Low-affinity Phase Research Articles

GluN2 Subunit-Dependent Redox Modulation of NMDA Receptor Activation by Homocysteine.

Homocysteine (HCY) molecule combines distinct pharmacological properties as an agonist of N-methyl-d-aspartate receptors (NMDARs) and a reducing agent. Whereas NMDAR activation by HCY was elucidated, whether the redox modulation contributes to its action is unclear. Here, using patch-clamp recording and imaging of intracellular Ca2+, we study dithiothreitol (DTT) effects on currents and Ca2+ responses activated by HCY through native NMDARs and recombinant diheteromeric GluN1/2A, GluN1/2B, and GluN1/2C receptors. Within a wide range (1–800 μM) of [HCY]s, the concentration–activation relationships for recombinant NMDARs revealed a biphasicness. The high-affinity component obtained between 1 and 100 µM [HCY]s corresponding to the NMDAR activation was not affected by 1 mM DTT. The low-affinity phase observed at [HCY]s above 200 μM probably originated from thiol-dependent redox modulation of NMDARs. The reduction of NMDAR disulfide bonds by either 1 mM DTT or 1 mM HCY decreased GluN1/2A currents activated by HCY. In contrast, HCY-elicited GluN1/2B currents were enhanced due to the remarkable weakening of GluN1/2B desensitization. In fact, cleaving NMDAR disulfide bonds in neurons reversed the HCY-induced Ca2+ accumulation, making it dependent on GluN2B- rather than GluN2A-containing NMDARs. Thus, estimated concentrations for the HCY redox effects exceed those in the plasma during intermediate hyperhomocysteinemia but may occur during severe hyperhomocysteinemia.

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.

Hepatic and intestinal glucuronidation of mono(2-ethylhexyl) phthalate, an active metabolite of di(2-ethylhexyl) phthalate, in humans, dogs, rats, and mice: an in vitro analysis using microsomal fractions.

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP) and has endocrine-disrupting effects. MEHP is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the hepatic and intestinal glucuronidation of MEHP in humans, dogs, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and dogs, and the biphasic model for rats and mice. The K m and V max values of human liver microsomes were 110µM and 5.8nmol/min/mg protein, respectively. The kinetics of intestinal microsomes followed the biphasic model for humans, dogs, and mice, and the Michaelis-Menten model for rats. The K m and V max values of human intestinal microsomes were 5.6µM and 0.40nmol/min/mg protein, respectively, for the high-affinity phase, and 430µM and 0.70nmol/min/mg protein, respectively, for the low-affinity phase. The relative levels of V max estimated by Eadie-Hofstee plots were dogs (2.0)>mice (1.4)>rats (1.0)≈humans (1.0) for liver microsomes, and mice (8.5)>dogs (4.1)>rats (3.1)>humans (1.0) for intestinal microsomes. The percentages of the V max values of intestinal microsomes to liver microsomes were mice (120%)>rats (57%)>dogs (39%)>humans (19%). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward MEHP markedly differed among species, and imply that these species differences are strongly associated with the toxicity of DEHP.

Glucuronidation of Edaravone by Human Liver and Kidney Microsomes: Biphasic Kinetics and Identification of UGT1A9 as the Major UDP-Glucuronosyltransferase Isoform

Edaravone was launched in Japan in 2001 and was the first neuroprotectant developed for the treatment of acute cerebral infarction. Edaravone is mainly eliminated as glucuronide conjugate in human urine (approximately 70%), but the mechanism involved in the elimination pathway remains unidentified. We investigated the glucuronidation of edaravone in human liver microsomes (HLM) and human kidney microsomes (HKM) and identified the major hepatic and renal UDP-glucuronosyltransferases (UGTs) involved. As we observed, edaravone glucuronidation in HLM and HKM exhibited biphasic kinetics. The intrinsic clearance of glucuronidation at high-affinity phase (CL(int1)) and low-affinity phase (CL(int2)) were 8.4 ± 3.3 and 1.3 ± 0.2 μl · min(-1) · mg(-1), respectively, for HLM and were 45.3 ± 8.2 and 1.8 ± 0.1 μl · min(-1) · mg(-1), respectively, for HKM. However, in microsomal incubations contained with 2% bovine serum albumin, CL(int1) and CL(int2) were 16.4 ± 1.2 and 3.7 ± 0.3 μl · min(-1) · mg(-1), respectively, for HLM and were 78.5 ± 3.9 and 3.6 ± 0.5 μl · min(-1) · mg(-1), respectively, for HKM. Screening with 12 recombinant UGTs indicated that eight UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B17) produced a significant amount of glucuronide metabolite. Thus, six UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A9, UGT2B7, and UGT2B17) expressed in human liver or kidney were selected for kinetic studies. Among them, UGT1A9 exhibited the highest activity (CL(int1) = 42.4 ± 9.5 μl · min(-1) · mg(-1)), followed by UGT2B17 (CL(int) = 3.3 ± 0.4 μl · min(-1) · mg(-1)) and UGT1A7 (CL(int) = 1.7 ± 0.2 μl · min(-1) · mg(-1)). Inhibition study found that inhibitor of UGT1A9 (propofol) attenuated edaravone glucuronidation in HLM and HKM. In addition, edaravone glucuronidation in a panel of seven HLM was significantly correlated (r = 0.9340, p = 0.0021) with propofol glucuronidation. Results indicated that UGT1A9 was the main UGT isoform involved in edaravone glucuronidation in HLM and HKM.

AtCIPK8, a CBL‐interacting protein kinase, regulates the low‐affinity phase of the primary nitrate response

Nitrate, the major nitrogen source for most plants, is not only a nutrient but also a signaling molecule. For almost two decades, it has been known that nitrate can rapidly induce transcriptional expression of several nitrate-related genes, a process that is referred to as the primary nitrate response. However, little is known about how plants actually sense nitrate and how the signal is transmitted in this pathway. In this study, a calcineurin B-like (CBL) -interacting protein kinase (CIPK) gene, CIPK8, was found to be involved in early nitrate signaling. CIPK8 expression was rapidly induced by nitrate. Analysis of two independent knockout mutants and a complemented line showed that CIPK8 positively regulates the nitrate-induced expression of primary nitrate response genes, including nitrate transporter genes and genes required for assimilation. Kinetic analysis of nitrate induction levels of these genes in wild-type plants indicated that there are two response phases: a high-affinity phase with a K(m) of approximately 30 mum and a low-affinity phase with a K(m) of approximately 0.9 mm. As cipk8 mutants were defective mainly in the low-affinity response, the high-affinity and low-affinity nitrate signaling systems are proposed to be genetically distinct, with CIPK8 involved in the low-affinity system. In addition, CIPK8 was found to be involved in long-term nitrate-modulated primary root growth and nitrate-modulated expression of a vacuolar malate transporter. Taken together, our results indicate that CBL-CIPK networks are responsible not only for stress responses and potassium shortage, but also for nitrate sensing.

Genetic Ablation of Caveolin‐1 does not Affect Pressure‐Induced Constriction but Alters Endothelin‐1 Pharmacology in Murine Cerebral Arteries

Numerous phenotypic changes have been reported in mice with knockout of caveolin‐1 (KO) expression. This study evaluated cerebroarterial reactivity to investigate the role of caveolin‐1 in vascular smooth muscle function. Arterial segments were isolated from KO and wild type (WT) mice of age 6–9 months, pressure‐mounted and diameter was monitored. No difference was observed in constriction to intraluminal pressure up to 110 mmHg, further increase in pressure caused forced dilation. Constrictions to 60 mM KCl, 10 μM phenylephrine and 10 μM serotonin and dilation to 10 μM bradykinin and 16mM KCl were not different in WT and KO arteries. Concentration response curves to endothelin‐1 in WT arteries were biphasic. In KO arteries, constriction to ET1 was significantly decreased and the second low affinity phase was not observed. Decreased ET1 constriction was not restored by 100 μM L‐NAME. Myogenic tone at 70 mmHg was reversed by SKF 96365 and 2APB, known blockers of transient receptor potential (TRP) channels, in WT but only partially decreased in KO arteries. ET1 constriction was equally sensitive to SKF 96365 and 2APB in both groups. Myogenic tone was reversed by Y27632, a rho‐kinase inhibitor in both groups. Decrease in ET1 constriction by Y27632 was significantly lower in KO arteries compared to WT arteries. Constriction to pressure and ET1 may be modulated by caveolin‐1 via activation of TRP channels and rho‐kinase.

The Mechanism of Carrier-Mediated Transport of Folates in BeWo Cells: The Involvement of Heme Carrier Protein 1 in Placental Folate Transport

The aim of this study was to elucidate the mechanism of folate transport in the placenta. A study of folate was carried out to determine which carriers transport folates in the human choriocarcinoma cell line BeWo, a model cell line for the placenta. We investigated the effects of buffer pH and various compounds on folate uptake. In the first part of the study, the expression levels of the mRNA of the folate receptor alpha (FRalpha), the reduced folate carrier (RFC), and heme carrier protein 1 (HCP1) were determined in BeWo cells by RT-PCR analysis. Folate uptake into BeWo cells was greater under an acidic buffer condition than under a neutral one. Structure analogs of folates inhibited folate uptake under all buffer pH conditions, but anion drugs (e.g., pravastatin) inhibited folate uptake only under an acidic buffer condition. Although thiamine pyrophosphate (TPP), a substrate of RFC, had no effect on folate uptake, hemin (a weak inhibitor of folate uptake via HCP1) decreased folate uptake to about 80% of the control level under an acidic buffer condition. Furthermore, kinetic analysis showed that hemin inhibited the low-affinity phase of folate uptake under an acidic buffer condition. We conclude that pH-dependent folate uptake in BeWo cells is mediated by at least two carriers. RFC is not involved in folate uptake, but FRalpha (high affinity phase) and HCP1 (low affinity phase) transport folate in BeWo cells.

Characterization of G-Protein Coupled Receptor Kinase Interaction with the Neurokinin-1 Receptor Using Bioluminescence Resonance Energy Transfer

To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET(2) saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.

Mg2+ and Ca2+ Differentially Regulate DNA Binding and Dimerization of DREAM

DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca(2+) binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca(2+) binding to the various mutants revealed that, in the absence of Mg(2+), Ca(2+) binds independently and sequentially to EF-3 (DeltaH = -2.4 kcal/mol), EF-4 (DeltaH = +5.2 kcal/mol), and EF-2 (DeltaH = +1 kcal/mol). By contrast, only two Ca(2+) bind to DREAM in the presence of physiological levels of Mg(2+) for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg(2+). ITC measurements demonstrate that one Mg(2+) binds enthalpically with high affinity (K(d) = 13 mum and DeltaH = -0.79 kcal/mol) and two or more Mg(2+) bind entropically in the millimolar range. Size-exclusion chromatography studies revealed that Mg(2+) stabilizes DREAM as a monomer, whereas Ca(2+) induces protein dimerization. Electrophoretic mobility shift assays indicated that Mg(2+) is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca(2+) levels, suggesting that binding of a single Ca(2+) at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca(2+) and Mg(2+) induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg(2+) binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca(2+)-induced protein dimerization disrupts DNA binding.

Zinc Potentiates an Uncoupled Anion Conductance Associated with the Dopamine Transporter

Binding of Zn(2+) to an endogenous binding site in the dopamine transporter (DAT) leads to inhibition of dopamine (DA) uptake and enhancement of carrier-mediated substrate efflux. To elucidate the molecular mechanism for this dual effect, we expressed the DAT and selected mutants in Xenopus laevis oocytes and applied the two-electrode voltage clamp technique together with substrate flux studies employing radiolabeled tracers. Under voltage clamp conditions we found that Zn(2+) (10 mum) enhanced the current induced by both DA and amphetamine. This was not accompanied by a change in the uptake rate but by a marked increase in the charge/DA flux coupling ratio as assessed from concomitant measurements of [(3)H]DA uptake and currents in voltage-clamped oocytes. These data suggest that Zn(2+) facilitates an uncoupled ion conductance mediated by DAT. Whereas this required substrate in the wild type (WT), we observed that Zn(2+) by itself activated such a conductance in a previously described mutant (Y335A). This signifies that the conductance is not strictly dependent on an active transport process. Ion substitution experiments in Y335A, as well as in WT, indicated that the uncoupled conductance activated by Zn(2+) was mainly carried by Cl(-). Experiments in oocytes under non-voltage-clamped conditions revealed furthermore that Zn(2+) could enhance the depolarizing effect of substrates in oocytes expressing WT. The data suggest that by potentiating an uncoupled Cl(-) conductance, Zn(2+) is capable of modulating the membrane potential of cells expressing DAT and as a result cause simultaneous inhibition of uptake and enhancement of efflux.

AtOPT6 transports glutathione derivatives and is induced by primisulfuron.

The oligopeptide transporter (OPT) family contains nine members in Arabidopsis. While there is some evidence that AtOPTs mediate the uptake of tetra- and pentapeptides, OPT homologs in rice (Oryza sativa; OsGT1) and Indian mustard (Brassica juncea; BjGT1) have been described as transporters of glutathione derivatives. This study investigates the possibility that two members of the AtOPT family, AtOPT6 and AtOPT7, may also transport glutathione and its conjugates. Complementation of the hgt1met1 yeast double mutant by plant homologs of the yeast glutathione transporter HGT1 (AtOPT6, AtOPT7, OsGT1, BjGT1) did not restore the growth phenotype, unlike complementation by HGT1. By contrast, complementation by AtOPT6 restored growth of the hgt1 yeast mutant on a medium containing reduced (GSH) or oxidized glutathione as the sole sulfur source and induced uptake of [3H]GSH, whereas complementation by AtOPT7 did not. In these conditions, AtOPT6-dependent GSH uptake in yeast was mediated by a high affinity (Km = 400 microm) and a low affinity (Km = 5 mm) phase. It was strongly competed for by an excess oxidized glutathione and glutathione-N-ethylmaleimide conjugate. Growth assays of yeasts in the presence of cadmium (Cd) suggested that AtOPT6 may transport Cd and Cd/GSH conjugate. Reporter gene experiments showed that AtOPT6 is mainly expressed in dividing areas of the plant (cambium, areas of lateral root initiation). RNA blots on cell suspensions and real-time reverse transcription-PCR on Arabidopsis plants indicated that AtOPT6 expression is strongly induced by primisulfuron and, to a lesser extent, by abscisic acid but not by Cd. Altogether, the data show that the substrate specificity and the physiological functions of AtOPT members may be diverse. In addition to peptide transport, AtOPT6 is able to transport glutathione derivatives and metal complexes, and may be involved in stress resistance.

Role of CYP3A4 and CYP2B6 in the in vitro N-demethylation of meperidine

Meperidine (MEP) is an opioid analgesic that exerts its chief pharmacological action in the brain. It is metabolized in the liver by N-demethylation to normeperidine, a potent CNS stimulant. We investigated the in vitro formation of normeperidine by pooled human liver microsomes (HLM) and recombinant human CYP450s. Normeperidine levels were measured by HPLC. Initial screening of 16 CYP isoforms (MEP=85 μM) showed CYP2B6>CYP2C19>CYP2C18>CYP3A4 as the enzymes with the highest MEP N-demethylase activity. Apparent kinetic parameters of MEP N-demethylase activity are described below.(See Table) Normeperidine formation in HLM was reduced by 42% in the presence of ketoconazole (CYP3A4 inhibitor, 2 μM). Treatment with anti-CYP2B and anti-2C inhibitory antibodies resulted in minor inhibition (<15%) of MEP N-demethylation in the liver. To further characterize the in vitro formation of normeperidine, future experiments will include correlation studies in HLM with probe substrates. Our studies suggest that (1) CYP3A4 (but not CYP3A5) has a dominant role in the hepatic metabolic clearance of MEP, and (2) CYP2B6 (a brain CYP) might contribute to normeperidine formation in the brain. Clinical Pharmacology & Therapeutics (2004) 75, P85–P85; doi: 10.1016/j.clpt.2003.11.325 Microsomes Km (μM) mean±SE) Vmax (pmol/min/pmol CYP) (mean±SE) Intrinsic clearance (μl/min/pmol CYP) HLM–high affinity phase 45.4 ± 12.8 124 ± 17 2.73 HLM–low affinity phase 1240 ± 185 1230 ± 60 0.99 CYP2B6 356 ± 34 138 ± 3 0.39 CYP2C19 71.4 ± 19.6 20.6 ± 1.1 0.29 CYP2C18 149 ± 47 17.0 ± 1.1 0.11 CYP3A4 355 ± 80 25.0 ± 1.3 0.07 CYP3A5 1260 ± 10 10.2 ± 0.3 0.01

SNX482 selectively blocks P/Q Ca 2+ channels and delays the inactivation of Na + channels of chromaffin cells

The effects of the toxin SXN482 on Ca 2+ channel currents ( I Ca), Na + currents ( I Na), and K + currents ( I K) have been studied in bovine adrenal medullary chromaffin cells voltage-clamped at −80 mV. Currents were elicited by depolarising pulses to 0–10 mV ( I Ca and I Na) or to +60 mV ( I K). SNX482 blocked I Ca in a concentration-dependent manner. The inhibition curve exhibited two phases. The first high-affinity phase comprised 28% of the whole-cell current and exhibited an IC 50 of 30.2 nM. The second low-affinity phase comprised over 70% of I Ca and had an IC 50 of 758.6 nM. Blockade was rapid and fully reversible upon washout of the toxin. Occlusion experiments showed additivity of blockade exerted by nifedipine plus SNX482 (0.3 μM) and by ω-conotoxin GVIA plus SNX482. In contrast, blockade exerted by combined ω-agatoxin IVA plus SNX482 (about 50% of the whole cell) did not show additivity. At 0.3 μM and higher concentrations, SNX482 delayed the inactivation of I Na. The time constant ( τ) for inactivation of I Na in control conditions doubled in the presence of 0.5 μM SNX482. At 0.3 μM, SNX482 did not affect I K. Our data demonstrate that: (i) SNX482 selectively blocks P/Q Ca 2+ channels at submicromolar concentrations; (ii) the toxin partially blocks Na + channels; (iii) SNX482 delays the inactivation of Na + channels. These results reveal novel properties of SNX482 and cast doubts on the claimed selectivity and specificity of the toxin to block the R-type Ca 2+ channel.

Metabolism of chloroform in the human liver and identification of the competent P450s.

The oxidative and reductive cytochrome P450 (P450)-mediated chloroform bioactivation has been investigated in human liver microsomes (HLM), and the role of human P450s have been defined by integrating results from several experimental approaches: cDNA-expressed P450s, selective chemical inhibitors and specific antibodies, correlation studies in a panel of phenotyped HLM. HLM bioactivated CHCl(3) both oxidatively and reductively. Oxidative reaction was characterized by two components, suggesting multiple P450 involvement. The high affinity process was catalyzed by CYP2E1, as clearly indicated by kinetic studies, correlation with chlorzoxazone 6-hydroxylation (r = 0.837; p < 0.001), and inhibition by monoclonal antihuman CYP2E1 and 4-methylpyrazole. The low affinity phase of oxidative metabolism was essentially catalyzed by CYP2A6. This conclusion was supported by the correlation with coumarin 7-hydroxylase (r = 0.777; p < 0.01), inhibition by coumarin and by the specific antibody, in addition to results with heterologously expressed P450s. Chloroform oxidation was poorly dependent on pO(2), whereas the reductive metabolism was highly inhibited by O(2). The production of dichloromethyl radical was significant only at CHCl(3) concentration > or =1 mM, increasing linearly with substrate concentration. CYP2E1 was the primary enzyme involved in the reductive reaction, as univocally indicated by all the different approaches. The reductive pathway seems to be scarcely relevant in the human liver, since it is active only at high substrate concentrations, and in strictly anaerobic conditions. The role of human CYP2E1 in CHCl(3) metabolism at low levels, typical of actual human exposure, provides insight into the molecular basis for eventual difference in susceptibility to chloroform-induced effects due to either genetic, pathophysiological, or environmental factors.

CYP-specific bioactivation of four organophosphorothioate pesticides by human liver microsomes

The bioactivation of azinphos-methyl (AZIN), chlorpyrifos (CPF), diazinon (DIA), and parathion (PAR), four widely used organophosphorothioate (OPT) pesticides has been investigated in human liver microsomes (HLM). In addition, the role of human cytochrome P450 (CYPs) in OPT desulfuration at pesticide levels representative of human exposure have been defined by means of correlation and immunoinhibition studies. CYP-mediated oxon formation from the four OPTs is efficiently catalyzed by HLM, although showing a high variability (>40-fold) among samples. Two distinct phases were involved in the desulfuration of AZIN, DIA, and PAR, characterized by different affinity constants ( K mapp1 = 0.13–9 μM and K mapp2 = 5– 269 μM). Within the range of CPF concentrations tested, only the high-affinity component was evidenced ( K mapp1 = 0.27–0.94 μM). Oxon formation in phenotyped individual HLM showed a significant correlation with CYP1A2-, 3A4-, and 2B6-related activities, at different levels depending on the OPT concentration. Anti-human CYP1A2, 2B6, and 3A4 antibodies significantly inhibited oxon formation, showing the same OPT concentration dependence. Our data indicated that CYP1A2 is mainly involved in OPT desulfuration at low pesticide concentrations, while the role of CYP3A4 is more significant to the low-affinity component of OPT bioactivation. The contribution of CYP2B6 to total hepatic oxon formation was relevant in a wide range of pesticide concentrations, being a very efficient catalyst of both the high- and low-affinity phase. These results suggest CYP1A2 and 2B6 as possible metabolic biomarkers of susceptibility to OPT toxic effect at the actual human exposure levels.

Relationship of isopentenyl diphosphate (IDP) isomerase activity to isoprene emission of oak leaves.

Oaks emit large amounts of isoprene, a compound that plays an important role in tropospheric chemistry. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) catalyzes the isomerization of isopentenyl diphosphate (IDP) to dimethylallyl diphosphate (DMADP), and in isoprene-emitting plants, isoprene synthase (IS) converts the DMADP to isoprene. To study the role of IDI in isoprene biosynthesis of oak leaves, we compared IDI and IS activities in pedunculate oak (Quercus robur L.) and pubescent oak (Quercus pubescens Willd.) with the isoprene emission rates of these species. We developed a non-radioactive enzyme assay to detect IDI activity in crude leaf extracts of Q. robur. The substrate dependency of IDI activity showed biphasic kinetics with Michaelis constants (K(m)(IDP)) of 0.7 +/- 0.2 micro M for a high-affinity phase and 39.5 +/- 6.9 micro M for a low-affinity phase, potentially attributable to different IDI isoforms. Under standard assay conditions, the temperature optimum for IDI activity was about 42 degrees C, but IDI activity was detectable up to 60 degrees C. A sharp pH optimum appeared around pH 7, with 20 mM Mg(2+) also required for IDI activity. Neither IDI activity nor IS activity showed diurnal variation in Q. robur leaves. The sum of IDI activities showed a significant linear correlation with IS activity in both Q. robur and Q. pubescens leaves, and both enzyme activities showed a linear relationship to isoprene emission factors in leaves of these oak species, indicating the possible involvement of IDI in isoprene biosynthesis by oak leaves.

Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, have different biochemical properties and functional roles

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (Km for ammonium and methylammonium of 8 μM and 31 μM, respectively) matched those of AtAMT1;1 expressed in yeast (Km for methylammonium of 32 μM and Ki for ammonium of 1–10 μM). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (Km for ammonium of 46 μM). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a Km of 36 μM, a low-affinity phase with a Km for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis.

Fourier Transform Infrared and Resonance Raman Studies of the Interaction of Azide with Cytochrome c Oxidase from Paracoccus denitrificans

The interaction of azide with oxidized cytochrome c oxidase from Paracoccus denitrificans has been studied by resonance Raman, Fourier transform infrared, and UV−vis spectroscopy. Azide binds in two phases: a high-affinity phase (Kd = 4.1 μM) in which it is bound to a nonmetal site near the binuclear center and a low-affinity phase (Kd = 11.4 mM) in which it is bound as a bridge to the binuclear center. The resonance Raman spectra of the low-affinity phase display one isotope-dependent vibrational mode at 417 cm-1. The FTIR spectra display two isotope-dependent bands at 2038 and 2056 cm-1. We assign the band at 417 cm-1 to ν(Fe−N3−CuB) and the bands at 2038 and 2056 cm-1 to νas(N3). We observe similar FTIR spectra for the azide complex of bovine heart oxidase and conclude that the binuclear center in this oxidase behaves in a manner analogous to the P. denitrificans enzyme. In contrast to mammalian cytochrome c oxidase (Li, W.; Palmer, G. Biochemistry 1993, 322, 1833−1843), the low-affinity phase observed in the interaction of azide with the P. denitrificans enzyme is not associated with binding of azide to heme a. The observation of two FTIR νas(N3) modes suggests that the azide ion binds to two different enzyme conformations, both forming bridging complexes with the binuclear center. Comparison of the UV−vis, resonance Raman, and FTIR data of the azide-bound cytochrome c aa3 from P. denitrificans and those of azide-bound quinol cytochrome bo3 suggest significant alterations in the interaction of azide with the oxidized forms of these bacterial oxidases resulting from specific structural differences within their respective heme a3−CuB and heme o3−CuB binuclear pockets.

Transition between different binding modes in rat DNA polymerase β-ssDNA complexes

Interactions of rat DNA polymerase β with a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence changes accompanying the binding, as a function of the thermodynamically rigorous binding density of rat pol β-ssDNA complexes, reveals the existence of two binding phases. In the first high affinity phase, rat pol β forms a complex with the ssDNA in which 16 nucleotides are occluded by the enzyme. In the second low affinity phase, a transition to a complex where the polymerase occludes only five nucleotides occurs. Thus, the data show that rat pol β binds the ssDNA in two binding modes which differ in the number of occluded nucleotides. We designate the first complex as the (pol β) 16 binding mode and the second as the (pol β) 5 binding mode. The formation of the (pol β) 16 and (pol β) 5 modes has been fully confirmed in experiments with short ssDNA oligomers, a 16mer which can form either the (pol β) 16 or the (pol β) 5 mode, and a 10mer which can form only the (pol β) 5 mode. Binding of rat pol β to the ssDNA has been analyzed using a statistical thermodynamic model which accounts for the existence of the two binding modes, cooperative interactions, and the overlap of potential binding sites. The results indicate that the 8 kDa domain of the enzyme is involved in ssDNA binding in both modes. Binding studies show that an isolated 8 kDa domain has the same intrinsic affinity for the ssDNA as the entire intact enzyme in its (pol β) 5 mode. However, the site size of the 8 kDa domain-ssDNA complex is ten nucleotides, suggesting that the formation of the (pol β) 5 mode is accompanied by a significant conformational transition of the intact protein. A higher intrinsic affinity, a higher net number of ions released, and a lower fluorescence change accompanying the formation of the (pol β) 16 than the (pol β) 5 mode indicate that the 31 kDa catalytic domain of the enzyme interacts with the ssDNA only in the (pol β) 16 mode. The significance of these results for understanding the functioning of rat pol β in the DNA metabolism is discussed.