for the presence of methionine at the N-terminus of nascent chains up to approx. 65 amino acid residues long. Thus protein synthesis in liver appears to be initiated also by methionine, but in this case cleavage takes place somewhat later during peptide elongation than in globin synthesis.

A model for the regulation of delta-aminolaevulinate synthetase induction in rat liver.

A reciprocal relationship exists between the cytochrome P-450 content and delta-aminolaevulinate synthetase activity in adult rats. In young rats the basal delta-aminolaevulinate synthetase activity is higher and the cytochrome P-450 content is lower compared with the adult rat liver. Administration of allylisopropylacetamide neither induces the enzyme nor causes degradation of cytochrome P-450 in the young rat liver, unlike adult rat liver. Allylisopropylacetamide fails to induce delta-aminolaevulinate synthetase in adrenalectomized-ovariectomized animals or intact animals pretreated with successive doses of the drug, in the absence of cortisol. The cortisol-mediated induction of the enzyme is sensitive to actinomycin D. Allylisopropylacetamide administration degrades microsomal haem but not nuclear haem. Haem does not counteract the decrease in cytochrome P-450 content caused by allylisopropylacetamide administration, but there is evidence for the formation of drug-resistant protein-bound haem in liver microsomal material under these conditions. Phenobarbital induces delta-aminolaevulinate synthetase under conditions when there is no breakdown of cytochrome P-450. On the basis of these results and those already published, a model is proposed for the regulation of delta-aminolaevulinate synthetase induction in rat liver.

A membrane-bound activity catalysing phosphatidylinositol breakdown to 1,2-diacylglycerol, D-myoinositol 1:2-cyclic phosphate an D-myoinositol 1-phosphate. Properties and subcellular distribution in rat cerebral cortex.

1. Breakdown of phosphatidylinositol was studied in homogenates and subcellular fractions of rat cerebral cortex by using both membrane-bound and externally added [(32)P]phosphatidylinositol as substrate. 2. In the presence of deoxycholate breakdown followed first-order kinetics at low substrate concentrations ([unk]1mm) and zero-order kinetics at higher concentrations (6-9mm). 3. Maximum breakdown by cerebral-cortex homogenates was approximately 0.5mumol/h per mg of protein and occurred at pH7.0 in the presence of 8mm-phosphatidylinositol, 2mm-CaCl(2) and 2mg of deoxycholate/ml. Activity was abolished by 1mm-ethanedioxybis(ethylamine)tetra-acetate. 4. The products of phosphatidylinositol breakdown were 1,2-diacylglycerol and a mixture of d-myoinositol 1:2-cyclic phosphate (55%) and d-myoinositol 1-phosphate (45%). The two phosphate esters appeared to be produced together and in constant proportions. 5. Some 51% of the activity was particle-bound, with the highest activities in small nerve endings, microsomal material and two synaptic membrane fractions (fractions Mic(20), Mic(100), M(1) 1.0 and M(1) 0.9 respectively), all of which were also rich in acetylcholinesterase and which have been shown to be rich in other surface-membrane enzymes. Much of the particle-bound activity therefore appears to be present in cerebral-cortex plasma membranes. 6. The results are discussed in relation to previously described soluble activities that catalyse the same reaction, and to a possible role of the membrane-bound enzyme in enhanced phosphatidylinositol turnover in externally stimulated cells.