Anionic Substrates Research Articles

Cofactor-independent oxygenases go it alone

Oxygenases have long been thought to require a cofactor for catalysis. The structure of a vancomycin biosynthetic enzyme in complex with a substrate analog, and with molecular oxygen bound in its active site, supports the idea that cofactor-independent oxygenases act by mediating direct single-electron transfer from a substrate anion to dioxygen.

Polyethylene Imine Derivatives (‘Synzymes') Accelerate Phosphate Transfer in the Absence of Metal

The efficient integration of binding, catalysis, and multiple turnovers remains a challenge in building enzyme models. We report that systematic derivatization of polyethylene imine (PEI) with alkyl (C(2)-C(12)), benzyl, and guanidinium groups gives rise to catalysts ('synzymes') with rate accelerations (k(cat)/k(uncat)) of up to 10(4) for the intramolecular transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate, HPNP, in the absence of metal. The synzymes exhibit saturation kinetics (K(M) approximately 250 microM, k(cat) approximately 0.5 min(-1)) and up to 2340 turnovers per polymer molecule. Catalysis can be specifically and competitively inhibited by anionic and hydrophobic small molecules. The efficacy of catalysis is determined by the PEI derivatization pattern. The derivatization reagents exert a synergistic effect, i.e., their combinations increase catalysis by more than the sum of each single modification. The pH-rate profile for k(cat)/K(M) is bell shaped with a maximum at pH 7.85 and can be explained as a combination of two effects that both have to be operative for optimal activity: K(M) increases at high pH due to deprotonation of PEI amines that bind the anionic substrate and kcat decreases as the availability of hydroxide decreases at low pH. Thus, catalysis is based on substrate binding by positively charged amine groups and the presence of hydroxide ion in active sites in an environment that is tuned for efficient catalysis. Inhibition studies suggest that the basis of catalysis and multiple turnovers is differential molecular recognition of the doubly negatively charged transition state (over singly charged ground state and product): this contributes a factor of at least 5-10-fold to catalysis and product release.

Mutational Analysis of a Highly Conserved Proline Residue in MRP1, MRP2, and MRP3 Reveals a Partially Conserved Function

The ATP-binding cassette multidrug resistance protein 1 MRP1 (ABCC1) mediates the cellular efflux of organic anions including conjugated metabolites, chemotherapeutic agents, and toxicants. We previously described a mutation in cytoplasmic loop 7 (CL7) of MRP1, Pro1150Ala, which reduced leukotriene C(4) (LTC(4)) transport but increased 17beta-estradiol 17beta-d-glucuronide (E(2)17betaG) and methotrexate (MTX) transport. Vanadate-induced trapping of [alpha-(32)P]8N(3)ADP by the Pro1150Ala mutant in the absence of substrate was also greatly reduced compared with wild-type MRP1 suggesting an uncoupling of ATP hydrolysis and transport activity. To determine whether the functional importance of MRP1-Pro(1150) is conserved, the analogous Pro(1158) and Pro(1147) residues in the MRP2 and MRP3 transporters, respectively, were mutated to Ala. Expression levels of the three mutants were unaffected; however, the vesicular transport activity of at least one organic anion substrate was significantly altered. As observed for MRP1-Pro1150Ala, LTC(4) transport by MRP2-Pro1158Ala was decreased. However, E(2)17betaG and MTX transport was comparable with that of wild-type MRP2 rather than increased as was observed for MRP1-Pro1150Ala. In the case of MRP3-Pro1147Ala, LTC(4) transport was increased, whereas E(2)17betaG transport was unaffected. MTX transport by MRP3-Pro1147Ala was also increased but to a lesser extent than for MRP1-Pro1150Ala. In contrast, all three mutants showed a marked reduction in levels of vanadate-induced trapped [alpha-(32)P]8N(3)ADP. We conclude that MRP1-Pro(1150), MRP2-Pro(1158), and MRP3-Pro(1147) in CL7 differ in their influence on substrate specificity but share a common role in the nucleotide interactions of these transporters.

Nonmammalian orthologs of prestin (SLC26A5) are electrogenic divalent/chloride anion exchangers

Individual members of the mammalian SLC26 anion transporter family serve two fundamentally distinct functions. Whereas most members transport different anion substrates across a variety of epithelia, prestin (SLC26A5) is special, functioning as a membrane-localized motor protein that generates electrically induced motions (electromotility) in auditory sensory hair cells of the mammalian inner ear. The transport mechanism of SLC26 proteins is not well understood, and a mechanistic relation between anion transport and electromotility has been suggested but not firmly established so far. To address these questions, we have cloned prestin orthologs from chicken and zebrafish, nonmammalian vertebrates that presumably lack electromotility in their auditory systems. Using patch-clamp recordings, we show that these prestin orthologs, but not mammalian prestin, generate robust transport currents in the presence of the divalent anions sulfate or oxalate. Transport is blocked by salicylate, an inhibitor of electromotility generated by mammalian prestin. The dependence of transport equilibrium potentials on sulfate and chloride concentration gradients shows that the prestin orthologs are electrogenic antiporters, exchanging sulfate or oxalate for chloride in a strictly coupled manner with a 1:1 stoichiometry. These data identify transport mode and stoichiometry of electrogenic divalent/monovalent anion exchange and establish a reliable and simple method for the quantitative determination of the various transport modes that have been proposed for other SLC26 transport proteins. Moreover, the sequence conservation between mammalian and nonmammalian prestin together with a common pharmacology of electromotility and divalent antiport suggest that the molecular mechanism behind electromotility is closely related to an anion transport cycle.

Towards the Design of Neutral Molecular Tweezers for Anion Recognition

Molecular tweezers are simple molecular receptors that can be characterized by the presence of two flat pincers separated by a more or less rigid tether. They have the ability to form complexes with a substrate molecule by gripping the substrate between the tips of the tweezers in a similar manner to that of mechanical tweezers. Klärner et al. synthesized one of the structurally simplest molecular tweezers, which is reported to bind electrodeficient aromatic and aliphatic substrates as well as organic cations. Complexes between these molecular tweezers and electron-rich aromatic, aliphatic, or anionic substrates have not been observed. Inspired by several recent reports that describe the interaction of hexafluorobenzene with electron-rich sites of molecules, we conducted a theoretical study to show the possibility of building molecular tweezers, based on those synthesized by Klärner, which were able to bind to anions and thus increase their potential as molecular receptors. We characterized complexes formed between several fluorinated derivatives of simple tweezers and an iodine anion, and analyzed the nature of the intermolecular interactions as well as the energetics for the process of complexation. The stabilization trend reflected by the energetic results when fluorine substituents were added to benzene rings confirms our hypothesis about the possibility of obtaining neutral tweezers composed of aromatic rings that can bind anions.

Regulation of Function by Dimerization through the Amino-terminal Membrane-spanning Domain of Human ABCC1/MRP1

Overexpression of some ATP-binding cassette (ABC) membrane transporters such as ABCB1/P-glycoprotein/MDR1 and ABCC1/MRP1 causes multidrug resistance in cancer chemotherapy. It has been thought that half-ABC transporters with one nucleotide-binding domain and one membrane-spanning domain (MSD) likely work as dimers, whereas full-length transporters with two nucleotide-binding domains and two or three MSDs function as monomers. In this study, we examined the oligomeric status of the human full-length ABC transporter ABCC1/MRP1 using several biochemical approaches. We found 1) that it is a homodimer, 2) that the dimerization domain is located in the amino-terminal MSD0L0 (where L0 is loop 0) region, and 3) that MSD0L0 has a dominant-negative function when coexpressed with wild-type ABCC1/MRP1. These findings suggest that ABCC1/MRP1 may exist and function as a dimer and that MSD0L0 likely plays some structural and regulatory functions. It is also tempting to propose that the MSD0L0-mediated dimerization may be targeted for therapeutic development to sensitize ABCC1/MRP1-mediated drug resistance in cancer chemotherapy.

Fluorescence Sensing of Anions Based on Inhibition of Excited-State Intramolecular Proton Transfer

Condensation of 2-(2'-aminophenyl)benzoxazole with p-toluenesulfonyl chloride and phenyl isocyanate yields two new anion sensors (TABO and PUBO), which can undergo excited-state intramolecular proton transfer (ESIPT) upon excitation. For the acid receptor TABO, the ESIPT process can be readily disturbed by basic anions such as F-, CH3COO-, and H2PO4- by deprotonating the sulfonamide unit, whereas in the case of PUBO, a good hydrogen-bonding donor, the ESIPT process is inhibited either by the fluoride-induced deprotonation of the urea unit or by the formation of a strong CH3COO--urea intermolecular hydrogen bond complex, and these two types of inhibition mechanisms consequently result in different ratiometric responses. But other anions with less hydrogen-bonding acceptor abilities cannot inhibit the ESIPT. Interestingly, the different inhibition abilities of F-, CH3COO-, and H2PO4- produce different spectral behaviors in PUBO, so this new sensor successfully distinguishes the subtle difference in these three anionic substrates of similar basicity and surface charge density.

Biosensing nitrite using the system nitrite redutase/Nafion/methyl viologen—A voltammetric study

This work describes the construction and voltammetric characterization of a nitrite biosensor based on a cytochrome c-type nitrite reductase (ccNiR) and the Nafion ionomeric matrix loaded with methyl viologen as redox mediator. Despite the potential electrostatic repulsions between the anionic substrate and the Nafion sulfonate groups, the resulting bioelectrode exhibited electrocatalytic activity toward nitrite. This phenomenon must be due to the nonuniformity of the enzyme/Nafion membrane, which allows the direct interaction between the substrate and numerous enzyme molecules. Nevertheless, the anionic nature of Nafion exerted a certain diffusion barrier to nitrite, as revealed by the unusually elevated limits of the linear dynamic range and k m app . The irregularity of the composite membrane also contributed to slow down the rate of charge transfer throughout the Nafion polymer. The level of viologens incorporated within the Nafion membrane had a strong influence in the analytical parameters: as much mediator was present, lower was the sensitivity and wider was the linear range. For an optimized ratio enzyme/mediator the sensitivity was 445 ± 8 mA M −1 cm −2, within the linear range 75–800 μM; the lowest detected nitrite concentration was 60 μM. The operational stability of the biosensor and the influence of some possible interferences were evaluated.

Transmembrane Domains 4, 5, 7, 8, and 10 of the Human Reduced Folate Carrier Are Important Structural or Functional Components of the Transmembrane Channel for Folate Substrates

The human reduced folate carrier (hRFC) facilitates membrane transport of folates and antifolates. hRFC is characterized by 12 transmembrane domains (TMDs). To identify residues or domains involved in folate binding, we used substituted cysteine (Cys) accessibility methods (SCAM) with sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES). We previously showed that residues in TMD11 of hRFC were involved in substrate binding, whereas those in TMD12 were not (Hou, Z., Stapels, S. E., Haska, C. L., and Matherly, L. H. (2005) J. Biol. Chem. 280, 36206-36213). In this study, 232 Cys-substituted mutants spanning TMDs 1-10 and conserved stretches within the TMD6-7 (residues 204-217) and TMD10-11 connecting loop domains were transiently expressed in hRFC-null HeLa cells. All Cys-substituted mutants showed moderate to high levels of expression on Western blots, and only nine mutants including R133C, I134C, A135C, Y136C, S138C, G163C, Y281C, R373C, and S313C were inactive for methotrexate transport. MTSES did not inhibit transport by any of the mutants in TMDs 1, 3, 6, and 9 or for positions 204-217. Whereas most of the mutants in TMDs 2, 4, 5, 7, 8, and 10, and in the TMD10-11 connecting loop were insensitive to MTSES, this reagent inhibited methotrexate transport (25-75%) by 26 mutants in these TMDs. For 13 of these (Y126C, S137C, V160C, S168C, W274C, S278C, V284C, V288C, A311C, T314C, Y376C, Q377C, and V380C), inhibition was prevented by leucovorin, another hRFC substrate. Combined with our previous findings, these results implicate amino acids in TMDs 4, 5, 7, 8, 10, and 11, but not in TMDs 1, 2, 3, 6, 9, or 12, as important structural or functional components of the putative hydrophilic cavity for binding of anionic folate substrates.

Probing the Chemical Steps of Nitroalkane Oxidation Catalyzed by 2-Nitropropane Dioxygenase with Solvent Viscosity, pH, and Substrate Kinetic Isotope Effects

Among the enzymes that catalyze the oxidative denitrification of nitroalkanes to carbonyl compounds, 2-nitropropane dioxygenase is the only one known to effectively utilize both the neutral and anionic (nitronate) forms of the substrate. A recent study has established that the catalytic pathway is common to both types of substrates, except for the initial removal of a proton from the carbon of the neutral substrates [Francis, K., Russell, B., and Gadda, G. (2005) J. Biol. Chem. 280, 5195-5204]. In the present study, the mechanistic properties of the enzyme have been investigated with solvent viscosity, pH, and kinetic isotope effects. With nitroethane or ethylnitronate, the kcat/Km and kcat values were independent of solvent viscosity, consistent with the substrate and product binding to the enzyme in rapid equilibrium. The abstraction of the proton from the alpha carbon of neutral substrates was investigated by measuring the pH dependence of the D(kcat/KNE) value with 1,1-[2H2]-nitroethane. The formation of the enzyme-bound flavosemiquinone formed during catalysis was examined by determining the pH dependence of the kcat/Km values with ethylnitronate and nitroethane and the inhibition by m-nitrobenzoate. Finally, alpha-secondary kinetic isotope effects with 1-[2H]-ethylnitronate were used to propose a non-oxidative tautomerization pathway, in which the enzyme catalyzes the interconversion of nitroalkanes between their anionic and neutral forms. The data presented suggest that enzymatic turnover of 2-nitropropane dioxygenase with neutral substrates is limited by the cleavage of the substrate CH bond at low pH, whereas that with anionic substrates is limited by the non-oxidative tautomerization of ethylnitroante to nitroethane at high pH.

Modulation of the Catalytic Behavior of α-Chymotrypsin at Monolayer-Protected Nanoparticle Surfaces

Amino-acid-functionalized gold clusters modulate the catalytic behavior of alpha-chymotrypsin (ChT) toward cationic, neutral, and anionic substrates. Kinetic studies reveal that the substrate specificity (k(cat)/K(M)) of ChT-nanoparticle complexes increases by approximately 3-fold for the cationic substrate but decreases by 95% for the anionic substrate as compared with that of free ChT, providing enhanced substrate selectivity. Concurrently, the catalytic constants (k(cat)) of ChT show slight augmentation for the cationic substrate and significant attenuation for the anionic substrate in the presence of amino-acid-functionalized nanoparticles. The amino acid monolayer on the nanoparticle is proposed to control both the capture of substrate by the active site and release of product through electrostatic interactions, leading to the observed substrate specificities and catalytic constants.

Functional Role of the C Terminus of Human Organic Anion Transporter hOAT1

Functional Role of the C Terminus of Human Organic Anion Transporter hOAT1

RhEPO (recombinant human eosinophil peroxidase): expression in Pichia pastoris and biochemical characterization.

A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast alpha-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN-, Br- and Cl-. On the basis of the estimated K(m) and kcat values it is evident that the pseudohalide SCN- is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.

Control of the Oxygen Dependence of an Implantable Polymer/Enzyme Composite Biosensor for Glutamate

Biosensors for glutamate (Glu) were fabricated from Teflon-coated Pt wire (cylinders and disks), modified with the enzyme glutamate oxidase (GluOx) and electrosynthesized polymer PPD, poly(o-phenylenediamine). The polymer/enzyme layer was deposited in two configurations: enzyme before polymer (GluOx/PPD) and enzyme after polymer (PPD/GluOx). These four biosensor designs were characterized in terms of response time, limit of detection, Michaelis-Menten parameters for Glu (J max and K(M)(Glu)), sensitivity to Glu in the linear response region, and dependence on oxygen concentration, K(M)(O2). Analysis showed that the two polymer/enzyme configurations behaved similarly on both cylinders and disks. Although the two geometries showed different behaviors, these differences could be explained in terms of higher enzyme loading density on the disks; in many analyses, the four designs behaved like a single population with a range of GluOx loading. Enzyme loading was the key to controlling the K(M)(O2) values of these first generation biosensors. The counterintuitive, and beneficial, behavior that biosensors with higher GluOx loading displayed a lower oxygen dependence was explained in terms of the effects of enzyme loading on the affinity of GluOx for its anionic substrate. Some differences between the properties of surface immobilized GluOx and glucose oxidase are highlighted.

High Resolution 31 P NMR Field Cycling Probes Phospholipid Dynamics and Interactions with Phosphatidylinositol‐Specific Phospholipase C

31P relaxation times for phospholipids in multicomponent unilamellar vesicles can be measured from 0.004 to 11.7 T by a novel high resolution field cycling technique (Proc. Natl. Acad. Sci. U.S.A. (2004) 101, 17066). The field dependence of the relaxation rate is modeled by a sum of dipolar and chemical shift anisotropy terms from which we extract correlation times for motion on three distinct time-scales: (i) μ s τ v reflecting overall rotational diffusion of the vesicle and by lateral diffusion of the lipids; (ii) ns τ c reflecting faster motions that effectively lead to rotational diffusion perpendicular to the membrane surface; and (iii) sub-ns τ reflecting fast motions of the ester esterified to the phosphate group. The analysis provides a CSA order parameter, Sc, and an effective P-H distance. The ability to monitor relaxation rates at very low fields also yields a good estimate of the time-average angle between the membrane surface and the vector connecting the phosphorus to the glycerol C3 protons (the primary contributors to dipolar relaxation in most phospholipids). Relative differences in P-H orientations can be obtained for different phospholipid headgroups in the same vesicle if smaller unilamellar vesicles are examined. We have used 31P field cycling to monitor association of Bacillus thuringiensis PI-PLC, a peripheral membrane protein, with both PC and several anionic phospholipid bilayers. Micellar diheptanoyl-PC forms complexes with the enzyme that can also be characterized. Differences in phospholipid dynamics induced by the protein binding shed light on how zwitterionic PC molecules activate the enzyme toward its anionic substrate. Supported by NIH GM 60418.

Understanding the Activation of Listeria monocytogenes Phosphatidylinositol‐Specific Phospholipase C

The phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes (a monomer in solution) shows unusual kinetic properties compared to other well-studied phospholipases: (i) increased specific activity with decreasing protein concentration, (ii) activation of the phosphotransferase step by salts, and (iii) activation of both the interfacial phosphotransferase and water-soluble phosphodiesterase steps by zwitterionic and neutral amphiphiles. A variety of biophysical studies (fluorescence, NMR, monolayer, vesicle binding) of enzyme/lipid complexes coupled with kinetics have allowed us to propose a model that accounts for these features. The enzyme binds tightly to anionic surfaces and much more weakly to a zwitterionic interface. The tight binding can be reduced by adding KCl at concentrations that activate the enzyme. In the crystal structure of the enzyme, many basic residues are clustered on the bottom side of TIM-barrel far away from the opening to the active site. These cause the enzyme to adopt a non-productive orientation on negatively charged membranes that leads to a reversible clustering of anionic lipids and vesicle aggregation. An increased surface concentration of zwitterionic / neutral amphiphiles along with the salt disperses the anionic substrate, shields charges on the protein, and enhances productive encounters of the protein with substrate molecules. This model has been tested by examining the behavior of enzyme with citraconylated lysines and mutants of neutral surface residues at the rim of the active site. The unusual kinetic behavior of this PI-PLC also appears to contribute to the escape of L. monocytogenes from primary vacuoles during infection. Supported by NIH GM 60418.

The efficiency of immobilised glutamate oxidase decreases with surface enzyme loading: an electrostatic effect, and reversal by a polycation significantly enhances biosensor sensitivity

The apparent Michaelis constant, K(M), for glutamate oxidase (GluOx) immobilised on Pt electrodes increased systematically with enzyme loading. The effect was due, at least in part, to electrostatic repulsion between neighbouring oxidase molecules and the anionic substrate, glutamate (Glu). This understanding has allowed us to increase the Glu sensitivity of GluOx-based amperometric biosensors in the linear response region (100+/-11 nA cm(-2)microM(-1) at pH 7.4; SD, n=23) by incorporating a polycation (polyethyleneimine, PEI) to counterbalance the polyanionic protein. Differences in the behaviour of glucose biosensors of a similar configuration highlight a limitation of using glucose oxidase as a model enzyme in biosensor design.

Ionization of Zwitterionic Amine Substrates Bound to Monomeric Sarcosine Oxidase

Monomeric sarcosine oxidase (MSOX) binds the L-proline zwitterion (pKa = 10.6). The reactive substrate anion is generated by ionization of the ES complex (pKa = 8.0). Tyr317 was mutated to Phe to determine whether this step might involve proton transfer to an active site base. The mutation does not eliminate the ionizable group in the ES complex (pKa = 8.9) but does cause a 20-fold decrease in the maximum rate of the reductive half-reaction. Kinetically determined Kd values for the ES complex formed with L-proline agree with results obtained in spectral titrations with the wild-type or mutant enzyme. Unlike the wild-type enzyme, Kd values with the mutant enzyme are pH-dependent, suggesting that the mutation has perturbed the pKa of a group that affects the Kd. As compared with the wild-type enzyme, an increase in charge transfer band energy is observed for mutant enzyme complexes with substrate analogues while a 10-fold decrease in the charge transfer band extinction coefficient is found for the complex with the L-proline anion. The results eliminate Tyr317 as a possible acceptor of the proton released upon substrate ionization. Since previous studies rule out the only other nearby base, we conclude that L-proline is the ionizable group in the ES complex and that amino acids are activated for oxidation upon binding to MSOX by stabilization of the reactive substrate anion. Tyr317 may play a role in substrate activation and optimizing binding, as judged by the effects of its mutation on the observed pKa, reaction rates, and charge transfer bands.

Hexaazamacrocycle Containing Pyridine and Its Dicopper Complex as Receptors for Dicarboxylate Anions

AbstractThe host–guest binding interactions of the hexaazamacrocycle [26]py2N4, in its tetraprotonated form H4[26]py2N44+ as well as in its dicopper(II) complex [Cu2([26]py2N4)(H2O)4]4+, with dicarboxylate anions of different stereoelectronicrequirements, such as oxalate (ox2–), malonate (mal2–), succinate (suc2–), fumarate (fu2–) and maleate (ma2–), were evaluated. The association constants were determined using potentiometric methods in aqueous solution, at 298.0 K and 0.10 mol·dm–3 KCl. These values for the tetraprotonated ditopic receptor with the dicarboxylate anions revealed that the main species in solution corresponds to the formation of {H4[26]py2N4(A)}2+ (pH ≈ 4–9), A being the substrate anion. The values determined are not especially high, but the receptor exhibits selectivity for the malonate anion. The study of the cascade complexes revealed several species in solution, involving mononuclear and dinuclear complexes, mainly protonated and hydrolysed species, as well as the expected complexes [Cu2([26]py2N4)(A)(H2O)x]2+ or [Cu2([26]py2N4)(A)2(H2O)y]. Ox2– and mal2– form cascade complexes with only one anion, which will necessarily bridge the two copper atoms because of the symmetrical arrangement of the dinuclear complex. The two other studied anions, suc2– and ma2–, form species involving two substrate anions, although species with only one suc2– anion were also found. UV/Vis and EPR spectroscopy have shown that the dicopper complex can operate as a sensor to detect and quantitatively determine oxalate spectrophotometrically because of the red shift of the maximum of the visible band observed by addition of ox2– to an aqueous solution of the dinuclear copper complex. However the selectivity of [Cu2([26]py2N4)(H2O)4]4+ as a receptor for ox2– in the studied series is not sufficiently high to detect ox2– spectrophotometrically in the presence of the other anions. Molecular dynamics simulations indicated that the H4[26]py2N44+ receptor provides a large and flexible cavity to accommodate the studied anions. Molecular recognition is based in electrostatic interactions rather than in multiple hydrogen‐bonding interactions acting cooperatively. By contrast, the [Cu2([26]py2N4)]4+ receptor has a well‐shaped cavity with adequate size to uptake these anions as bridging ligands with formation of four Cu–O bonds. The ox2– anion is encapsulated within the cascade complex while the remaining anions are located above the N6 macrocyclic plane, suggesting a selective coordination behaviour of this receptor. In spite of our molecular simulation being carried out in gas phase, the modelling results are consistent with the solution studies. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Sex Differences in the mRNA, Protein, and Functional Expression of Organic Anion Transporter (Oat) 1, Oat3, and Organic Cation Transporter (Oct) 2 in Rabbit Renal Proximal Tubules

Sex differences in transport of the organic anion (OA) substrate p-aminohippurate (PAH) and the organic cation (OC) substrate tetraethylammonium (TEA) have been recognized for some time. In the rat kidney, androgens up-regulate and estrogens down-regulate PAH and TEA transport, which correlate with similar changes in mRNA and protein expression for the renal basolateral membrane transporters organic anion transporter (Oat) 1 and organic cation transporter (Oct) 2. However, these sex differences are not readily demonstrated in other species. The present study characterizes the kinetics of basolateral membrane PAH, estrone sulfate (ES), and TEA uptake in renal proximal tubule (RPT) suspensions isolated from female and male rabbits to compare functional expression of transport with mRNA and protein expression for rbOat1, rbOat3, and rbOct2. Although rbOat1-rbOat3 mRNA expression exhibited developmental differences, no sex differences in mRNA levels were observed. Oat1 and Oat3 protein expression in RPT suspensions also was similar between adult female and male rabbits. In contrast, rbOct1 and rbOct2 mRNA levels did not show developmental differences, but rbOct2 mRNA expression was greater in adult male than female rabbits. However, the sex difference in rbOct2 mRNA level did not translate to rbOct2 protein expression. Importantly, functional expression of Oat1, Oat3, and Oct2 transport as measured by kinetics (J(max) and K(t)) of PAH, ES, and TEA uptake was similar between adult male and female rabbits, and correlated with rbOat1, rbOat3, and rbOct2 protein expression. Thus, unlike rodents, rabbit renal OA and OC transport does not exhibit sex differences, pointing to the need for caution in extrapolating transport-related sex differences between species.