Low-affinity Process Research Articles

Breaking the Concentration Limit in Fluorescence Fluctuation Spectroscopy with Camera-Based Detection.

Fluorescence correlation spectroscopy (FCS) is an extremely versatile tool that has been widely used to measure chemical reaction rates, protein binding, nanoparticle-protein interactions, and biomolecular dynamics in vitro and in vivo. As an inherently micro-sized approach, FCS is compatible with high-throughput screening applications, as demanded for drug design, but typically limited to nanomolar concentrations, which restricts possible applications. Here, we show how massively parallel camera-based detection with side illumination can extend the usable concentration range of FCS more than 100-fold to measure low affinity processes. Our line illumination (LIM) approach is robust, fast (1 s acquisition times), and does not require any reference measurements to characterize the observation volume size.

Unified comprehension of the kinetics of isothermal ceramic processes under field-free and resonant wave-field conditions

Abstract This paper proposes a unified comprehension of the isothermal ceramic process kinetics stemming from mesoscopic irreversible thermodynamics. Accordingly, a unified process kinetic equation (UPKE) is derived, which predicts that the global isothermal process rate of any ceramic process is, in general, nonlinearly related both to its activation energy and its affinity. Nevertheless, for a low-affinity ceramic process conducted either in a field-free or resonant wave-field condition, its global isothermal rate, according to the proposed UPKE, is approximately linearly related to its affinity in the spirit of Fick’s diffusion law. Therefore, the rate enhancement of a low-affinity process occurring in any resonant wave-field may be caused either by a reduction in activation energy, as in microwave-enhanced sintering, or by activation energy reduction along with an affinity augmentation, as in microwave-assisted glass-crystallization. Conversely, for a high-affinity ceramic process, e.g., a chemical reaction, the ‘degenerate’ UPKE predicts that its kinetics is exclusively dictated by the activation energy in the spirit of Arrhenius’s rate law. Hence, isothermal rate enhancements of chemical reactions in ceramic processing under resonant wave-field conditions are believed to predominantly result from a field-induced reduction in activation energy.

Role of ABC and Solute Carrier Transporters in the Placental Transport of Lamivudine.

Lamivudine is one of the antiretroviral drugs of choice for the prevention of mother-to-child transmission (MTCT) in HIV-positive women. In this study, we investigated the relevance of drug efflux transporters P-glycoprotein (P-gp) (MDR1 [ABCB1]), BCRP (ABCG2), MRP2 (ABCC2), and MATE1 (SLC47A1) for the transmembrane transport and transplacental transfer of lamivudine. We employed in vitro accumulation and transport experiments on MDCK cells overexpressing drug efflux transporters, in situ-perfused rat term placenta, and vesicular uptake in microvillous plasma membrane (MVM) vesicles isolated from human term placenta. MATE1 significantly accelerated lamivudine transport in MATE1-expressing MDCK cells, whereas no transporter-driven efflux of lamivudine was observed in MDCK-MDR1, MDCK-MRP2, and MDCK-BCRP monolayers. MATE1-mediated efflux of lamivudine appeared to be a low-affinity process (apparent Km of 4.21 mM and Vmax of 5.18 nmol/mg protein/min in MDCK-MATE1 cells). Consistent with in vitro transport studies, the transplacental clearance of lamivudine was not affected by P-gp, BCRP, or MRP2. However, lamivudine transfer across dually perfused rat placenta and the uptake of lamivudine into human placental MVM vesicles revealed pH dependency, indicating possible involvement of MATE1 in the fetal-to-maternal efflux of the drug. To conclude, placental transport of lamivudine does not seem to be affected by P-gp, MRP2, or BCRP, but a pH-dependent mechanism mediates transport of lamivudine in the fetal-to-maternal direction. We suggest that MATE1 might be, at least partly, responsible for this transport.

Excitatory Amino Acid Transporter 1-Mediated L-Glutamate Transport at the Inner Blood-Retinal Barrier: Possible Role in L-Glutamate Elimination from the Retina.

The purpose of this study was to elucidate the transport mechanism(s) of L-glutamate (L-Glu), a neuroexcitatory neurotransmitter, in the inner blood-retinal barrier (BRB). The L-Glu transport was evaluated by an in vitro uptake study with a conditionally-immortalized rat retinal capillary endothelial cell line, TR-iBRB2 cells. L-Glu uptake by TR-iBRB2 exhibited time- and concentration-dependence, and was composed of high- and low-affinity processes with Michaelis-Menten constants (Km) of 19.3 µM and 275 µM, respectively. Under Na(+)-free conditions, L-Glu uptake by TR-iBRB2 involved one-saturable kinetics with a Km of 190 µM, which is similar to that of the low-affinity process of L-Glu uptake under normal conditions. Moreover, substrates/inhibitors of system Xc(-), which is involved in blood-to-retina transport of compounds across the inner BRB, strongly inhibited the L-Glu uptake under Na(+)-free conditions, suggesting that Na(+)-independent low-affinity L-Glu transport at the inner BRB is carried out by system Xc(-). Regarding the Na(+)-dependent high affinity process of L-Glu transport at the inner BRB, L-Glu uptake by TR-iBRB2 under normal conditions was significantly inhibited by substrates/inhibitors of excitatory amino acid transporter (EAAT) 1-5, but not alanine-serine-cysteine transporters. Reverse-transcription polymerase chain reaction (RT-PCR) analysis and immunoblot analysis demonstrated that mRNA and protein of EAAT1 are expressed in TR-iBRB2 cells, whereas mRNAs and/or proteins of EAAT2-5 are not. Immunohistochemical analysis revealed that EAAT1 protein is localized on the abluminal membrane of the retinal capillaries. In conclusion, EAAT1 most likely mediates Na(+)-dependent high-affinity L-Glu transport at the inner BRB and appears to take part in L-Glu elimination from the retina across the inner BRB.

A novel sucrose/H+ symport system and an intracellular sucrase in Leishmania donovani

The flagellated form of pathogenic parasitic protozoa Leishmania, resides in the alimentary tract of its sandfly vector, where sucrose serves as a major nutrient source. In this study we report the presence of a sucrose transport system in Leishmania donovani promastigotes. The kinetics of sucrose uptake in promastigotes are biphasic in nature with both high affinity Km (Km of ∼75μM) and low affinity Km (Km∼1.38mM) components. By contrast the virulent amastigotes take up sucrose via a low affinity process with a Km of 2.5mM. The transport of sucrose into promastigotes leads to rapid intracellular acidification, as indicated by changes in the fluorescence of the pH indicator 2′,7′-bis-(2-carboxyethyl)-5-(6) Carboxyfluorescein (BCECF). In experiments with right side-out plasma membrane vesicles derived from L. donovani promastigotes, an artificial pH gradient was able to drive the active accumulation of sucrose. These data are consistent with the operation of a H+-sucrose symporter. The symporter was shown to be independent of Na+ and to be insensitive to cytochalasin B, to the flavonoid phloretin and to the Na+/K+ ATPase inhibitor ouabain. However, the protonophore carbonylcyanide P- (trifluromethoxy) phenylhydrazone (FCCP) and a number of thiol reagents caused significant inhibition of sucrose uptake. Evidence was also obtained for the presence of a stable intracellular pool of the sucrose splitting enzyme, sucrase, in promastigote stage parasites. The results are consistent with the hypothesis that L. donovani promastigotes take up sucrose via a novel H+–sucrose symport system and that, on entering the cell, the sucrose is hydrolysed to its component monosaccharides by an intracellular sucrase, thereby providing an energy source for the parasites.

Glycine and l-arginine transport in cultured Müller glial cells (TR-MUL)

We have reported previously that creatine is biosynthesized from glycine and l-arginine in Müller cells, but the mechanism responsible for glycine and l-arginine uptake by Müller cells remains elusive. To explore this issue, [ 14C]glycine and [ 3H] l-arginine uptake by Müller cells was characterized using a conditionally immortalized rat Müller cell line (TR-MUL5 cells). [ 14C]Glycine uptake by TR-MUL5 cells was Na +- and Cl −-dependent, and a saturable process with Michaelis–Menten constants of 48.6 μM and 4.53 mM, and inhibited by glycine transporter 1 (GlyT1) and system A substrates. Under Cl −-free conditions, the high-affinity process was abolished. RT-PCR analysis demonstrated that GlyT1 and system A (encoding ATA1 and ATA2) mRNA are expressed in TR-MUL5 cells. These uptake studies suggest that GlyT1 and system A are involved in [ 14C]glycine uptake for the high- and low-affinity processes, respectively, in TR-MUL5 cells. [ 3H] l-Arginine uptake by TR-MUL5 cells was primarily an Na +-independent and saturable process with Michaelis–Menten constants of 15.0 and 403 μM. This process was inhibited by substrates of cationic amino acid transporter (CAT)s, such as l-arginine, l-lysine, and l-ornithine. The expression of CAT1 protein was detected in TR-MUL5 cells. These results suggest that CAT1 is at least involved in [ 3H] l-arginine uptake by TR-MUL5 cells. In conclusion, GlyT1 and CAT1 most likely mediate glycine and l-arginine uptake, respectively, by Müller cells and are expected to play an important role in supplying precursors for creatine biosynthesis in Müller cells.

Site-Specific Interactions of Cu(II) with α and β-Synuclein: Bridging the Molecular Gap between Metal Binding and Aggregation

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. Protein-metal interactions play a critical role in AS aggregation and might represent the link between the pathological processes of protein aggregation and oxidative damage. Our previous studies established a hierarchy in AS-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. In this work, we have addressed unresolved structural details related to the binding specificity of Cu(II) through the design of site-directed and domain-truncated mutants of AS and by the characterization of the metal-binding features of its natural homologue beta-synuclein (BS). The structural properties of the Cu(II) complexes were determined by the combined application of nuclear magnetic resonance, electron paramagnetic resonance, UV-vis, circular dichroism spectroscopy, and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Two independent, noninteracting copper-binding sites with significantly different affinities for the metal ion were detected in the N-terminal regions of AS and BS. MALDI MS provided unique evidence for the direct involvement of Met1 as the primary anchoring residue for Cu(II) in both proteins. Comparative spectroscopic analysis of the two proteins allowed us to deconvolute the Cu(II) binding modes and unequivocally assign the higher-affinity site to the N-terminal amino group of Met1 and the lower-affinity site to the imidazol ring of the sole His residue. Through the use of competitive chelators, the affinity of the first equivalent of bound Cu(II) was accurately determined to be in the submicromolar range for both AS and BS. Our results prove that Cu(II) binding in the C-terminal region of synucleins represents a nonspecific, very low affinity process. These new insights into the bioinorganic chemistry of PD are central to an understanding of the role of Cu(II) in the fibrillization process of AS and have implications for the molecular mechanism by which BS might inhibit AS amyloid assembly.

Purine nucleobase transport in the intraerythrocytic malaria parasite

Hypoxanthine, a nucleobase, serves as the major source of the essential purine group for the intraerythrocytic malaria parasite. In this study we have measured the uptake of hypoxanthine, and that of the related purine nucleobase adenine, by mature blood-stage Plasmodium falciparum parasites isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasitophorous vacuole membranes. The uptake of both [ 3H]hypoxanthine and [ 3H]adenine was comprised of at least two components; in each case there was a rapid equilibration of the radiolabel between the intra- and extracellular solutions via a low-affinity transport mechanism, and an accumulation of radiolabel (such that the estimated intracellular concentration exceeded the extracellular concentration) via a higher-affinity process. The uptake of [ 3H]adenine was studied in more detail. The rapid, low-affinity equilibration of [ 3H]adenine between the intra-and extracellular solution was independent of the energy status of the parasite whereas the higher-affinity accumulation of the radiolabel was ATP-dependent. A kinetic analysis of adenine uptake revealed that the low-affinity (equilibrative) process had a K m of ∼1.2 mM, similar to the value of 0.82 mM estimated here (using the Xenopus laevis oocyte expression system) for the K m for the transport of adenine by PfENT1, a parasite-encoded member of the ‘equilibrative nucleoside/nucleobase transporter’ family. The results indicate that nucleobases enter the intraerythrocytic parasite via a rapid, equilibrative process that has kinetic characteristics similar to those of PfENT1.

Quantitative Analysis of Binding of Single-Stranded DNA by Escherichia coli DnaB Helicase and the DnaB·DnaC Complex

DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.

Modeling the concentration-dependent permeation modes of the KcsA potassium ion channel.

The potassium channel from Streptomyces lividans (KcsA) is an integral membrane protein with sequence similarity to all known potassium channels, particularly in the selectivity filter region. A recently proposed model for ion channels containing either n or (n-1) single-file ions in their selectivity filters [P. H. Nelson, J. Chem. Phys. 177, 11396 (2002)] is applied to published KcsA channel K+ permeation data that exhibit a high-affinity process at low concentrations and a low-affinity process at high concentrations [M. LeMasurier et al., J. Gen. Physiol. 118, 303 (2001)]. The kinetic model is shown to provide a reasonable first-order explanation for both the high- and low-concentration permeation modes observed experimentally. The low-concentration mode ([K+]<200 mM) has a 200-mV dissociation constant of 56 mM and a conductance of 88 pS. The high-concentration mode ([K+]>200 mM) has a 200-mV dissociation constant of 1100 mM and a conductance of 500 pS. Based on the permeation model, and x-ray analysis [J. H. Morais-Cabral et al., Nature (London) 414, 37 (2001)], it is suggested that the experimentally observed K+ permeation modes correspond to an n=3 mechanism at high concentrations and an n=2 mechanism at low concentrations. The ratio of the electrical dissociation distances for the high- and low-concentration modes is 3:2, also consistent with the proposed n=3 and n=2 modes. Model predictions for K+ channels that exhibit asymmetric current-voltage (I-V) curves are presented, and further validation of the kinetic model via molecular simulation and experiment is discussed. The qualitatively distinct I-V characteristics exhibited experimentally by Tl+, NH+4, and Rb+ ions at 100 mM concentration can also be explained using the model, but more extensive experimental tests are required for quantitative validation of the model predictions.

Recovering antibody secretion using a hapten ligand as a chemical chaperone.

Engineered antibodies have come to the forefront as research reagents and clinical therapeutics. However, reduced stability or expression levels pose a major problem with many engineered antibodies. As a model for understanding functional consequences of variable region mutation, we have studied the assembly and trafficking of anti-phenylphosphocholine antibodies. Previously, we identified severe secretion defects because of mutations in the heavy chain second complementarity determining region, which is involved in antigen binding. Here we demonstrate that immunoglobulin secretion is increased up to 27-fold by incubating stably transfected PCG1-1 cells with cognate hapten p-nitrophenylphosphocholine. Secretion was unaffected by nonbinding analogs. Radiotracer and metabolic labeling experiments demonstrated specific cellular uptake of p-nitrophenylphosphocholine and increased intracellular heavy and light chain assembly. Brefeldin A inhibited hapten-mediated immunoglobulin secretion but not assembly, indicating that assembly occurs early within the biosynthetic pathway. Recovery of secretion correlated with antigen binding capacity, suggesting that the rescue mechanism involves stabilization of heavy and light chain variable domains. This model system provides the first demonstration that cognate ligands can increase intracellular assembly of functional anti-hapten antibody within mammalian cells and suggests that small molecules of appropriate specificity and affinity acting as chemical chaperones may find application for increasing or regulating immunoglobulin expression.

KcsA: it's a potassium channel.

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) >> Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) >> Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.

Kinetic profile of overall elimination of 5-methyltetrahydropteroylglutamate in rats.

The in vivo biliary and urinary excretion kinetics of 5-methyltetrahydropteroylglutamate (5-CH3-H4PteGlu) were studied in rats. During infusion at various rates (48-965 nmol. h-1. kg-1), the total body clearance (CLtotal) of 5-CH3-H4PteGlu could be attributed almost entirely to the sum of the biliary and urinary (CLurine,p) excretion clearances. After a 4-h infusion at the highest rate, the 5-CH3-H4PteGlu in the liver was 10 times higher than the endogenous level, whereas its polyglutamate form did not increase, suggesting that most of the infused 5-CH3-H4PteGlu is not incorporated in the polyglutamate pool but is eliminated by excretion. The parallel increase in CLtotal and CLurine,p with the increase in infusion rate might result from saturation of reabsorption at the renal proximal tubules, since the urinary excretion clearance, defined with respect to the kidney concentration, also increased while the biliary excretion clearance, defined with respect to the liver concentration, remained almost constant. We conclude that the hepatobiliary excretion is a relatively low-affinity process with a constant clearance, whereas the renal tubular reabsorption is saturated at higher plasma 5-CH3-H4PteGlu concentration ( approximately 0.5 microM). Urinary excretion becomes the predominant elimination route for any excess 5-CH3-H4PteGlu in the body.

Measurement of the kinetics of protein uptake by proximal tubular cells using an optical biosensor

The affinity and specificity of protein reabsorption by proximal tubular cells have been investigated using techniques for monitoring endocytosis, demonstrating a high capacity but low affinity process. It is not known whether uptake is through binding to a single binding site/receptor with differing affinities, or if there are several classes of binding sites receptors, each specific for differing proteins or groups, such as, high or low molecular weight proteins. We have developed a novel technique for analyzing the kinetics of protein binding to tubular cells using a optical biosensor system. We have studied the binding of cultured LLCPK cells to albumin and RBP immobilized onto the sensor. By adding increasing concentrations of competing proteins [varying in molecular weight from 66,000 to 11,800 D and pI from 4.6 to 9.2 as represented by albumin, alpha1-microglobulin (alpha1M), retinol binding protein (RBP), cystatin C and beta2-microglobulin (beta2m)], specific and inhibitable cell binding was demonstrated. Equilibrium constants, KA, could be calculated from the reciprocal of the protein concentration causing 50% inhibition in binding rate. These were: albumin = 8.0 x 10(4) M(-1), alpha1M = 2.0 x 10(5) M(-1), RBP = 2.7 x 10(4) M(-1), cystatin C = 2.0 x 10(4) M(-1), beta2m = 4.2 x 10(3) M(-1). There were no significant differences between the measured KA's whether RBP or albumin were immobilized on the surface. All the proteins gave similar shaped inhibition profiles, suggesting that there is one binding site/receptor for all proteins studied, regardless of molecular weight or charge, but there are differing affinities for each protein.

Na+-DependentL-Arginine Transport in the Pigmented Rabbit Conjunctiva

The objective of this study was to characterize Na+-coupledL-arginine (L-Arg) transport in the pigmented rabbit conjunctiva. The excised pigmented rabbit conjunctiva was mounted in the modified Ussing chamber for measurement of short-circuit current (Isc),3H-L-arginine (3H-L-Arg) flux, and22Na flux.L-Arg when added to the mucosal side led to 0.32–2.65 μA cm−2increases in the Isc at 37°C, but not at 4°C or in a Na+-free solution.L-Arg at 1 mMstimulated net Na+absorption by 0.12 μEq cm−2h−1. The evidence for carrier-mediated transport ofL-Arg includes: (1) temperature dependence and saturability over 0.01–10 mM, (2) Na+dependence and ouabain sensitivity, (3) 84±2% reduction in the apparent permeability coefficient (Papp) of3H-L-Arg in the presence of excess unlabeledL-Arg (1 mM), and (4) 16-fold difference inL-Arg transport (at 0.1 mM) between the mucosal-to-serosal and the serosal-to-mucosal direction. Moreover,L-Arg transport was inhibited by basic amino acids, large neutral amino acids, and nitric oxide synthase inhibitors, but not by acidic and small neutral amino acids. Kinetic analysis revealed the possible existence of both high and low affinity processes forL-Arg transport. A half maximal concentration (Km) and maximalL-Arg flux (Jmax) values of the low and high affinity processes were 5.90 and 0.07 mM, and 1,248 and 111 pmol cm−2min−1, respectively. Hill analysis ofL-Arg transport at 0.1 mMin the presence of varying Na+concentrations in the mucosal bathing fluid yielded a Hill coefficient of 0.93, suggesting a 1:1 coupling between Na+andL-Arg. In conclusion, Na+-coupled transport process(es) forL-Arg in accordance with a 1:1 stoichiometry appear to be present on the mucosal side of the pigmented rabbit conjunctiva. The pattern of inhibition by basic and large neutral amino acids and Na+dependency are suggestive of system B0,+-mediatedL-Arg transport.

Non-competitive binding of the nonpeptide antagonist BIBP3226 to rat forebrain neuropeptide Y 1 receptors

[ 3 H ]Neuropeptide Y labelled neuropeptide Y receptors in rat forebrain membranes as a homogenous class of high-affinity sites. Between 80 and 85% of these receptors showed high affinity for Y 1-selective antagonists such as (R)- N 2-(diphenylacetyl)- N-[(4-hydroxy-phenyl)methyl]- d-arginine amide (BIBP3226). While competitive in functional studies, BIBP3226 produced parallel shifts of the Scatchard plots of [ 3 H ]neuropeptide Y saturation binding in rat forebrain membranes. Mechanisms which are routinely invoked to explain non-competitive binding do not apply to BIBP3226. Wash-out experiments, involving successive treatment of the membranes with BIBP3226, buffer (wash-out step) and [ 3 H ]neuropeptide Y, argue against irreversible or a pseudo-irreversible binding of the antagonist. Allosteric inhibition is also unlikely since BIBP3226 did not affect the rate of dissociation of [ 3 H ]neuropeptide Y in isotope dilution experiments. The non-hydrolyzable guanine nucleotide, 5′-guanylylimidodiphosphate (Gpp(NH)p), abolished the binding of [ 3 H ]neuropeptide Y and increased its rate of dissociation in isotope dilution experiments. This suggests that the initial [ 3 H ]neuropeptide Y-receptor association is a low affinity process and that the observed binding of [ 3 H ]neuropeptide Y is related to the formation of a ternary [ 3 H ]neuropeptide Y-receptor-G protein complex. Two- or even multistate models (in which BIBP3226 could potentially behave as an inverse agonist) could therefore be needed to explain the non-competitive antagonism of BIBP3226 in broken cell preparations.

Sodium-dependent proline and glutamate uptake by hippocampal synaptosomes during postnatal development

Na +-dependent uptake of proline and glutamate by hippocampal synaptosomes was studied during postnatal development. At all ages from 9 days to adulthood, hippocampal synaptosomes transported proline by both a high-affinity and a low-affinity process, whereas glutamate was always transported predominantly by a high-affinity process. During the period of rapid synaptogenesis, the K T for high-affinity proline transport overshot the adult value, whereas the K T for glutamate transport increased steadily toward the adult value. The ratio of K T values for proline and glutamate was 2–3 times the adult value between 12 and 24 days of age. Although high-affinity transporters for proline and glutamate are expressed by nearly the same hippocampal pathways, they are differentially regulated during postnatal development.

Iron transport mechanisms in reticulocytes and mature erythrocytes

The mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with 59Fe-labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration-dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin-bound Fe(II) by two mechanisms. One is a high-affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low-affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly associated with exchange of Na+. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)-sucrose and Fe(II)-ascorbate using a variety of incubation conditions. No major differences were observed.

Spodoptera frugiperda (Sf9) cells express novel purine- and pyrimidine-selective nucleoside transporters

Nucleoside fluxes in mammalian cells are mediated by a family of plasma membrane transporters that function by equilibrative or concentrative mechanisms. Members of this family can be identified on the basis of their permeant selectivities and sensitivities to various inhibitors. This study was initiated to characterize nucleoside transport processes of cultured armyworm ovary (Sf9) cells in anticipation of using Sf9 cells for the functional expression of recombinant mammalian nucleoside transporter proteins. Kinetic analysis of zero- trans influx of 3H-nucleosides in Sf9 cells revealed the presence of high ( K m; 10–40 μM) and low ( K m; ⩾0.4 mM) affinity transport processes. Influx of [ 3H]adenosine at 1 μM (high-affinity process) was inhibited only by purine nucleosides (adenosine > 3′-deoxyadenosine > formycin B > guanosine > inosine), and that of [ 3H]uridine was inhibited only by pyrimidine nucleosides (uridine > 3′-deoxyuridine > thymidine). By contrast, fluxes of [ 3H]adenosine and [ 3H]uridine at 100 μM (low-affinity process) were inhibited by both pyrimidine and purine nucleosides. These results suggested the presence of at least two high-affinity transport processes with selectivity for either purine or pyrimidine nucleosides and a low-affinity process with selectivity for both. None of the transport processes were sodium-dependent since uptake of adenosine and uridine was unaffected by elimination of the sodium gradient, and assays that employed formycin B, a non-metabolized permeant of the high-affinity purine-selecive nucleoside transport process, revealed that transport was equilibrative. Nitrobenzylthioinosine, dipyridamole, and dilazep, potent inhibitors of equilibrative nucleoside transport processes in mammalian cells, were poor inhibitors of adenosine and uridine fluxes in Sf9 cells.

Characterization of a Mg-Dependent, Na-Inositol Co-Transport Process in Cardiac Sarcolemmal Vesicles

[ 3H]- myo-Inositol transport into cardiac sarcolemmal vesicles is both a time- and Na +-dependent process. Transport was stimulated 3-fold by 1 μM valinomycin suggesting the process is electrogenic. myo-Inositol transport was dependent upon the presence of Mn 2+ or Mg 2+ but not Ca 2+. Kinetic analysis revealed a high affinity transport process that was saturable (V max, 650 ± 71 pmoles myo-inositol/mg protein/min; K m, 37.7 ± 1.3 μM) and a low affinity process that was unsaturable up to 1.0 mM substrate. Transport was not inhibited by 1.0 mM of either L-glucose, D- or L-galactose. However, D-glucose and L-fucose at 1.0 mM were inhibitory. Higher concentrations (30 mM) of each of the sugars inhibited transport. myo -Inositol transport was also inhibited by the inositol isomers (1.0 mM), D- chiro-inositol, epi-inositol and scyllo-inositol with scyllo-inositol being most effective. Kinetic analysis established sycllo -inisitol as a competitive inhibitor of cardiac myo -inositol transport, increasing the K m for substrate three-fold (122 ± 21 μM). These data indicate that inositol transport across cardiac sarcolemma is a Mg 2+-dependent Na + co-transport process that is electrogenic and stereospecific.