Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas foetus has been proven to be a target for potential anti-tritrichomonial chemotherapy. Using a structure-based approach, the base-binding region of the active site of this enzyme, which confers unique purine base specificity, was characterized using site-directed mutagenesis. Determining the roles of different active-site residues in purine specificity would form the basis for designing specific inhibitors toward the parasitic enzyme. A D163N mutant converts the HGXPRTase into a HGPRTase, which no longer recognizes xanthine as a substrate, whereas specificities toward guanine and hypoxanthine are unaffected. Apparently, the side-chain carboxyl of Asp163 forms a hydrogen bond through a water molecule with the C2-carbonyl of xanthine, which constitutes the critical force enabling the enzyme to recognize xanthine as a substrate. Mutations of Arg155, which orients and stacks the neighboring Tyr156 onto the bound purine base by forming a salt bridge between itself and Glu11, result in drastic increases in the Kms for GMP and XMP (but not IMP). This change leads to increased kcats for the forward reactions with guanine and xanthine as substrates without affecting the conversion of hypoxanthine to IMP. Thus, the apparent dislocation of Tyr156, resulted from mutations of Arg155, bring little effect on the hydrophobic interactions between Tyr156 and the purine ring. But the forces involved in recognizing the exocyclic C2-substituents of the purine ring, which involve the Tyr156 hydroxyl, Ile157 backbone carbonyl, and Asp163 side-chain carboxyl, may be weakened by the shifted conformation of the peptide backbone resulted from loss of the Glu11-Arg155 salt bridge. The conserved Lys134 was proven to be the primary determinant in conferring the specificity of the enzyme toward 6-oxopurines. By substituting the lysine residue for a serine, which can potentially hydrogen bond to either an amino or an oxo-group, we have successfully augmented the purine specificity of the enzyme. The K134S mutant recognizes adenine in addition to hypoxanthine, guanine, and xanthine as its substrates. Adenine and hypoxanthine are equivalent substrates for the mutant enzyme with similar Kms of 34.6 and 38.0 microM, respectively. The catalysis of an adenine phosphoribosyltransferase reaction by this mutant enzyme was further demonstrated by the competitive inhibition of AMP with an estimated Kis of 25.4 microM against alpha-D-5-phosphoribosyl-pyrophosphate (PRPP) in converting hypoxanthine to IMP. We have thus succeeded in using site-directed mutagenesis to convert T. foetusHGXPRTase into either a HGPRTase or a genuine AHGXPRTase.     

Energy characteristics of an ATP-hydrolase reaction catalyzed by solubilized Ca2+,Mg2+-ATPase from smooth muscle cell membrane

The effects of temperature, dielectric permeability and ionic strength on the activity of purified Ca2+, Mg(2+)-ATPase solubilized from myometrial sarcolemma have been studied under saturation of the enzyme with Ca2+, Mg2+ and ATP. The values of activation energy calculated from Arrhenius plots for both ATP hydrolase reactions catalysed by solubilized and reconstituted into azolectin liposomes Ca2+, Mg(2+)-ATPase and Mg2+, ATP-dependent Ca2+ transport by the reconstituted enzyme were 56.4 +/- 1.5, 68.0 +/- 5.1 and 63.1 +/- 2.9 kJ/mol, respectively. Analysis of experimental data in terms of the Laidler-Scatchard and Bronsted-Bjerrum theories revealed that the separation of the reaction products--the chelate MgADP complex--from the active site of the enzyme bearing one unity positive charge is the limiting step of the Ca2+, Mg(2+)-dependent enzymatic ATP-hydrolysis under conditions of substrate saturation. The values of the electrostatic components of the free energy, enthalpy and entropy of activation of the ATP hydrolase reaction were 46.6 +/- 0.3 kJ/mol, -(20.5 +/- 0.4) kJ/mol and -(214.2 +/- 4.3) J/(mol.degrees K), respectively. The nonelectrostatic component of activation enthalpy was 76.9 kJ/mol. The results obtained suggest that changes in polarity of the incubation medium markedly affect the activity of transport Ca2+, Mg(2+)-ATPase solubilized from smooth muscle cell plasma membranes and that the electrostatic interactions between the enzyme active site and specific reagents (MgADP, in particular) significantly contribute to the energetics of the ATP hydrolase reaction.     

Mathematical modelling of unsteady-state transport of metal ions through supported liquid membrane

In this study, a mathematical model for unsteady-state transport of metal ions from aqueous solution through supported liquid membrane containing carrier is presented. For a transport process using supported liquid membrane, the three resistances to consider are the membrane resistance and the aqueous boundary layer resistances at the side of feed and stripping phases. The transport of species in the feed, the membrane and the stripping phases is modelled by Fick's second law. Time-dependent boundary conditions for the feed, the membrane and the stripping phases are obtained by means of kinetics of chemical reaction in the feed–membrane and the membrane–stripping interface. Partial differential equations obtained for the feed, the membrane and the stripping phases are analytically solved by using Duhamel's Theorem. Concentration distribution within the aqueous film layers and membrane as a function of position and time was derived from models obtained. The obtained model is checked against experimental data corresponding to the transport of copper ions from aqueous solution through supported liquid membrane by LIX 984.     

Structure-based redesign of corepressor specificity of the Escherichia coli purine repressor by substitution of residue 190

Guanine or hypoxanthine, physiological corepressors of the Escherichia coli purine repressor (PurR), promote formation of the ternary PurR-corepressor-operator DNA complex that functions to repress pur operon gene expression. Structure-based predictions on the importance of Arg190 in determining 6-oxopurine specificity and corepressor binding affinity were tested by mutagenesis, analysis of in vivo function, and in vitro corepressor binding measurements. Replacements of Arg190 with Ala or Gln resulted in functional repressors in which binding of guanine and hypoxanthine was retained but specificity was relaxed to permit binding of adenine. X-ray structures were determined for ternary complexes of mutant repressors with purines (adenine, guanine, hypoxanthine, and 6-methylpurine) and operator DNA. These structures indicate that R190A binds guanine, hypoxanthine, and adenine with nearly equal, albeit reduced, affinity in large part because of a newly made compensatory hydrogen bond between the rotated hydroxyl side chain of Ser124 and the exocyclic 6 positions of the purines. Through direct and water-mediated contacts, the R190Q protein binds adenine with a nearly 75-fold higher affinity than the wild type repressor while maintaining wild type affinity for guanine and hypoxanthine. The results establish at the atomic level the basis for the critical role of Arg190 in the recognition of the exocyclic 6 position of its purine corepressors and the successful redesign of corepressor specificity.     

Dam methyltransferase from Escherichia coli: kinetic studies using modified DNA oligomers: nonmethylated substrates

Steady-state kinetics of the N6-adenine Dam methyltransferase have been measured using as substrates non-self-complementary tetradecanucleotide duplexes that contain the GATC target sequence. Modifications in the GATC target sequence of one or both of the strands included substitution of guanine by hypoxanthine, thymine by uracil or 5-ethyl-uracil and adenine by diamino-purine (2-amino-adenine). Thermodynamic parameters for the 14-mer duplexes were also determined. DNA methylation of duplexes containing single dl for dG substitution of the Dam recognition site was little perturbed compared with the canonical substrate. Replacement of dG residues by dl in both strands resulted in a decrease of the specificity constant. Substitution in both strands appears to be cumulative. Substitution of the methyl-accepting adenine residues by 2-amino-adenine resulted in surprisingly little perturbation. Dam methyltransferase is rather tolerant to different substitutions. The results show much less spread than those for the analogous hemimethylated substrates studied previously (Marzabal et al., 1995). The absence of the methylation marker appears to be deleterious to the specificity of the transition state of the active complex, while the binding of the DNA substrate to the enzyme appears to be mostly determined by the thermodynamic stability of the DNA duplex.     

Intramitochondrial localization and proposed metabolic significance of serine transhydroxymethylase

Serine transhydroxymethylase is a latent enzyme of intact rat liver mitochondria. The enzyme is neither solubilized by the selective removal of the outer membrane with digitonin, nor inactivated by concentrations of diazobenzenesulfonate that do not penetrate the inner membrane, but that do inhibit solubilized serine transhydroxymethylase. Swelling of mitochondria was studied in isoosmotic solutions of substrates under conditions that would define transport as neutral uniport, anion-hydroxyl exchange, anion-anion exchange, or electrophoretic. L-Serine and glycine appear to be rapidly taken up by a nonelectrophoretic uniport mechanism, while folate and tetrahydrofolate are not tranported. The results localize the enzyme in the matrix and indicate that the latent activity results from a lack of tetrahydrofolate transport across the inner membrane. Based on these results, the dual localization of serine transhydroxymethylase in the mitochondria and the cytosol is proposed to provide a one-carbon shuttle system to link one-carbon metabolism in the two-cellular compartments.     

Prolidase as a prodrug converting enzyme I. Synthesis of proline analogue of chlorambucil and its susceptibility to the action of prolidase

The feasibility to targeting prolidase as an antineoplastic prodrug-converting enzyme has been examined. The synthesis of proline analogue of chlorambucil (well known antineoplastic agent) conjugated through imido-bond (potential target for prolidase action) has been performed. It was found that the product of synthesis, N-[4-[4-(N,N-bis(2-chloroethyl)amino) phenyl]butyryl]-L-proline is insoluble in aqueous solutions but it may be solubilized in methanol. The methanol in 30% concentration reduces catalytic activity of prolidase to 40% of values found in aqueous solution, although it allows in such conditions the measurement of substrate susceptibility to the action of this enzyme. It has been presented that product of synthesis is weakly susceptible to the action of purified prolidase, comparable to the susceptibility of glycyl-L-hydroxyproline. Although insolubility of the proline analogue of chlorambucil in aqueous solutions limit its potential therapeutic value, the presented data suggest that prolidase may have a broader substrate specificity. It suggests that targeting of prolidase as a prodrug-converting enzyme may serve as a novel strategy in therapy of various diseases.     

Active secretion of hypoxanthine and xanthine by guinea pig jejunum in vitro

Isolated epithelium of guinea pig jejunum secretes hypoxanthine and xanthine by a transport process that is capable of uphill transport and dependent on metabolic energy supply. Unidirectional influx of hypoxanthine across both the luminal and the contraluminal cell membrane appears to be saturable; influx across the contraluminal membrane is inhibited by 2,4-dinitrophenol (DNP). Efflux across the luminal membrane is diminished by DNP; efflux across the contraluminal membrane is increased by DNP. This evidence suggests the existence of a mediated transport system both in the luminal and the contraluminal cell membrane. Additionally, intracellular metabolism of hypoxanthine seems to regulate transepithelial permeation: increased hypoxanthine salvage by the phosphoribosyltransferase reduces the rate of secretion. However, the incorporation of hypoxanthine into the nucleotides is limited when the hypoxanthine is added to the luminal side of the epithelium, and the permeation rate in the absorptive direction is not markedly influenced by the rate of hypoxanthine salvage. These findings are a further example of the functional orientation of the jejunal epithelial cells with respect to enzymic activity and transepithelial transport properties.     

Possible enzyme deficiency in bovine lymphosarcoma cells grown in vitro

A culture of bovine lymphosarcoma cells was unable to grow in HAT medium, suggesting that the cells were unable to use either exogenous hypoxanthine or exogenous thymidine. Further work showed that they were able to incorporate tritiated thymidine but not tritiated guanine. This indicated that failure to grow in HAT medium was due to the cells being deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase which is responsible for incorporation of the bases hypoxanthine and guanine. This finding may have application in the therapy of bovine lymphosarcoma.     

Concentration of europium(III) with supported liquid membrane containing a xylene solution of di-2-ethyihexyl phosphoric acid

The mechanism of europium transport through a supported liquid membrane is presented. The membrane consisted of a Teflon filter membrane with xylene solution of di-2-ethylhexyl phosphoric acid (HDEHP) as a mobile carrier held within the pores by capillary forces. Interposing the liquid membrane between two aqueous solutions of different pH, europium was transported and concentrated from the high pH solution to the low pH solution across the liquid membrane. The experiments were carried out to investigate the effects of the concentration of europium in the aqueous phase and HDEHP concentration in the membrane solution on the permeation rates of europium. The experimental results have been compared with a transport model for concentrating europium across the liquid membrane. The permeation rates of europium can be explained approximately by the diffusion process of the complex formed between europium ion and HDEHP at the membrane interface through the membrane in addition to the diffusion process of europium in the aqueous film adjacent to the membrane interface. Formerly Postdoctoral Fellow, Ames Laboratory, USDOE     

Effect of partial urinary outlet obstruction in the rabbit on the incorporation of adenine into adenine nucleotides in bladder smooth muscle

Bladder outlet obstruction induces marked morphological, functional, and metabolic changes within the urinary bladder. Recent studies indicate that there is a close correlation between the contractile dysfunction induced by partial outlet obstruction and a marked decrease in mitochondrial oxidative activity of the hypertrophied bladder tissue. The current study investigates the effect of partial outlet obstruction on adenine metabolism within the bladder tissue. After transport into the cell, adenine becomes available as a substrate for adenine phosphoribosyl transferase (APRT), the enzyme that catalyses the non-mitochondrial conversion of adenine into AMP. Subsequently, AMP is phosphorylated to ADP, the phosphate acceptor in mitochondrial oxidative phosphorylation. The results of these studies demonstrate that partial outlet obstruction induces a significant increase in 14C-adenine uptake into the urinary bladder smooth muscle which in turn provides substrate for APRT and results in an increase in 14C-AMP synthesis. In contrast, the rate of incorporation of adenine into ATP+ADP was similar for both control and obstructed tissue. The activity of APRT was not significantly different in control and obstructed tissue.     

Fc epsilon RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling

Detergent-resistant plasma membrane structures, such as caveolae, have been implicated in signalling, transport, and vesicle trafficking functions. Using sucrose gradient ultracentrifugation, we have isolated low-density, Triton X-100-insoluble membrane domains from RBL-2H3 mucosal mast cells that contain several markers common to caveolae, including a src-family tyrosine kinase, p53/56lyn. Aggregation of Fc epsilon RI, the high-affinity IgE receptor, causes a significant increase in the amount of p53/56lyn associated with these low-density membrane domains. Under our standard conditions for lysis, IgE-Fc epsilon RI fractionates with the majority of the solubilized proteins, whereas aggregated receptor complexes are found at a higher density in the gradient. Stimulated translocation of p53/56lyn is accompanied by increased tyrosine phosphorylation of several proteins in the low-density membrane domains as well as enhanced in vitro tyrosine kinase activity toward these proteins and an exogenous substrate. With a lower detergent-to-cell ratio during lysis, significant Fc epsilon RI remains associated with these membrane domains, consistent with the ability to coimmunoprecipitate tyrosine kinase activity with Fc epsilon RI under similar lysis conditions [Pribluda, V. S., Pribluda, C. & Metzger, H. (1994) Proc. Natl. Acad. Sci. USA 91, 11246-11250]. These results indicate that specialized membrane domains may be directly involved in the coupling of receptor aggregation to the activation of signaling events.     

Effect of some operating variables on cobalt(II) transport across a bulk liquid membrane

An experimental study is presented on facilitated transport of cobalt(II) cations through a bulk liquid membrane containing di(2-ethylhexyl)phosphoric acid (D2EHPA). The effects on the kinetics of cobalt(II) transport of stirring rate of the receiving phase and its acidity, mobile carrier (D2EHPA) concentration, emulsifier (Span 80) concentration, initial cobalt concentration, interfacial area, and membrane thickness have been investigated. It has been found that cobalt transport increased with both stirring of the receiving phase and its acidity, while the presence of emulsifier reduced this transport, particularly at the membrane/receiving phase interface. Moreover, the mean cobalt transport rate was practically independent of membrane/feed solution interfacial area (under experimental conditions), while this rate substantially increased with the initial cobalt concentration in aqueous feed solution.     

Isolation, sequence, and bioactivity of FMRFamide-related peptides from the locust ventral nerve cord

The electrochemical behaviour of the bioreductive redox active nitroimidazole drug metronidazole has been examined in the presence and absence of the DNA bases using three electrochemical techniques, all of which indicate the capacity for interaction between reduced products and DNA bases. The 4-electron metronidazole (RNO2) metronidazole-hydroxylamine (RNHOH) couple in an aqueous medium shows a positive shift in reduction potential upon addition of thymine, adenine and guanine, but a negative shift for cytosine. Interpretation of these results for an irreversible process is, however, inconclusive. In dimethylformamide/H2O the presence of DNA base on the one-electron addition product, the nitro radical anion, was examined by cyclic voltammetry. All except guanine resulted in interaction with the metronidazole nitro radical anion (RNO2-), as measured by the decrease in the return-to-forward peak current ratio, in the following order of increasing reactivity: cytosine, adenine and thymine (at a metronidazole: base ratio of 1:1). The increase in the stability of the radical anion by increasing the pH of the dimethylformamide/H2O medium resulted in a decreased reaction with thymine.     

Topology of allosteric regulation of lactose permease

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.     

Catalysis in human hypoxanthine-guanine phosphoribosyltransferase: Asp 137 acts as a general acid/base

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes the reversible formation of IMP and GMP from their respective bases hypoxanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phosphoribosyl-1-pyrophosphate (PRPP). The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine moiety, allowing enzymic catalysis to reduce the energy barrier associated with the reaction. The pH profile of kcat for IMP pyrophosphorolysis revealed an essential acidic group with pKa of 7.9 whereas those for IMP or GMP formation indicated involvement of essential basic groups. Based on the crystal structure of human HGPRTase, protonation/deprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have constructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis. D137N displayed an 18-fold decrease in kcat for nucleotide formation with Hx as substrate, a 275-fold decrease in kcat with Gua, and a 500-fold decrease in kcat for IMP pyrophosphorolysis. D137N also showed lower KD values for nucleotides and PRPP. The pH profiles of kcat for D137N were severely altered. In contrast to D137N, the kcat for K165Q was decreased only 2-fold in the forward reaction and was slightly increased in the reverse reaction. The Km and KD values showed that K165Q interacts with substrates more weakly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type. In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (3000-fold reduction) was affected much more than hypoxanthine (32-fold reduction). In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrates.     

The isolation and characterization of Saccharomyces cerevisiae mutants that constitutively express purine biosynthetic genes

In response to an external source of adenine, yeast cells repress the expression of purine biosynthesis pathway genes. To identify necessary components of this signalling mechanism, we have isolated mutants that are constitutively active for expression. These mutants were named bra (for bypass of repression by adenine). BRA7 is allelic to FCY2, the gene encoding the purine cytosine permease and BRA9 is ADE12, the gene encoding adenylosuccinate synthetase. BRA6 and BRA1 are new genes encoding, respectively, hypoxanthine guanine phosphoribosyl transferase and adenylosuccinate lyase. These results indicate that uptake and salvage of adenine are important steps in regulating expression of purine biosynthetic genes. We have also shown that two other salvage enzymes, adenine phosphoribosyl transferase and adenine deaminase, are involved in activating the pathway. Finally, using mutant strains affected in AMP kinase or ribonucleotide reductase activities, we have shown that AMP needs to be phosphorylated to ADP to exert its regulatory role while reduction of ADP into dADP by ribonucleotide reductase is not required for adenine repression. Together these data suggest that ADP or a derivative of ADP is the effector molecule in the signal transduction pathway.     

Toxoplasma gondii: characterization of a mutant resistant to 6-thioxanthine

6-Thioxanthine caused 50% inhibition of the growth of Toxoplasma gondii in human fibroblasts at a concentration of 5 micrograms/ml. A mutant induced by treatment with ethylnitrosourea (ThxR-1) was 20-fold more resistant than the wildtype. Wild-type parasites grown in Lesch-Nyhan fibroblasts efficiently incorporated hypoxanthine, guanine, and xanthine, but ThxR-1 incorporated each of these precursors less than 2% as well as the wildtype did. Soluble extracts of wild-type parasites had potent phosphoribosyltransferase activities for hypoxanthine, guanine, and xanthine, while extracts of ThxR-1 had barely detectable activity with any of these substrates. The basis for the resistance of ThxR-1 to 6-thioxanthine is, therefore, the lack of the enzyme hypoxanthine-guanine phosphoribosyltransferase. Thus, salvage pathways that employ this enzyme are not essential for the acquisition of purines, which the parasite must obtain from the host cell. Incubation in a medium containing mycophenolic acid and xanthine allowed the efficient recovery of wild-type T. gondii in the presence of many ThxR-1 parasites. Together with the use of 6-thioxanthine to detect resistant mutants in the presence of many wild-type parasites, this procedure provides a simple selection and back-selection for mutations that affect the hypoxanthine-guanine phosphoribosyltransferase gene of T. gondii.     

Rational design of novel antimicrobials: blocking purine salvage in a parasitic protozoan

All parasitic protozoa obtain purine nucleotides solely by salvaging purine bases and/or nucleosides from their host. This observation suggests that inhibiting purine salvage may be a good way of killing these organisms. To explore this idea, we attempted to block the purine salvage pathway of the parasitic protozoan Tritrichomonas foetus. T. foetus is a good organism to study because its purine salvage depends primarily on a single enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), and could provide a good model for rational drug design through specific enzyme inhibition. Guided by the crystal structure of T. foetus HGXPRTase, we used structure-based drug design to identify several non-purine compounds that inhibited this enzyme without any detectable effect on human HGPRTase. One of these compounds, 4-[N-(3, 4-dichlorophenyl)carbamoyl]phthalic anhydride (referred to as TF1), was selected for further characterization. TF1 was shown to be a competitive inhibitor of T. foetus HGXPRTase with respect to both guanine (in the forward reaction; Ki = 13 microM) and GMP (in the reverse reaction; Ki = 10 microM), but showed no effect on the homologous human enzyme at concentrations of up to 1 mM. TF1 inhibited the in vitro growth of T. foetus with an EC50 of approximately 40 microM. This inhibitory effect was associated with a decrease in the incorporation of exogenous guanine into nucleic acids, and could be reversed by supplementing the growth medium with excess exogenous hypoxanthine or guanine. Thus, rationally targeting an essential enzyme in a parasitic organism has yielded specific enzyme inhibitors capable of suppressing that parasite's growth.     

Okadaic acid and its interaction with sodium, potassium, magnesium and calcium ions: complex formation and transport across a liquid membrane

Okadaic acid, a macrocyclic polyether compound, was shown to mediate the transfer of Na+, K+, Mg2+ and Ca2+ ions from aqueous solution to an organic phase, with a preference for Na+ ions. A kinetic study of the transport of these ions across a liquid membrane showed that the Na+ ion was more rapidly transported than the other ions and that the Na+ ion flux was dependent on the okadaic acid concentration.