Native Microsomes Research Articles

Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate.

Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTMcharacterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.

AUTOIMMUNE MARKERS IN MEASLES INFECTION OF VARYING SEVERITY

Measles is an acute viral infectious disease caused by an RNA-containing virus of Morbilliviridae genus. This infectious disease is characterized by the development of catarrhal syndrome, general intoxication syndrome and specific exanthema. The virus is transmitted from person to person by airborne mechanism, when coughing, sneezing and exhaling air droplets during conversation. It is shown that measles is characterized by long-term immunosuppression, which persists for a long period after the patient's clinical recovery and normalization of blood parameters that can lead to an exacerbation of various chronic and autoimmune diseases. Taking into account the impairment of cytokine and immunological reactivity observed in adult patients with measles infection, violations of the dynamics of interferon levels elevation and immunosuppression caused by the virus itself, as well as the lack of studies on autoantibody levels in patients with measles of varying severity, investigating autoimmune markers is of great relevance as can be used to predict and prevent the development and activation of autoimmune responses. The purpose of the study is to determine the involvement of autoimmune reactions in the immunopathogenesis of measles infection in individuals with various degrees of severity of the disease. The study included 65 patients with a diagnosis of measles who were treated at the Kharkiv region clinical infectious hospital in the period for 2017 – 2019. Depending on the severity of the disease and the presence of complications the patients included in the study were divided into 4 groups. The control group consisted of 20 voluntary donors who at the time of blood sampling had no clinical signs of measles, had no contact with infected patients, and had no exclusion criteria.
 All patients included in the study were tested for serum levels of antibodies to cell membrane phospholipids, IgG to liver and kidney microsomes (anti-LKM-1), and IgG to native DNA (ADNA 2) on the 1st and 10th day of hospital stay. These indicators were assessed by the method of solid-phase enzyme immunoassay (ELISA) using test systems manufactured by “Granum” LLC (Ukraine), EUROIMMUNE (Germany) and BioRad (USA). Statistical processing of the obtained results was carried out using the Statistica 6.0 software. When interpreting the significance of the difference in results, the Student's criterion was used; the critical value of the significance level was considered to be p ˂ 0.05. Results and discussion. It was found that on the day of admission to the hospital there was an increase in the level of autoantibodies to native DNA, liver and kidney microsomes, as well as cell membrane phospholipids in all groups of patients. In dynamics (on the 10th day of hospital stay), the patients of groups 1-4 demonstrated a decrease in the level of all types of autoantibodies; in the patients who had a severe course of the disease and developed complications development, the levels was higher than in the control group. The obtained data indicate that with the development of measles infection against the background of activation of general and specific immune response, there is an activation of autoimmune reactions, whose activity fades as the infectious process goes away. Autoimmune reactions under a favourable clinical course of measles infection, most likely, are not aggressive but protective by their nature and are aimed at eliminating infected and damaged cells. Patients with measles of varying severity have increased levels of autoantibodies to native DNA, liver and kidney microsomes, as well as cell membrane phospholipids. It has been found out that the levels of autoantibodies do not go beyond the reference values, but in severe disease, borderline values of autoantibodies are recorded in a significant number of patients. The study of autoantibody levels can be recommended in cases of severe measles infection, both with and without complications, to predict and prevent the development of autoimmune pathology.

Glucuronidation of mono(2-ethylhexyl) phthalate in humans: roles of hepatic and intestinal UDP-glucuronosyltransferases.

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP), which is an endocrine-disrupting chemical. In the present study, MEHP glucuronidation in humans was studied using recombinant UDP-glucuronosyltransferases (UGTs) and microsomes of the liver and intestine. Among the recombinant UGTs examined, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, and UGT2B7 glucuronidated MEHP. The kinetics of MEHP glucuronidation by UGT1A3, UGT1A7, UGT1A8, UGT1A10, UGT2B4, and UGT2B7 followed the Michaelis-Menten model, whereas that by UGT1A9 fit the negative allosteric model. CLint values were in the order of UGT1A9>UGT2B7>UGT1A7>UGT1A8≥UGT1A10>UGT1A3>UGT2B4. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model. Diclofenac (20µM) and raloxifene (20µM) decreased CLint values to 43 and 36% that of native microsomes, respectively. The kinetics of MEHP glucuronidation by intestine microsomes fit the biphasic model. Diclofenac (150 and 450µM) decreased CLint values to 32 and 13% that of native microsomes for the high-affinity phase, and to 28 and 21% for the low-affinity phase, respectively. Raloxifene (2.5 and 7.0µM) decreased CLint values to 35 and 4.1% that of native microsomes for the high-affinity phase, and to 48 and 53% for the low-affinity phase, respectively. These results suggest that MEHP glucuronidation in humans is catalyzed by UGT1A3, UGT1A9, UGT2B4, and/or UGT2B7 in the liver, and by UGT1A7, UGT1A8, UGT1A9, UGT1A10, and/or UGT2B7 in the intestine, and also that these UGT isoforms play important and characteristic roles in the detoxification of DEHP.

In vitro metabolism of the 5-hydroxytryptamine1B receptor antagonist elzasonan

The metabolism of elzasonan has been examined in vitro using hepatic microsomes from human and recombinant heterologously expressed P450 enzymes (rCYP). Metabolism occurs primarily via oxidative N-demethylation to form M4 and oxidation reactions to form elzasonan N-oxide (M5) and 5-hydroxyelzasonan metabolite (M3). Additionally, elzasonan was shown to be metabolized to the novel cyclized indole metabolite (M6) which undergoes subsequent oxidation to form the iminium ion metabolite (M3a). The rCYP data was normalized relative to the levels of each CYP form in native human liver microsomes to better assess the contribution of each rCYP in the metabolism of elzasonan. Results demonstrated the involvement of CYP3A4 in the pathways leading to M3a, M3, M5 and M6 and CYP2C8 in the formation of M4. Kinetic constants for the formation of M3 were determined and correlation and inhibition studies suggested that CYP3A4 is primarily responsible for the formation of M3 and CYP2C19 plays a very minor role in its formation. Cytochrome b5 has shown to be an essential component in P450 3A4 catalyzed 5-hydroxyelzasonan formation and provides insights on the disconnect between human liver microsomes data and that of rCYP. Furthermore, rCYP3A4 containing b5 are useful models for predicting the rates for liver microsomes P450-dependent drug oxidations and should be utilized routinely.

Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via beta-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor beta-oxidized in mitochondria. We thus examined whether omega-hydroxylation of 3-hydroxy fatty acids (3-OHFAs), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their omega-hydroxylated metabolites, 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS. Rates of 3,18-dihydroxystearate and 3,16-dihydroxypalmitate formation were 1.23 +/- 0.5 and 1.46 +/- 0.30 nmol product formed/min/mg protein, respectively (mean +/- SD; n = 13). Polyspecific CYP4F antibodies markedly inhibited microsomal omega-hydroxylation of 3-hydroxystearate (68%) and 3-hydroxypalmitate (99%), whereas CYP4A11 and CYP2E1 antibodies had little effect. Upon reconstitution, CYP4F11 and, to a lesser extent, CYP4F2 catalyzed omega-hydroxylation of 3-hydroxystearate, whereas CYP4F3b, CYP4F12, and CYP4A11 exhibited negligible activity. CYP4F11 was the lone CYP4F/A enzyme that effectively oxidized 3-hydroxypalmitate. Kinetic parameters of microsomal 3-hydroxystearate metabolism were K(m) = 55 microM and V(max) = 8.33 min(-1), whereas those for 3-hydroxypalmitate were K(m) = 56.4 microM and V(max) = 14.2 min(-1). CYP4F11 kinetic values resembled those of native microsomes, with K(m) = 53.5 microM and V(max) = 13.9 min(-1) for 3-hydroxystearate and K(m) = 105.8 microM and V(max) = 70.6 min(-1) for 3-hydroxypalmitate. Our data show that 3-hydroxystearate and 3-hydroxypalmitate are converted to omega-hydroxylated 3-OHDCA precursors in human liver and that CYP4F11 is the predominant catalyst of this reaction. CYP4F11-promoted omega-hydroxylation of 3-OHFAs may modulate the disposition of these compounds in pathological states in which enhanced fatty acid mobilization or impairment of mitochondrial fatty acid beta-oxidation increases circulating 3-OHFA levels.

Role of cysteine residues in the function of human UDP glucuronosyltransferase isoform 1A1 (UGT1A1)

Bilirubin glucuronidation, catalysed by UGT1A1 [UGT (UDP glucuronosyltransferase) isoform 1A1, EC 2.4.1.17], is critical for biliary elimination of bilirubin. UGT1A1 deficiency causes CN-1 (Crigler-Najjar syndrome type 1), which is characterized by potentially lethal unconjugated hyperbilirubinaemia. Nucleotide sequence analysis of UGT1A1 in two CN-1 patients revealed that patient A was homozygous for a nt 530 G-->A (where nt 530 G-->A means guanine to adenine transition at nucleotide 530) mutation, predicting a C177Y substitution, and patient B had a nt 466 T-->C mutation on one allele and a nt 1070 A-->G mutation on the other, predicting a C156R and a Q357R substitution respectively. All 11 cysteine residues of mature human UGT1A1 are highly conserved in other human UGT isoforms and in rat, mouse and Rhesus monkey UGT1A1, suggesting their functional importance. Expression of mutagenized UGT1A1 plasmids showed that substitution of any of the seven cysteine residues located within the endoplasmic reticulum cisternae (including those mutated in patients A and B) abolished UGT1A1 activity or markedly increased its apparent K(m) for bilirubin. Substitution of the three cysteine residues within the C-terminal cytosolic tail had minimal effect on basal UGT1A1 activity, but prevented UGT1A1 activation by UDP-GlcNAc. N-Ethylmaleimide did not inhibit UGT1A1 activity in native microsomes, but prevented UGT1A1 activation by UDP-GlcNAc and inhibited the activity in digitonin-permeabilized microsomes. Dithiothreitol did not affect UGT1A1 activity in human liver microsomes. Together, the results suggested that free thiol groups, but not disulphide bonding, of seven cysteine residues within the intracisternal region of human UGT1A1 are important for its catalytic activity, while cysteine residues in the cytosolic domain may be involved in its physiological activation by UDP-GlcNAc.

Solubilization, purification and reconstitution of Ca 2+-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca 2+-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C 12E 8) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca 2+-ATPase with a greater specific activity than solubilization with C 12E 8 or Triton X-100. DHPC was determined to be superior to C 12E 8; while that the C 12E 8 was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca 2+-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C 12E 8 and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca 2+ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C 12E 8 and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca 2+-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C 12E 8 and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca 2+ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca 2+-ATPase retained more organized and native secondary conformation compared to C 12E 8 and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C 12E 8 and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca 2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C 12E 8 and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.

Antioxidant effect of conjugated linoleic acid and vitamin A during non enzymatic lipid peroxidation of rat liver microsomes and mitochondria.

In the study reported here the effect of conjugated linoleic acid (CLA) and vitamin A on the polyunsaturated fatty acid composition, chemiluminescence and peroxidizability index of microsomes and mitochondria isolated from rat liver was analyzed. The effect of CLA on the polyunsaturated fatty acid composition of native microsomes was evidenced by an statistically significant p < 0.007 decrease of linoleic acid C18:2 n6, whereas in mitochondria it was observed a decrease p < 0.0001 of arachidonic acid C20:4 n6 when compared with vitamin A and control groups. Docosahexaenoic acid C22:6 n3 in mitochondria was reduced p < 0.04 in CLA and vitamin A groups when compared with control. After incubation of microsomes or mitochondria in an ascorbate (0.4 mM)-Fe++ (2.15 microM) system (120 min at 37 degrees C) it was observed that the total cpm/mg protein originated from light emission: chemiluminescence was lower in liver microsomes or mitochondria obtained from CLA group (received orally: 12.5 mg/daily during 10 days) than in the vitamin A group (received intraperitoneal injection: daily 0.195 g/ kg during 10 days). CLA reduced significantly maximal induced chemiluminescence in microsomes relative to vitamin A and control groups, whereas in mitochondria the effect was observed relative to control group. The polyunsaturated fatty acid composition of liver microsomes or mitochondria changed by CLA and vitamin A treatment. The polyunsaturated fatty acids mainly affected when microsomes native and peroxidized from control group were compared were linoleic, linolenic and arachidonic acids, while in vitamin A group linoleic and arachidonic acid were mainly peroxidized, whereas in CLA group only arachidonic acid was altered. In mitochondria obtained from the three groups arachidonic acid and docosahexaenoic acid showed a significant decrease when native and peroxidized groups were compared. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of fatty acids, show significant changes in the CLA group compare vitamin A and control groups. The simultaneous analysis of peroxidizability index, chemiluminescence and fatty acid composition demonstrated that CLA is more effective than vitamin A protecting microsomes or mitochondria from peroxidative damage.

In vitro-in vivo correlations for drugs eliminated by glucuronidation: investigations with the model substrate zidovudine.

To investigate the effects of incubation conditions on the kinetic constants for zidovudine (AZT) glucuronidation by human liver microsomes, and whether microsomal intrinsic clearance (CLint) derived for the various conditions predicted hepatic AZT clearance by glucuronidation (CLH) in vivo. The effects of incubation constituents, particularly buffer type (phosphate, Tris) and activators (Brij58, alamethacin, UDP-N-acetylglucosamine (UDP-NAcG)), on the kinetics of AZT glucuronidation by human liver microsomes was investigated. AZT glucuronide (AZTG) formation by microsomal incubations was quantified by h.p.l.c. Microsomal CLint values determined for the various experimental conditions were extrapolated to a whole organ CLint and these data were used to calculate in vivo CLH using the well-stirred, parallel tube and dispersion models. Mean CLint values for Brij58 activated microsomes in both phosphate (3.66 +/- 1.40 micro l min-1 mg-1, 95% CI 1.92, 5.39) and Tris (3.79 +/- 0.74 micro l min-1 mg-1, 95% CI 2.87, 4.71) buffers were higher (P < 0.05) than the respective values for native microsomes (1.04 +/- 0.42, 95% CI 0.53, 1.56 and 1.37 +/- 0.30 micro l min-1 mg-1, 95% CI 1.00, 1.73). Extrapolation of the microsomal data to a whole organ CLint and substitution of these values in the expressions for the well-stirred, parallel tube and dispersion models underestimated the known in vivo blood AZT clearance by glucuronidation by 6.5- to 23-fold (3.61-12.71 l h-1vs 82 l h-1). There was no significant difference in the CLH predicted by each of the models for each set of conditions. A wide range of incubation constituents and conditions were subsequently investigated to assess their effects on GAZT formation, including alamethacin, UDP-NAcG, MgCl2, d-saccharic acid 1,4-lactone, ATP, GTP, and buffer pH and ionic strength. Of these, only decreasing the phosphate buffer concentration from 0.1 m to 0.02 m for Brij58 activated microsomes substantially increased the rate of GAZT formation, but the extrapolated CLH determined for this condition still underestimated known AZT glucuronidation clearance by more than 4-fold. AZT was shown not to bind nonspecifically to microsomes. Analysis of published data for other glucuronidated drugs confirmed a trend for microsomal CLint to underestimate in vivo CLH. AZT glucuronidation kinetics by human liver microsomes are markedly dependent on incubation conditions, and there is a need for interlaboratory standardization. Extrapolation of in vitro CLint underestimates in vivo hepatic clearance of drugs eliminated by glucuronidation.

Propranolol as an inhibitor of some cytochrome P450-dependent monooxygenase activities in native and induced rat liver microsomes.

The in vitro effect of propranolol (10(-3) M and 10(-4) M), a nonselective and extensively metabolized beta-adrenergic blocking agent, on rat liver drug metabolism in native and induced (with phenobarbital and beta-naphthoflavone [beta-NF]) microsomes was studied. The type of inhibition and the inhibitory constants of some cytochrome P450-dependent microsomal enzyme reactions (hexobarbital oxidation [HBO], ethylmorphine-N-demethylation [EMND], aniline hydroxylation [AH], ethoxycoumarin-O-deethylation [ECOD], ethoxyresorufin-O-dealkylation [EROD] and penthoxyresorufin-O-dealkylation [PROD]) were estimated. The results showed that propranolol competitively inhibited AH activity in native microsomes. The type of inhibition was changed from competitive to noncompetitive in all other enzyme activities studied. This inhibition was more pronounced after phenobarbital induction in PROD (Ki = 0.11 +/- 0.01 mM), ECOD (Ki = 0.40 +/- 0.09 mM) and EMND (Ki = 0.59 +/- 0.1 mM), and after beta-NF induction in AH (Ki = 0.28 +/- 0.05 mM) and in HBO (Ki = 0.35 +/- 0.1 mM) in native microsomes. It was assumed that the noncompetitive type of inhibition is due to the covalent binding of reactive metabolites derived from propranolol to hepatic microsomal proteins. The competitive type of inhibition of AH suggested a common P450 isoenzyme in the metabolism of propranolol and aniline. Thus, in this study, propranolol has been found to be not only a selective inhibitor of CYP2D6 isoenzyme-dependent reactions, but also a nonspecific inhibitor of other cytochrome P450 isoenzymes.

Comparative study of the metabolism of drug substrates by human cytochrome P450 3A4 expressed in bacterial, yeast and human lymphoblastoid cells

1. The aim was to compare the metabolic activity of human CYP3A4 expressed in bacteria (E. coli), yeast (S. cerevisiae) and human lymphoblastoid cells (hBl), with the native CYP3A4 activity observed in a panel of human livers. 2. Three CYP3A4 substrates were selected for study: dextromethorphan (DEM), midazolam (MDZ) and diazepam (DZ). The substrate metabolism in each of the four systems was characterized by deriving the kinetic parameters Km or S50, Vmax and intrinsic clearance (CLint) or maximum clearance (CLmax) from the kinetic profiles; the latter differing by 100-fold across the three substrates. 3. The Km or S50 for the formation of metabolites 3-methoxymorphinan (MEM), 1'-hydroxymidazolam (1'-OH MDZ) and 3-hydroxydiazepam (3HDZ) compared well in all systems. For CYP3A4-mediated metabolism of DEM, MDZ and DZ, the Vmax for hBl microsomes were generally 2-9-fold higher than the respective yeast and human liver microsomes and E. coli membrane preparations, resulting in greater CLint or CLmax. In the case of 3HDZ formation, non-linear kinetics were observed for E. coli, hBl microsomes and human liver microsomes, whereas the kinetics observed for S. cerevisiae were linear. 4. The use of native human liver microsomes for drug metabolic studies will always be preferable. However, owing to the limited availability of human tissues, we find it is reasonable to use any of the recombinant systems described herein, since all three recombinant systems gave good predictions of the native human liver enzyme activities.

Aldehydic products of lipid peroxidation inactivate cytochrome P-450

The effects of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), on the structure of rat liver microsomal membrane and cytochrome P-450 was studied. MDA (15-30 microM) similarly to p-chlormercuribenzoate decreased the cytochrome P-450 content by 50 % and lowered microviscosity of lipid surrounding of the spin label OTMB bound to SH-groups of membrane proteins. OTMB was effectively reduced by K3Fe(CN)6 in microsomes preincubated with MDA (20 (M), but not in native microsomes. HNE (10 microM) decreased the cytochrome P-450 content by 90 %. Reduced glutathione and cysteine (5 mM) prevented the decrease of cytochrome P-450 under influence of both MDA or HNE, whereas cytochrome P-420 formation remains unchanged. MDA and HNE decreased activities of NADPH oxidase and NADPH cytochrome c reductase. HNE increased microviscosity of the OTMB lipid environment. The further increase of HNE concentration did not affect this parameter. Both MDA and HNE increased the absorbance at 420 nm, which indicated inactivation of cytochrome P-450 by changes in hydrophobicity of lipid surrounding. We suggest that HNE and aliphatic aldehydes at low concentrations can enter into hydrophobic environment of cytochrome P-450 binding to its SH-groups, which led to inactivation of cytochrome P-450. At the same time, the modification of membrane surface layer and subsequent decrease of hydrophobicity of cytochrome P-450 environment preceded the binding of MDA to SH-groups of cytochrome P-450 to develop its inactivating effect.

Tungstate: A Potent Inhibitor of Multifunctional Glucose-6-Phosphatase

The insulin-like action of tungstate in diabetic rats (A. Barberàet al.,1994,J. Biol. Chem.269, 20047–20053) prompted us to examine the effects of tungstate on the glucose-6-phosphatase system. Our results indicate that tungstate is a potent inhibitor of glucose-6-phosphatase, with aKiin the 10–25 μM range determined with native microsomes and in the 1–7 μM range determined with detergent-treated microsomes. With both preparations, simple linear competitive inhibition was observed versus glucose 6-phosphate (glucose-6-P) as substrate with the glucose-6-P phosphohydrolase activity of the enzyme. Tungstate was a simple linear competitive inhibitor versus carbamyl phosphate (carbamyl-P) and a linear noncompetitive inhibitor versus glucose with the carbamyl-P:glucose phosphotransferase activity of the glucose-6-phosphatase system. These findings, in addition to the observation that tungstate protected the enzyme against thermal inactivation, indicate that tungstate binds with high affinity and competes at the active site of the enzyme where the substrates glucose-6-P and carbamyl-P bind prior to catalysis. Our results suggest that potent inhibition of glucose-6-P hydrolysis by tungstate is likely responsible, at least in part, for the normalization of glycemia and the rebound in hepatic glucose-6-P levels observed in earlier studies in which tungstate exhibited insulin-like action in diabetic rats.

Ascorbate-Fe2+ lipid-peroxidation of rat liver microsomes: effect of vitamin E and cytosolic proteins.

In the present study, we examined the effect of the intraperitoneal administration of vitamin E (100 mg/kg weight/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of rat liver microsomes . We also analyzed the effect of hepatic cytosolic proteins on this process. The results indicate that the ascorbate induced light emission was 76% lower in microsomes (1 mg protein) obtained from vitamin E treated animals when compared with controls. In the presence of cytosolic protein (1 mg) the chemiluminescence of control microsomes diminished 55.8 and 59.5% when cytosol from controls and treated animals was used, respectively. The chemiluminescence of vitamin E microsomes diminished 25.03 and 22.08% when both types of cytosol were added to the medium. Dialyzed or treated at 70 degrees C cytosol was also able to inhibit the lipid peroxidation of either control or vitamin E rat liver microsomes. By means of gas chromatography we analyzed the fatty acid composition of native and peroxidated microsomes from both animal groups. The peroxidation affected principally arachidonic acid and its diminution was more evident in the control microsomes than in the microsomes from the vitamin E treated group. By HPLC we analyzed the vitamin E content in all subcellular fractions employed. In microsomes from the vitamin E-group, the content of vitamin was 11 times higher than in the control ones (0.678 +/- 0.1038 vs. 0.062 +/- 0.0045 microg alpha-tocopherol/mg protein, respectively), while levels in the cytosol from the vitamin E-group were only 2 times higher than in the control cytosol (0.057 +/- 0.0051 vs. 0.025 +/- 0.0015 microg alpha-tocopherol/mg protein, respectively).

Inhibition of glucuronidation by an acyl-CoA-mediated indirect mechanism

The mechanism of the inhibition of glucuronidation by long-chain fatty acyl-CoAs was studied in rat liver microsomal membranes and in isolated hepatocytes. Palmitoyl- and oleoyl-CoA did not affect p-nitrophenol UDP-glucuronosyltransferase activity in native microsomes but were inhibitory in permeabilised vesicles. The extent of inhibition was dependent on the effectiveness of permeabilisation and was constant in time in fully permeabilised microsomes. Fatty acyl-CoAs mobilised calcium from calcium-loaded microsomes. Elevation of the intracellular acyl-CoA level by the addition of palmitate or oleate inhibited the glucuronidation of p-nitrophenol in isolated hepatocytes. This effect could be abolished by emptying the intracellular calcium stores. Therefore, it is concluded that fatty acyl-CoAs inhibit glucuronidation indirectly, presumably via calcium mobilisation.

Inhibition of the glucose-6-phosphatase system by N-bromoacetylethanolamine phosphate, a potential affinity label for auxiliary proteins

N-Bromoacetylethanolamine phosphate (BAEP) has been used previously as an affinity label to study the hexose phosphate binding sites of fructose-6- P, 2-kinase:fructose-2, 6-bisphosphatase (Sakakibara et al. (1984) J. Biol. Chem. 259, 14023–14028). We have employed this compound to probe components of the glucose-6-phosphatase system using a combination of time-dependent and immediate inhibition kinetic techniques. Inhibition of d-glucose-6-phosphate (G6 P) phosphohydrolase activity of native microsomes was irreversible and time- and inhibitor-concentration-dependent. Only a partial time-dependent, irreversible inhibition of the PP i phosphohydrolase activity of native microsomes was observed. BAEP inhibited PP i:glucose phosphotransferase activity of native microsomes in a concentration-dependent, irreversible manner which was more extensive than that seen with PP i phosphohydrolase, but less extensive than was observed with G6 P phosphohydrolase. Disruption of microsomal integrity by detergent-treatment either prior to incubation with BAEP or subsequent to preliminary incubation with BAEP but prior to assay for activity abolished the time-dependent inhibition. These irreversible, time- and concentration-dependent inhibitory actions of BAEP thus are manifest at a site or sites where the intact membrane-bound enzyme first makes contact with substrates G6 P and PP i. An additional site of inhibition by BAEP, through relatively weak, reversible competitive inhibition at the active catalytic site, is indicated by classical steady-state kinetic analysis. The irreversible, time- and concentration-dependent inhibitions by BAEP seen with G6 P and PP i as substrates strongly suggest the potential utility of radio-labeled BAEP as an affinity label for the identification and ultimate isolation and study of uncharacterized auxiliary components of the glucose 6-phosphatase system.

Role of molecular species catabolism in the temperature-induced restructuring of phosphatidylcholines in liver microsomes of thermally-acclimated rainbow trout (Oncorhynchus mykiss)

Most previous studies of the temperature-induced restructuring of phospholipid molecular species composition have examined steps in the biosynthesis of phospholipids to explain the accumulation of unsaturated fatty acids in membranes of cold-acclimated poikilotherms. In contrast, the present study explores the role of phospholipases in this restructuring process by determining the rates of degradation of specific molecular species of phosphatidylcholine, using enzymes (microsomes) freshly isolated from the liver of rainbow trout. (Oncorhynchus mykiss) acclimated to either 5° or 20°C. The substrate preparation employed to assay phospholipase activity possessed a range of molecular species, all radiolabeled with 1-(14)C-palmitic acid at thesn-1 position, similar to that present in native trout liver microsomes. After defined periods of incubation (120 and 240 min at 5°C; 60 and 120 min at 20°C), phospholipids were extracted from the reaction mixture and the distribution of radioactivity among the molecular species of phosphatidylcholine was determined by HPLC/liquid scintillation counting. In general, molecular species catabolism was not significantly influenced by either assay or acclimation temperature. Only in 20°C-acclimated fish did a reduction in assay temperature (from to 20 to 5°C) result in significantly increased proportions of radioactivity being recovered in one polyunsaturated fatty acid-containing species (16:0/22:6-PC). It is concluded: 1) that phospholipase specificity, assayed under conditions approximating thosein situ, is not significantly influenced by temperature; and 2), that the increased proportions of unsaturated fatty acid-containing molecular species of phosphatidylcholine observed at low temperatures must reflect the specificity of biosynthetic rather than degradative processes.

Fatty acyl-CoA esters inhibit glucose-6-phosphatase in rat liver microsomes.

In native rat liver microsomes glucose 6-phosphatase activity is dependent not only on the activity of the glucose-6-phosphatase enzyme (which is lumenal) but also on the transport of glucose-6-phosphate, phosphate and glucose through the respective translocases T1, T2 and T3. By using enzymic assay techniques, palmitoyl-CoA or CoA was found to inhibit glucose-6-phosphatase activity in intact microsomes. The effect of CoA required ATP and fatty acids to form fatty acyl esters. Increasing concentrations (2-50 microM) of CoA (plus ATP and 20 microM added palmitic acid) or of palmitoyl-CoA progressively decreased glucose-6-phosphatase activity to 50% of the control value. The inhibition lowered the Vmax without significantly changing the Km. A non-hydrolysable analogue of palmitoyl-CoA also inhibited, demonstrating that binding of palmitoyl-CoA rather than hydrolysis produces the inhibition. Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer demonstrated that palmitoyl-CoA alone or CoA plus ATP and palmitic acid altered the microsomal permeability to glucose 6-phosphate, but not to glucose or phosphate, indicating that T1 is the site of palmitoyl-CoA binding and inhibition of glucose-6-phosphatase activity in native microsomes. The type of inhibition found suggests that liver microsomes may comprise vesicles heterogeneous with respect to glucose-6-phosphate translocase(s), i.e. sensitive or insensitive to fatty acid ester inhibition.

Effect of oral administration of ursodeoxycholic acid on rat hepatic and intestinal UDP-glucuronosyltransferase.

The effect of oral administration of the bile acid ursodeoxycholic acid on rat hepatic and intestinal microsomal UDP-glucuronosyltransferase was studied. The bile acid was administered during 8 days at a daily dose of 500 mg/kg body weight. Enzyme activity was assessed in native and activated microsomes, using bilirubin and p-nitrophenol as substrates. Activation was achieved either by including UDP-N-acetylglucosamine in the incubation mixture or by preincubating native microsomes with an optimal concentration of Lubrol Px. Irrespective of activation status of the microsomes, ursodeoxycholic acid treatment increased enzyme activities toward both substrates in intestine, but not in liver. The analysis of the degree of activation by Lubrol Px revealed that, at least for bilirubin, ursodeoxycholic acid decreased the latency of the intestinal enzyme. The analysis of the lipid composition of microsomes showed several changes in response to ursodeoxycholic acid in intestine but not in liver. Thus, a decrease in cholesterol/phospholipid ratio and an increase in the unsaturation index of total-lipid fatty acids, which correlated well with a membrane "fluidification," were observed. These modifications appear to be related to the lower latency of bilirubin UDP-glucuronosyltransferase in intestine from treated rats and could be responsible, at least in part, for the improvement of enzyme activity in this group. Whatever the mechanism involved, the increment of intestinal UDP-glucuronosyltransferase activities toward both substrates may be relevant as a complement to the hepatic enzymes in those liver diseases in which ursodeoxycholic acid is used as a therapeutic agent.

Antioxidant effects of estradiol and 2-hydroxyestradiol on iron-induced lipid peroxidation of rat liver microsomes

In the present study, the antioxidant effects of estradiol (E 2) and 2-hydroxyestradiol (2-OHE 2) on microsomal lipid peroxidation induced by Fe 3+/ADP/NADPH and Fe 2+/ascorbate are described. The extent of lipid peroxidation was measured by thiobarbituric acid reactive substances (TBARS) detection, low-level chemiluminescence, and oxygen consumption. 2-OHE 2 had a potent antioxidant activity, which in all cases was higher than that of E 2. In the Fe 2+/ascorbate model, 2-OHE 2 showed a similar pattern of inhibition, irrespective of the presence of NADPH or the functionality of microsomes. However, E 2 produced only a slight inhibition when either denatured microsomes or native microsomes without NADPH were used, whereas it protective effect increased considerably when microsomal E 2 metabolism was favored. During enzymic Fe 3+/ADP/NADPH-induced lipid peroxidation, both E 2 and 2-OHE 2 were found to provide good protection. Results underline the importance of the chemical structure of these compounds and the role of estradiol metabolism in its antioxidant effects. (Steroids 59:383–388, 1994)