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Apparent Affinity (apparent + affinity)
Selected AbstractsTapping the nucleotide pool of the host: novel nucleotide carrier proteins of Protochlamydia amoebophilaMOLECULAR MICROBIOLOGY, Issue 6 2006Ilka Haferkamp Summary Protochlamydia amoebophila UWE25 is related to the Chlamydiaceae comprising major pathogens of humans, but thrives as obligate intracellular symbiont in the protozoan host Acanthamoeba sp. The genome of P. amoebophila encodes five paralogous carrier proteins belonging to the nucleotide transporter (NTT) family. Here we report on three P. amoebophila NTT isoforms, PamNTT2, PamNTT3 and PamNTT5, which possess several conserved amino acid residues known to be critical for nucleotide transport. We demonstrated that these carrier proteins are able to transport nucleotides, although substrate specificities and mode of transport differ in an unexpected manner and are unique among known NTTs. PamNTT2 is a counter exchange transporter exhibiting submillimolar apparent affinities for all four RNA nucleotides, PamNTT3 catalyses an unidirectional proton-coupled transport confined to UTP, whereas PamNTT5 mediates a proton-energized GTP and ATP import. All NTT genes of P. amoebophila are transcribed during intracellular multiplication in acanthamoebae. The biochemical characterization of all five NTT proteins from P. amoebophila in this and previous studies uncovered that these metabolically impaired bacteria are intimately connected with their host cell's metabolism in a surprisingly complex manner. [source] A dimeric 5- enol -pyruvyl-shikimate-3-phosphate synthase from the cyanobacterium Spirulina platensisNEW PHYTOLOGIST, Issue 2 2001Giuseppe Forlani Summary ,,Isolation and biochemical characterization is reported here of 5- enol -pyruvyl-shikimate-3-phosphate (EPSP) synthase, the enzyme that catalyses the sixth step in the common prechorismate pathway of aromatic amino acid biosynthesis and the target of the widely used herbicide glyphosate, from the cyanobacterium Spirulina platensis. ,,Homogeneous enzyme preparations were obtained by ammonium sulphate fractionation, anion-exchange and substrate-elution chromatography, and chromatofocusing. Protein characterization was carried out by conventional kinetic analysis, PAGE and gel permeation. ,,A 2800-fold purification was achieved, with a recovery of 20% of initial activity. Unusually low apparent affinities for both substrates, phosphoenolpyruvate and shikimate-3-phosphate, did not correspond to decreased glyphosate sensitivity. During SDS-PAGE, the protein migrated as a single band corresponding to a molecular mass of c. 49 kDa. The behaviour of the protein upon gel permeation chromatography under nondenaturing conditions was, however, consistent with a mass of c. 91 kDa. ,,The native enzyme appears to be homodimeric, a remarkable feature that has not been previously reported for EPSP synthases from either cyanobacteria or higher plants. The presence of mono- and dimeric EPSP synthases could represent an important tool for cyanobacterial classification. [source] Expression and regulation of interleukin-10 and interleukin-10 receptor in rat astroglial and microglial cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2002Annemarie Ledeboer Abstract Activated glial cells crucially contribute to brain inflammatory responses. Interleukin-10 (IL-10) is an important modulator of glial cell responses in the brain. In the present study we describe the expression of IL-10 and the IL-10 receptor (IL-10R1) in primary cocultures of rat microglial and astroglial cells. Using quantitative RT-PCR and ELISA, we show that IL-10 mRNA expression and subsequent IL-10 secretion is time-dependently induced by lipopolysaccharide (LPS). IL-10R1, however, is constitutively expressed in glial cell cocultures, as shown by RT-PCR and immunocytochemistry. Radioligand binding studies using 125I-IL-10 reveal that rat glial cells express a single binding site with an apparent affinity of approximately 600 pm for human IL-10. Observations in enriched cultures of either microglial or astroglial cells indicate that both cell types express IL-10 mRNA and are capable of secreting IL-10. Both cell types also express IL-10R1 mRNA and protein. However, in glial cell cocultures immunoreactive IL-10R1 protein is predominantly observed in astrocytes, suggesting that microglial expression of IL-10R1 in cocultures is suppressed by astrocytes. In addition, exogenous IL-10 is highly potent in down-regulating LPS-induced IL-1, and IL-10 mRNA, and, at a higher dose, IL-10R1 mRNA in untreated and LPS-treated cultures, suggesting that IL-10 autoregulates its expression and inhibits that of IL-1, at the transcriptional level. Together the findings support the concept that IL-10, produced by activated microglial and astroglial cells, modulates glia-mediated inflammatory responses through high-affinity IL-10 receptors via paracrine and autocrine interactions. [source] The CO2 -concentrating mechanism in the bloom-forming cyanobacterium Microcystis aeruginosa (Cyanophyceae) and effects of UVB radiation on its operation,JOURNAL OF PHYCOLOGY, Issue 5 2007Yanfang Song The bloom-forming cyanobacterium Microcystis aeruginosa (Kütz.) Kütz. 854 was cultured with 1.05 W · m,2 ultraviolet-B radiation (UVBR) for 3 h every day, and the CO2 -concentrating mechanism (CCM) within this species as well as effects of UVBR on its operation were investigated. Microcystis aeruginosa 854 possessed at least three inorganic carbon transport systems and could utilize external HCO3, and CO2 for its photosynthesis. The maximum photosynthetic rate was approximately the same, but the apparent affinity for dissolved inorganic carbon was significantly decreased from 74.7 ,mol · L,1 in the control to 34.7 ,mol · L,1 in UVBR-treated cells. At 150 ,mol · L,1 KHCO3 and pH 8.0, Na+ -dependent HCO3, transport contributed 43.4%,40.2% to the photosynthesis in the control and 34.5%,31.9% in UVBR-treated cells. However, the contribution of Na+ -independent HCO3, transport increased from 8.7% in the control to 18.3% in UVBR-treated cells. The contribution of CO2 -uptake systems showed little difference: 47.9%,51.0% in the control and 49.8%,47.2% in UVBR-treated cells. Thus, the rate of total inorganic carbon uptake was only marginally affected, although UVBR had a differential effect on various inorganic carbon transporters. However, the number of carboxysomes in UVBR-treated cells was significantly decreased compared to that in the control. [source] Isoxsuprine hydrochloride in the horse: a reviewJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2002R. S. ERKERT Isoxsuprine hydrochloride has been suggested for use in horses for treatment of navicular syndrome and laminitis. The drug has been shown to be a , -adrenoreceptor antagonist with , -adrenoreceptor agonistic properties, with both characteristics contributing to vasodilation and uterine relaxation. In addition, the drug is capable of decreasing blood viscosity and platelet aggregation. Studies have shown i.v. isoxsuprine to have a plasma half-life of <3 h with a large apparent volume of distribution. Cardiovascular effects resolve rapidly following i.v. administration, but are absent with oral dosing. Oral bioavailability is 2.2% with a high first pass effect. Isoxsuprine has an apparent affinity for melanin that may contribute to extended renal excretion. Clinical trials appear to support the use of isoxsuprine for treatment of navicular disease. However, poor bioavailability, lack of cardiovascular effects following oral administration, superficial support in clinical trials, and new evidence regarding the pathogenesis of navicular syndrome indicate that the use of isoxsuprine for treatment of navicular syndrome or laminitis is questionable at best. [source] Subunit-specific desensitization of heteromeric kainate receptorsTHE JOURNAL OF PHYSIOLOGY, Issue 4 2010David D. Mott Kainate receptor subunits can form functional channels as homomers of GluK1, GluK2 or GluK3, or as heteromeric combinations with each other or incorporating GluK4 or GluK5 subunits. However, GluK4 and GluK5 cannot form functional channels by themselves. Incorporation of GluK4 or GluK5 into a heteromeric complex increases glutamate apparent affinity and also enables receptor activation by the agonist AMPA. Utilizing two-electrode voltage clamp of Xenopus oocytes injected with cRNA encoding kainate receptor subunits, we have observed that heteromeric channels composed of GluK2/GluK4 and GluK2/GluK5 have steady state concentration,response curves that were bell-shaped in response to either glutamate or AMPA. By contrast, homomeric GluK2 channels exhibited a monophasic steady state concentration,response curve that simply plateaued at high glutamate concentrations. By fitting several specific Markov models to GluK2/GluK4 heteromeric and GluK2 homomeric concentration,response data, we have determined that: (a) two strikingly different agonist binding affinities exist; (b) the high-affinity binding site leads to channel opening; and (c) the low-affinity agonist binding site leads to strong desensitization after agonist binding. Model parameters also approximate the onset and recovery kinetics of desensitization observed for macroscopic currents measured from HEK-293 cells expressing GluK2 and GluK4 subunits. The GluK2(E738D) mutation lowers the steady state apparent affinity for glutamate by 9000-fold in comparison to GluK2 homomeric wildtype receptors. When this mutant subunit was expressed with GluK4, the rising phase of the glutamate steady state concentration,response curve overlapped with the wildtype curve, whereas the declining phase was right-shifted toward lower affinity. Taken together, these data are consistent with a scheme whereby high-affinity agonist binding to a non-desensitizing GluK4 subunit opens the heteromeric channel, whereas low-affinity agonist binding to GluK2 desensitizes the whole channel complex. [source] Properties of glycine receptors underlying synaptic currents in presynaptic axon terminals of rod bipolar cells in the rat retinaTHE JOURNAL OF PHYSIOLOGY, Issue 15 2009Svein Harald Mørkve The excitability of presynaptic terminals can be controlled by synaptic input that directly targets the terminals. Retinal rod bipolar axon terminals receive presynaptic input from different types of amacrine cells, some of which are glycinergic. Here, we have performed patch-clamp recordings from rod bipolar axon terminals in rat retinal slices. We used whole-cell recordings to study glycinergic inhibitory postsynaptic currents (IPSCs) under conditions of adequate local voltage clamp and outside-out patch recordings to study biophysical and pharmacological properties of the glycine receptors with ultrafast application. Glycinergic IPSCs, recorded in both intact cells and isolated terminals, were strychnine sensitive and displayed fast kinetics with a double-exponential decay. Ultrafast application of brief (,1 ms) pulses of glycine (3 mm) to patches evoked responses with fast, double-exponential deactivation kinetics, no evidence of desensitization in double-pulse experiments, relatively low apparent affinity (EC50,100 ,m), and high maximum open probability (,0.9). Longer pulses evoked slow, double-exponential desensitization and double-pulse experiments indicated slow, double-exponential recovery from desensitization. Non-stationary noise analysis of IPSCs and patch responses yielded single-channel conductances of ,41 pS and ,64 pS, respectively. Directly observed single-channel gating occurred at ,40,50 pS and ,80,90 pS in both types of responses, suggesting a mixture of heteromeric and homomeric receptors. Synaptic release of glycine leads to transient receptor activation, with about eight receptors available to bind transmitter after release of a single vesicle. With a low intracellular chloride concentration, this leads to either hyperpolarizing or shunting inhibition that will counteract passive and regenerative depolarization and depolarization-evoked transmitter release. [source] Activation of olfactory-type cyclic nucleotide-gated channels is highly cooperativeTHE JOURNAL OF PHYSIOLOGY, Issue 1 2005Vasilica Nache Cyclic nucleotide-gated (CNG) ion channels play a key role in the sensory transduction of vision and olfaction. The channels are opened by the binding of cyclic nucleotides. Native olfactory CNG channels are heterotetramers of CNGA2, CNGA4, and CNGB1b subunits. Upon heterologous expression, only CNGA2 subunits can form functional homotetrameric channels. It is presently not known how the binding of the ligands to the four subunits is translated to channel opening. We studied activation of olfactory CNG channels by photolysis-induced jumps of cGMP or cAMP, two cyclic nucleotides with markedly different apparent affinity. It is shown that at equal degree of activation, the activation time course of homotetrameric channels is similar with cGMP and cAMP and it is also similar in homo- and heterotetrameric channels with the same cyclic nucleotide. Kinetic models were globally fitted to activation time courses of homotetrameric channels. While all models containing equivalent binding sites failed, a model containing three binding sites with a ligand affinity high,low,high described the data adequately. Only the second binding step switches from a very low to a very high open probability. We propose a unique gating mechanism for homotetrameric and heterotetrameric channels that involves only three highly cooperative binding steps. [source] | ||||||||||