Nmol Min-1 Mg-1 Protein Research Articles

Role of Human Aldehyde Oxidase in the Generation of Reactive Oxygen Species during the Metabolism of Nicotine

Nicotine is the major addictive constituent of tobacco and alternative tobacco products including electronic cigarettes (e‐cigs). While e‐cig vaping has been proposed as safer than traditional tobacco smoking, recent studies has demonstrated that these products can also trigger oxidant stress and inflammation with resultant pulmonary and cardiovascular disease. E‐cig liquids contain high levels of nicotine typically of 12 to 24 mg/ml or more and with the use of high power delivery devices high, poorly controlled levels of nicotine can be delivered. When inhaling tobacco cigarette smoke or e‐cig vape, nicotine is rapidly absorbed through the lungs. The major pathway of nicotine metabolism in humans consists of the C‐oxidation of nicotine to produce cotinine involving a two‐step process: the first step is CYP2A6‐catalyzed 5′‐oxidation to nicotine‐Δ1′(5′)‐iminium ion (NI), which is in equilibrium with 5′‐hydroxynicotine; and the second step is aldehyde oxidase (AO) catalyzed oxidation of NI to cotinine.Using both cytochrome‐c reduction and EPR spin trapping techniques, we show for the first time that the oxidation of NI by human AO produces superoxide radical anion (O2• −) with high flux rates. Purified recombinant human AO (hAO) was prepared and characterized by SDS gel electrophoresis and optical absorption spectroscopy. hAO, 40 nM was incubated in the presence of NI concentrations from 1‒50 µM. The kinetic parameters, Km and Vmax values, for superoxide generation from NI were determined to be 2.7 ± 0.4 μM and 694 ± 24 nmol min−1 mg−1 of protein, respectively. From quantitation of the measured superoxide and the amount of NI consumed, we estimate that ~60% of the oxygen consumed in this process is converted to superoxide with ~40% hydrogen peroxide. Superoxide generation was almost completely quenched by either 0.5 μM raloxifene, a potent inhibitor of AO, or 0.1 μM Copper‐Zinc‐superoxide dismutase (SOD1) with no superoxide generation detected either in the absence of either AO or nicotine‐Δ1′(5′)‐iminium ion.Therefore, human AO efficiently catalyzes the oxidation of NI to cotinine with concomitant production of reactive oxygen species. Due to the high expression levels of AO in lung tissue as well as its rapid rate of NI metabolism with low Km, AO rapidly and efficiently converts NI to cotinine with the formation of superoxide and hydrogen peroxide. Thus, nicotine metabolism will be associated with the formation of superoxide, hydrogen peroxide and secondary reactive oxygen species that can in turn cause lung oxidative stress and inflammation.

Characterization of a Selenium-resistance-enhancing Homocysteine S-methyltransferase from Aegilops tauschii

In this study, the cDNA of homocysteine S-methyltransferase was isolated from Aegilops tauschii Coss., with the gene accordingly designated as AetHMT1. Similar to other methyltransferases, AetHMT1 contains a GGCCR consensus sequence for a possible zinc-binding motif near the C-terminal and a conserved cysteine residue upstream of the zinc-binding motif. Analysis of AetHMT1 uncovered no obvious chloroplast or mitochondrial targeting sequences. We functionally expressed AetHMT1 in Escherichia coli and confirmed its biological activity, as evidenced by a positive HMT enzyme activity of 164.516 ± 17.378 nmol min-1 mg-1 protein when catalyzing the transformation of L-homocysteine. Compared with the bacterium containing the empty vector, E. coli harboring the recombinant AetHMT1 plasmid showed much higher tolerance to selenate and selenite. AetHMT1 transcript amounts in different organs were increased by Na2SeO4 treatment, with roots accumulating higher amounts than stems, old leaves and new leaves. We have therefore successfully isolated HMT1 from Ae. tauschii and characterized the biochemical and physiological functions of the corresponding protein.

Comparative Investigation of Glutathione S-transferase (GST) in Different Crops and Purification of High Active GSTs from Onion (Allium cepa L.)

Glutathione S-transferases (GSTs; EC 2.5.1.18) are abundant proteins encoded by a highly divergent ancient gene family with protective functions through detoxification and non-catalytic roles as carriers of cytotoxins. In this work, we investigated the GST activities in seedlings of 38 crops to obtain a plant with high active GST for further study. In screening of 38 crops, onion seedling showed the highest GST activity (483.54 nmol min-1mg-1 protein) followed by wheat(372.89 nmol min-1mg-1 protein), barley (253.44 nmol min-1mg-1 protein), rice (244.12 nmol min-1mg-1 protein) and proso millet (173.34 nmol min-1mg-1 protein). Carrot seedling showed the lowest activity (3.63 nmol min-1mg-1 protein).In onion plants, both root and leaf showed high GST activity. Onion bulb GSTs were separated DEAE cellulose column chromatography, and purified by affinity chromatography (S-hexyl glutathione-agarose). Three GST peaks were found to elute at 67, 107 and 140mM of KCl gradient solution, and were named as GSTa, GSTb and GSTc. Among the three GSTs, GSTa, GSTb and GSTc contained 9.58, 61.45 and 28.97% of total activity, respectively. In purification, GSTa, GSTb and GSTc had specific activities of 9075, 17259 and 19868 nmol min-1 mg-1 protein, respectively, along with yield of 2.48, 3.17 and 1.28,and purification fold of 15.8, 29.9 and 34.5, respectively. The purity and molecular mass of the fraction was examined by SDS-PAGE. The silver staining of the purified GSTa, GSTb and GSTc indicated that final product of GSTb and GSTc were highly purified and migrated as a single band on SDS-PAGE with an apparent molecular mass of 27 kDa. However, GSTa eluted with Glyoxalase-I (Gly-I) and to purify GSTa, more methodological application are suggested.

Alpha chymotrypsin coated clusters of Fe3O4nanoparticles for biocatalysis in low water media

BackgroundEnzymes in low water containing non aqueous media are useful for organic synthesis. For example, hydrolases in such media can be used for synthetic purposes. Initial work in this area was carried out with lyophilized powders of enzymes. These were found to have poor activity. Drying (removing bulk water) by precipitation turned out to be a better approach. As enzymes in such media are heterogeneous catalysts, spreading these precipitates over a large surface gave even better results. In this context, nanoparticles with their better surface to volume ratio provide obvious advantage. Magnetic nanoparticles have an added advantage of easy separation after the reaction. Keeping this in view, alpha chymotrypsin solution in water was precipitated over a stirred population of Fe3O4 nanoparticles in n-propanol. This led to alpha chymotrypsin activity coated over clusters of Fe3O4 nanoparticles. These preparations were found to have quite high transesterification activity in low water containing n-octane.ResultsPrecipitation of alpha chymotrypsin over a stirred suspension of Fe3O4 nanoparticles (3.6 nm diameter) led to the formation of enzyme coated clusters of nanoparticles (ECCNs). These clusters were also magnetic and their hydrodynamic diameter ranged from 1.2- 2.6 microns (as measured by dynamic light scattering). Transmission electron microscopy (TEM), showed that these clusters had highly irregular shapes. Transesterification assay of various clusters in anhydrous n-octane led to optimization of concentration of nanoparticles in suspension during precipitation. Optimized design of enzyme coated magnetic clusters of nanoparticles (ECCN 3) showed the highest initial rate of 465 nmol min-1 mg-1protein which was about 9 times higher as compared to the simple precipitates with an initial rate of 52 nmol min-1 mg-1 protein.Circular Dichroism (CD)(with a spinning cell accessory) showed that secondary structure content of the alpha Chymotrypsin in ECCN 3 [15% α-helix, 37% β-sheet and 48% random coil] was identical to the simple precipitates of alpha chymotrypsin.ConclusionA strategy for obtaining a high activity preparation of alpha chymotrypsin for application in low water media is described. Such high activity biocatalysts are useful in organic synthesis.

The reductase domain in a Type I fatty acid synthase from the apicomplexan Cryptosporidium parvum: Restricted substrate preference towards very long chain fatty acyl thioesters

BackgroundThe apicomplexan Cryptosporidium parvum genome possesses a 25-kb intronless open reading frame (ORF) that predicts a multifunctional Type I fatty acid synthase (CpFAS1) with at least 21 enzymatic domains. Although the architecture of CpFAS1 resembles those of bacterial polyketide synthases (PKSs), this megasynthase is predicted to function as a fatty acyl elongase as our earlier studies have indicated that the N-terminal loading unit (acyl-[ACP] ligase) prefers using intermediate to long chain fatty acids as substrates, and each of the three internal elongation modules contains a complete set of enzymes to produce a saturated fatty acyl chain. Although the activities of almost all domains were confirmed using recombinant proteins, that of the C-terminal reductase domain (CpFAS1-R) was yet undetermined. In fact, there were no published studies to report the kinetic features of any reductase domains in bacterial PKSs using purified recombinant or native proteins.ResultsIn the present study, the identity of CpFAS1-R as a reductase is confirmed by in silico analysis on sequence similarity and characteristic motifs. Phylogenetic analysis based on the R-domains supports a previous notion on the bacterial origin of apicomplexan Type I FAS/PKS genes. We also developed a novel assay using fatty acyl-CoAs as substrates, and determined that CpFAS1-R could only utilize very long chain fatty acyl-CoAs as substrates (i.e., with activity on C26 > C24 > C22 > C20, but no activity on C18 and C16). It was capable of using both NADPH and NADH as electron donors, but prefers NADPH to NADH. The activity of CpFAS1-R displayed allosteric kinetics towards C26 hexacosanoyl CoA as a substrate (h = 2.0; Vmax = 32.8 nmol min-1 mg-1 protein; and K50 = 0.91 mM).ConclusionsWe have confirmed the activity of CpFAS1-R by directly assaying its substrate preference and kinetic parameters, which is for the first time for a Type I FAS, PKS or non-ribosomal peptide synthase (NRPS) reductase domain. The restricted substrate preference towards very long chain fatty acyl thioesters may be an important feature for this megasynthase to avoid the release of product(s) with undesired lengths.

Use of Biochemical Markers for the Assessment of Organic Pollutant Contamination of the Vltava river, Czech Republic

In this study, selected biochemical markers - cytochrome P450 (CYP 450), ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST) and glutathione (GSH) - were measured in chub (Leuciscus cephalus L.) liver samples obtained from three locations on the Vltava river (Podolí, Podbaba and Vraňany) and from a control location nmicals commonly known as their inducers. These include polychlorinated biphenyls, hexachlorobenzene and octachlorostyrene measured in chub muscle and polyaromatic hydrocarbons measured in bear Vodňany. The levels of selected biomarkers should correlate with the concentration of cheottom sediments obtained from the same locations. The highest EROD activity (median 101.37 pmol min-1 mg-1 protein), GST activity (median 42.82 nmol min-1 mg-1 protein), and GSH concentration (median 8.01 nmol mg-1 protein) were found in fish liver from the Podbaba location. There were no significant differences in CYP P450 level or EROD activity among the different locations. In Podbaba, GST activity (P < 0.01) and GSH concentrations (P < 0.001) were significantly higher than in the control location. The results of these analyses were correlated and a significant correlation was found between biochemical markers and their inducers. The results show that the use of biochemical markers in water quality assessment is a convenient method that can supplement classical chemical monitoring.

Glucan, water dikinase phosphorylates crystalline maltodextrins and thereby initiates solubilization

Starch phosphorylation by glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step in the breakdown of native starch particles, but the underlying mechanisms have remained obscure. In this paper, the initial reactions of starch degradation were analyzed using crystallized maltodextrins as model carbohydrates. As revealed by X-ray diffraction analysis, the crystallized maltodextrins represent the B-type starch allomorph. Recombinant GWD phosphorylated crystalline maltodextrins with a high specific activity (55-60 nmol mg-1 protein min-1), but exhibited very little activity with the same maltodextrins that had been solubilized by heat treatment. Recombinant phosphoglucan, water dikinase (PWD; EC 2.7.9.5) utilized the crystalline maltodextrins only when pre-phosphorylated by GWD. Phosphorylation of crystalline maltodextrins, as catalyzed by GWD, initiated solubilization of neutral as well as phosphorylated glucans. In both the insoluble and the soluble state, mono-, di- and triphosphorylated alpha-glucans were observed, with wide and overlapping ranges of degree of polymerization. Thus, the substrate specificity of the GWD is defined by the physical arrangement of alpha-glucans rather than by structural parameters, such as the distribution of branching points or degree of polymerization. Unlike GWD and PWD, recombinant beta-amylase isozyme 3 (BAM3), which has been shown to be essential for plastidial starch degradation, preferentially degraded soluble maltodextrins rather than crystallized glucans. In summary, two conclusions were reached. Firstly, carbohydrate targets of GWD are primarily defined by the molecular order of glucan helices. Secondly, GWD-catalyzed phosphorylation mediates the phase transition of glucans from a highly ordered to a less ordered and hydrated state.

Biomarkers of Contaminant Exposure in Chub (Leuciscus cephalus L.) - Biomonitoring of Major Rivers in the Czech Republic.

Biochemical analysis of organisms to assess exposure to environmental contaminants is of great potential use. Biochemical markers, specifically liver enzymes of the first and the second phase of xenobiotic transformation - cytochrome P450 (CYP 450), ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST) and tripeptide reduced glutathione (GSH) - were used to assess contamination of the aquatic environment at 12 locations near the mouths of major rivers in the Czech Republic. These rivers were the Lužnice, Otava, Sázava, Berounka, Vltava, Labe, Ohře, Svratka, Dyje, Morava and Odra. The indicator species selected was the Chub (Leuciscus cephalus L.). The highest levels of CYP 450 and EROD catalytic activity were found in livers of fish from the Labe (Obříství) (0.32±0.10 nmol mg-1 protein and 1061.38±545.51 pmol min-1 mg-1 protein, respectively). The highest levels of GST catalytic activity and GSH content were found in fish from the Otava (35.39±13.35 nmol min-1 mg-1 protein and 4.29±2.10 nmol GSH mg-1 protein, respectively). They were compared with levels of specific inductors of these biochemical markers in muscle. The results confirmed contamination of some river locations (Labe Obříství, Svratka).

Reactive Nitrogen Species-Dependent Effects on Soybean Chloroplasts

Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was studied by electron paramagnetic resonance (EPR) spin-trapping technique1. Both nitrite and L-arginine (arg) are the required substrates for enzymatic activities considered as possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min-1 mg-1 protein in the presence of 1 mM NaNO2. Chloroplasts incubated with 1 mM arg showed a NO production of 0.76 ± 0.04 nmol min-1 mg-1 protein. This production was inhibited when chloroplasts were incubated in presence of NOS-inhibitors L-NAME and L-NNA. In vitro exposure of chloroplasts to a NO-donor (GSNO) decreased both ascorbyl radical content and the activity of ascorbate peroxidase, without modification of the total ascorbate content. Exposure of the isolated chloroplasts to a NO-donor decreased lipid radical content in membranes, however, incubation in the presence of 25 μM peroxynitrite (ONOO-) led to an increase in lipid-derived radicals (34%). The effect of ONOO- on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to control. Taken as a whole, NO seems to be an endogenous metabolite in soybean chloroplasts and reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplasts, since both a decreased lipid radical content in membranes and a decrease in the activity of ascorbate peroxidase were observed after exposure to a NO donor.

The Osmotic Activation of Transporter ProP Is Tuned by Both Its C-terminal Coiled-coil and Osmotically Induced Changes in Phospholipid Composition

Transporter ProP of Escherichia coli (ProPEc) senses extracellular osmolality and mediates osmoprotectant uptake when it is rising or high. A replica of the ProPEc C terminus (Asp468-Arg497) forms an intermolecular alpha-helical coiled-coil. This structure is implicated in the osmoregulation of intact ProPEc, in vivo. Like that from Corynebacterium glutamicum (ProPCg), the ProP orthologue from Agrobacterium tumefaciens (ProPAt) sensed and responded to extracellular osmolality after expression in E. coli. The osmotic activation profiles of all three orthologues depended on the osmolality of the bacterial growth medium, the osmolality required for activation rising as the growth osmolality approached 0.7 mol/kg. Thus, each could undergo osmotic adaptation. The proportion of cardiolipin in a polar lipid extract from E. coli increased with extracellular osmolality so that the osmolality activating ProPEc was a direct function of membrane cardiolipin content. Group A ProP orthologues (ProPEc, ProPAt) share the C-terminal coiled-coil domain and were activated at low osmolalities. Like variant ProPEc-R488I, in which the C-terminal coiled-coil is disrupted, ProPEc derivatives that lack the coiled-coil and Group B orthologue ProPCg required a higher osmolality to activate. The amplitude of ProPEc activation was reduced 10-fold in its deletion derivatives. The coiled-coil structure is not essential for osmotic activation of ProP per se. However, it tunes Group A orthologues to osmoregulate over a low osmolality range. Coiled-coil lesions may impair both coiled-coil formation and interaction of ProPEc with amplifier protein ProQ. Cardiolipin may contribute to ProP adaptation by altering bulk membrane properties or by acting as a ProP ligand.

Sucrose metabolizing enzymes in cell suspension cultures of Bauhinia forficata, Curcuma zedoaria and Phaseolus vulgaris

The objective of this work was to study the activity of sucrose metabolizing enzymes in extracts of cell suspension cultures of Bauhinia forficata Link, Curcuma zedoaria Roscoe and Phaseolus vulgaris L. Invertase pathway was identified in the three studied species. Sucrose synthase pathway was also responsible for sucrose metabolism in Curcuma zedoaria and Phaseolus vulgaris cells. Activity values higher than 300 nmol min-1 mg-1 of protein were found for acid and neutral invertases, UDPglucose pyrophosphorylase and phosphoglucomutase in the cell extract of the three plant species. Sucrose synthase showed low activity in Bauhinia forficata cells. As sucrose concentration in the culture medium decreased, sucrose synthase activity increased in C. zedoaria and P. vulgaris cells. The glycolytic enzymes activity gradually reduced at the end of the culture period, when carbohydrate was limited.

Benzydamine N-oxidation as an index reaction reflecting FMO activity in human liver microsomes and impact of FMO3 polymorphisms on enzyme activity.

The role of flavin containing monooxygenases (FMO) on the disposition of many drugs has been insufficiently explored. In vitro and in vivo tests are required to study FMO activity in humans. Benzydamine (BZD) N-oxidation was evaluated as an index reaction for FMO as was the impact of genetic polymorphisms of FMO3 on activity. BZD was incubated with human liver microsomes (HLM) and recombinant enzymes. Human liver samples were genotyped using PCR-RFLP. BZD N-oxide formation rates in HLM followed Michaelis-Menten kinetics (mean Km = 64.0 microM, mean Vmax = 6.9 nmol mg-1 protein min-1; n = 35). N-benzylimidazole, a nonspecific CYP inhibitor, and various CYP isoform selective inhibitors did not affect BZD N-oxidation. In contrast, formation of BZD N-oxide was almost abolished by heat treatment of microsomes in the absence of NADPH and strongly inhibited by methimazole, a competitive FMO inhibitor. Recombinant FMO3 and FMO1 (which is not expressed in human liver), but not FMO5, showed BZD N-oxidase activity. Respective Km values for FMO3 and FMO1 were 40.4 microM and 23.6 microM, and respective Vmax values for FMO3 and FMO1 were 29.1 and 40.8 nmol mg-1 protein min-1. Human liver samples (n = 35) were analysed for six known FMO3 polymorphisms. The variants I66M, P135L and E305X were not detected. Samples homozygous for the K158 variant showed significantly reduced Vmax values (median 2.7 nmol mg-1 protein min-1) compared to the carriers of at least one wild type allele (median 6.2 nmol mg-1 protein min-1) (P < 0.05, Mann-Whitney-U-test). The V257M and E308G substitutions had no effect on enzyme activity. BZD N-oxidation in human liver is mainly catalysed by FMO3 and enzyme activity is affected by FMO3 genotype. BZD may be used as a model substrate for human liver FMO3 activity in vitro and may be further developed as an in vivo probe reflecting FMO3 activity.

Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L‐glutamate transporter

1. In this study we have examined the use of the ecdysone-inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. 2. HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using the inducible vector pIND and a clone designated HEK/EAAT2 was selected for further characterization. 3. Na+-dependent L-glutamate uptake activity (3.2 pmol min-1 mg-1) was observed in EcR 293 cells and this was increased approximately 2 fold in the uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity in the absence of exogenous inducing agent. Exposure of HEK/EAAT2 cells to the ecdysone analogue Ponasterone A (10 microM for 24 h) resulted in a > or = 10 fold increase in the Na+-dependent activity. 4. L-glutamate uptake into induced HEK/EAAT2 cells followed first-order Michaelis-Menten kinetics and Eadie-Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7+/-7.5 microM, Vmax 3.8+/-0.9 nmol min-1 mg-1 protein. 5. The pharmacological profile of the EAAT2 subtype was characterized using a series of L-glutamate transport inhibitors and the rank order of inhibitory potency was similar to that described previously for the rat homologue GLT-1 and in synaptosomal preparations from rat cortex. 6. Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155+/-5% control in the presence of 30 microM) of the L-glutamate transport capacity in the induced HEK/EAAT2 cells. 7. This study demonstrates that the expression of EAAT2 can be regulated in a mammalian cell line using the ecdysone-inducible mammalian expression system.

Nitrogen, carbon, and sulfur metabolism in natural thioploca samples

Filamentous sulfur bacteria of the genus Thioploca occur as dense mats on the continental shelf off the coast of Chile and Peru. Since little is known about their nitrogen, sulfur, and carbon metabolism, this study was undertaken to investigate their (eco)physiology. Thioploca is able to store internally high concentrations of sulfur globules and nitrate. It has been previously hypothesized that these large vacuolated bacteria can oxidize sulfide by reducing their internally stored nitrate. We examined this nitrate reduction by incubation experiments of washed Thioploca sheaths with trichomes in combination with 15N compounds and mass spectrometry and found that these Thioploca samples produce ammonium at a rate of 1 nmol min-1 mg of protein-1. Controls showed no significant activity. Sulfate was shown to be the end product of sulfide oxidation and was observed at a rate of 2 to 3 nmol min-1 mg of protein-1. The ammonium and sulfate production rates were not influenced by the addition of sulfide, suggesting that sulfide is first oxidized to elemental sulfur, and in a second independent step elemental sulfur is oxidized to sulfate. The average sulfide oxidation rate measured was 5 nmol min-1 mg of protein-1 and could be increased to 10.7 nmol min-1 mg of protein-1 after the trichomes were starved for 45 h. Incorporation of 14CO2 was at a rate of 0.4 to 0.8 nmol min-1 mg of protein-1, which is half the rate calculated from sulfide oxidation. [2-14C]acetate incorporation was 0.4 nmol min-1 mg of protein-1, which is equal to the CO2 fixation rate, and no 14CO2 production was detected. These results suggest that Thioploca species are facultative chemolithoautotrophs capable of mixotrophic growth. Microautoradiography confirmed that Thioploca cells assimilated the majority of the radiocarbon from [2-14C]acetate, with only a minor contribution by epibiontic bacteria present in the samples.

Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis.

Biosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two-step oxidation process of choline, with glycine betaine aldehyde as the intermediate. We have exploited the sensitivity of B. subtilis strains defective in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate. These strains were also defective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC) were required for these phenotypes. Molecular analysis allowed us to demonstrate that the opuB and opuC operons each encode a binding protein-dependent ABC transport system that consists of four components. The presumed binding proteins of both ABC transporters were shown to be lipoproteins. Kinetic analysis of [14C]-choline uptake via OpuB (K(m) = 1 microM; Vmax = 21 nmol min-1 mg-1 protein) and OpuC (K(m) = 38 microM; Vmax = 75 nmol min-1 mg-1 protein) revealed that each of these ABC transporters exhibits high affinity and substantial transport capacity. Western blotting experiments with a polyclonal antiserum cross-reacting with the presumed substrate-binding proteins from both the OpuB and OpuC transporter suggested that the expression of the opuB and opuC operons is regulated in response to increasing osmolality of the growth medium. Primer extension analysis confirmed the osmotic control of opuB and allowed the identification of the promoter of this operon. The opuB and opuC operons are located close to each other on the B. subtilis chromosome, and their high sequence identity strongly suggests that these systems have evolved from a duplication event of a primordial gene cluster. Despite the close relatedness of OpuB and OpuC, these systems exhibit a striking difference in substrate specificity for osmoprotectants that would not have been predicted readily for such closely related ABC transporters.

Inactivation of toluene 2-monooxygenase in Burkholderia cepacia G4 by alkynes.

High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C5 to C10) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O2 uptake activities, while acetate- and 3-methylcatechol-dependent O2 uptake activities were unaffected. Toluene-dependent O2 uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min-1 to 2.45 min-1. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H2O2, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne-ethylene and propylene-were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent Ks and Vmax values of 39.7 microM and 112.3 nmol min-1 mg of protein-1 for ethylene and 32.3 microM and 89.2 nmol min-1 mg of protein-1 for propylene.

In vitro modulation of L‐arginine transport in trophoblast cells by glucose

The uptake of the semi-essential amino acid, L-arginine, into trophoblast cells was measured with the aim of determining the effect of different glucose concentrations on L-arginine influx kinetics. This study used a novel superfused microcarrier culture system of BeWo cells (an established choriocarcinoma cell line with many characteristics of normal human trophoblast) and a rapid, paired-tracer dilution technique to measure unidirectional influx into the cells. At 10 mmol L-1 D-glucose, L-arginine unidirectional influx across the microvillous border of the cells was saturable with a Km of 1.14 +/- 0.14 mmol L-1 and a Vmax of 121. 36 +/- 5.89 nmol mg-1 protein min-1. When cells were preincubated for 24 h in the presence of 30 mmol L-1 D-glucose, there was a significant increase in the Vmax for L-arginine of nearly 30%. Similarly, preincubation in the presence of 1 mmol L-1 D-glucose and 12.5 mIU mL-1 human insulin reduced the Km for L-arginine influx by over 55%. These data suggest that the modulation of placental transport of L-arginine by glucose and insulin could contribute to the fetal macrosomia observed in diabetic mothers.

Calcium uptake by the sarcoplasmic reticulum, high energy content and histological changes in ischemic cardiomyopathy.

Sarcoplasmic reticulum (SR) Ca2+ uptake, myocardial high energy content and histology were examined in different zones of hearts from patients with ischemic cardiomyopathy. Unfractionated homogenates were prepared from left ventricular samples obtained in three zones of each heart: an infarct-remote zone, an outer peri-infarct zone, and an inner peri-infarct zone. Oxalate-supported 45Ca2+ uptake was measured at 37 degrees C using a filtration method. Maximum rate (Vmax) of uptake in absence or in presence of ryanodine was lower in inner peri-infarct (7.4 +/- 0.7 and 9.5 +/- 0.8 nmol min-1 mg-1 of protein, respectively; mean +/- SEM) and outer peri-infarct tissues (8.8 +/- 0.8 and 12.0 +/- 0.8 nmol min-1 mg-1) than in infarct-remote myocardium (12.7 +/- 2.1 and 15.8 +/- 2.2 nmol min-1 mg-1). The apparent affinity constants for Ca2+ (KCa) as well as the Hill coefficients were not different. Homogenate DNA (1.6 +/- 0.1, 1.6 +/- 0.1 and 1.7 +/- 0.1 mg/g of remote, inner peri-infarct and outer peri-infarct myocardium, respectively) and adenine nucleotides contents (ATP: 15 +/- 1.3, 14 +/- 0.8 and 15 +/- 1.0 mumol/g dry weight, respectively) were similar in all tissues. Fibrosis was increased in inner peri-infarct tissue (37 +/- 6%; vs. 13 +/- 2% and 12 +/- 2% in both remote and outer peri-infarct tissues, respectively), but the number of abnormal cells was not significantly different. The decrease of Ca2+ uptake in ischemic cardiomyopathy is not homogeneous in the ventricular wall, and reflects a decreased number/activity of SR Ca(2+)-ATPase, without altered Ca(2+)-affinity or increased Ca2+ leakage through ryanodine receptors.

Functional co-expression of CYP2D6 and human NADPH-cytochrome P450 reductase in Escherichia coli.

The polymorphic human CYP2D6 has been co-expressed with human NADPH-cytochrome P450 oxidoreductase in Escherichia coli in order to generate a functional recombinant monooxygenase system for the study of xenobiotic metabolism. The two cDNAs were co-expressed from separate, compatible plasmids with different antibiotic selection markers. The CYP2D6 could be detected in bacterial cells at levels up to 700 nmol I-1 culture by Fe(2+)-CO versus Fe2+ difference spectroscopy, exhibiting the characteristic absorbance peak at 450 nm. Immunoblotting demonstrated the presence of both proteins in bacterial membranes, where they were expressed at levels significantly higher than those found in human liver microsomes. Membrane content was 150-200 pmol CYP2D6 (determined spectrally) and 100-230 pmol CYP-reductase (determined enzymatically) per mg protein. Critically, the two co-expressed proteins were able to couple to form a NADPH-dependent monooxygenase which metabolized the CYP2D6 substrate bufuralol (Vmax 3.30 nmol min-1 mg-1 protein; K(m) 11.1 microM) in isolated membrane fractions. This K(m) value was similar to the K(m) determined in human liver microsomes. Activity could be inhibited by the specific inhibitor quinidine. Of greater significance however, was the finding that intact E. coli cells, even in the absence of exogenous NADPH, were able to metabolize bufuralol at rates almost as high as those measured in membranes (4.6 +/- 0.4 min-1 versus 5.7 +/- 0.2 min-1 at 50 microM substrate). Such recombinant strains will greatly facilitate the molecular characterization of allelic variants of cytochrome P450 isoenzymes.

Properties of the membranes containing the particulate methane monooxygenase from Methylosinus trichosporium OB3b.

The particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was partially purified and characterized by measuring the effects of reducing agents and additives, and the stability of pMMO was studied. Duroquinol was a suitable reducing agent, and pMMO was stabilized by bovine serum albumin (BSA). Among the additives, the copper (II) ion stimulated pMMO at low concentration and inhibited at high concentration. The optimum conditions for pMMO activity were as follows: 45 degrees C, pH 6.5 and 55 mM 3-morpholinopropanesulfonic acid (MOPS) buffer, and the rate of propene epoxide formation was 13.6 nmol min-1 mg-1 protein. ESR spectra indicate that the copper cluster in the membrane fraction is reduced by duroquinol and oxidized by dioxygen. The result suggests that the copper cluster is contained in the active site of pMMO.