Transport of ethanolamine and its incorporation into the variant surface glycoprotein of bloodstream forms of Trypanosoma brucei

The membrane-attached form of the variant surface glycoprotein (mf-VSG) of bloodstream forms of Trypanosoma brucei is anchored to the plasma membrane by a hydrophobic C-terminal lipo-oligosaccharide containing ethanolamine. Analysis by polyacrylamide gel electrophoresis showed that several different cloned T. brucei strains (strain EATRO 110 and variants 117 and 118 of strain 427) incorporated [3H]ethanolamine into both mf-VSG and the soluble VSG derived from it, but not into other proteins. Other trypanosomatids, e.g. Leishmania mexicana promastigotes, T. cruzi epimastigotes, and T. brucei procyclic forms, did not incorporate ethanolamine into cellular proteins. Thus, [3H]ethanolamine can be used as a specific biosynthetic label for T. brucei VSG polypeptides. The time course of incorporation of [3H]ethanolamine into VSG showed a lag period of about 15 min. Double-labelling experiments using [3H]ethanolamine and H3[32P]O4 demonstrated that ethanolamine labelled only the C-terminal moiety and was not incorporated into other portions of the VSG molecule. Cellular uptake of ethanolamine occurred via a specific carrier-mediated transport system having a Vmax of 132 pmol min-1 mg-1 protein and a Km of 3.7 microM. The properties of this transport system are consistent with the possibility that ethanolamine is derived entirely from the host.     

L-arginine transport at the fetal side of human placenta: effect of aspirin in pregnancy

L-Arginine transport by the fetal side of human placenta was investigated through the characterization of L-[3H]arginine uptake in isolated perfused cotyledon. Competitive inhibition experiments suggest the presence of at least two transport systems: a Na+-independent, pH-insensitive system inhibitable by cationic amino acids, similar to system y+, and a Na+-dependent system which recognizes both cationic and neutral amino acids only in the presence of Na+, i.e. a Bo,+-like system. The kinetic analysis of L-arginine uptake in the presence of Na+ revealed that the process is mediated by saturable components: a high-affinity system (Km = 167 +/- 18.0 microM; Vmax = 0.174 +/- 0.012 micromol min-1) and a low-affinity carrier (Km = 980 +/- 112 microM; Vmax = 1.60 +/- 0.12 micromol min-1). In the absence of Na+, L-arginine uptake was fitted by one model with a Michaelis-Menten constant of 200 +/- 24.8 microM. These results suggest that the high-affinity component corresponds to the Na+-independent system y+, whilst the low-affinity system may represent the activity of the Na+-dependent Bo,+ transporter. Kinetic studies in placentae taken from aspirin-treated pregnancies showed that L-arginine is transported with a significantly higher affinity (Km = 42.5 +/- 5.7 microM), but with a lower capacity (Vmax = 0.064 +/- 0.003 micromol min-1) than in the non-treated group. The latter finding suggests that aspirin would facilitate the uptake of the NO precursor only at very low arginine concentrations.     

Functional characterization of Na+-independent choline transport in primary cultures of neurons from mouse cerebral cortex

We report here the functional characteristics of Na+-independent choline transport system in primary cultures of neurons from mouse cerebral cortex. Na+-independent choline transport was saturable with a Michaelis constant (Kt) of 26.7+/-1.2 microM and a maximal velocity (Vmax) of 1.04+/-0.02 nmol/mg protein/10 min. Choline uptake was significantly influenced by extracellular pH and by membrane depolarization. This uptake system was inhibited by various organic cations including unlabeled choline, guanidine, diphenhydramine and the choline analog hemicholinium-3. However, the prototypical organic cation tetraethylammonium and cimetidine showed very little affinity for the Na+-independent choline uptake system in neurons. These results indicate that mouse cerebrocortical neurons express a Na+-independent, high-affinity choline transport system. RT-PCR revealed that choline transporter-like protein 1 (CTL1) and its spliced variant CTL1a, which have been reported to be novel Na+-independent choline transporter, are expressed in mouse cerebrocortical neurons. The Na+-independent transport properties of choline in mouse neurons is similar or identical to that of CTL1 and/or CTL1a. This choline transport system seems to have relevance not only for neuronal physiology but also for the uptake of pharmacologically important organic cation drugs.     

Copper transport kinetics by isolated rat hepatocytes

Uptake and efflux of 64Cu were examined to determine whether hepatic parenchymal cells exhibit the kinetic criteria of a specific transport system for copper and related trace metals. Saturation kinetics were clearly indicated by both v versus [Cu] and 1/v versus 1/[Cu] plots (Km = 11 +/- 0.6 microM and Vmax = 2.7 nmol Cu X min-1 X mg prot-1). Identical results were obtained by cold-copper analyses, and contributions from simple diffusion or nonspecific binding were not detected. Virtually all of the accumulated 64Cu was intracellular by 0.5 min (the initial velocity period), with approximately 40% in the cytosolic fraction. Several related trace metals inhibited 64Cu uptake, but Ni(II) at a 10:1 molar excess did not. Zn(II) acted as a simple competitive inhibitor of 64Cu uptake (Ki = 16 microM). Efflux from preloaded cells was biphasic, with an initial rapid phase of approximately 5 min. Approximately 35% of preloaded 64Cu was transported out of the cells by 40 min, and little efflux occurred thereafter. Thus, hepatocytes exhibit saturation kinetics, competition by related substrates, and countertransport criteria of specific facilitated transport. A wide variety of metabolic inhibitors have no effect on 64Cu uptake under the same conditions that inhibit the active transport of bile acids. Specific inhibitor tests for electrogenic coupling were also negative. Because the identical kinetic parameters were obtained for free 64Cu and the 1:1 64Cu-histidine complex, it is inferred that copper is probably transported as the free ion. Cells incubated with greater than or equal to 10 microM 64Cu showed a net loss of copper after 40- to 60-min incubation, which may involve specific hepatic mechanisms in copper homeostasis.     

Putrescine transport system in Leishmania infantum promastigotes

A transport system for putrescine in Leishmania infantum (= L. donovani infantum) promastigotes has been identified and characterized by measuring the uptake of radioactively labelled putrescine. Putrescine uptake was time- and temperature-dependent without any accumulation taking place at 0 degrees C. Uptake of putrescine was maximal at pH values near neutrality. Putrescine uptake showed an apparent Km = 1.08 +/- 0.12 microM and a Vmax = 1.74 + 0.62 pmol min-1 (10(6) cells)-1. The effect of metabolic poisons and uncoupling agents suggests that the putrescine uptake was energy-dependent. The transport system seemed to be highly specific for putrescine as neither aminoacids nor related compounds tested showed any competition with putrescine uptake. The trypanocide Berenil inhibited almost completely the putrescine uptake with non-competitive kinetics and a value of Ki = 43 microM.     

Influence of ATP on sarcoplasmic reticulum function of vascular smooth muscle

A vesicular fraction isolated from bovine aorta and enriched in fragmented sarcoplasmic reticulum (FSR) exhibited active calcium transport and ATPase activity. By use of a hypotonic NaHCO3 extraction solution, an active preparation was isolated that retained activity for up to 4 days. A small but significant (P less than 0.05) Ca2+-stimulated, Mg2+-dependent ATPase associated with calcium transport was demonstrated with a specific activity of 0.33 mumol inorganic phosphate (Pi).mg-1.min-1. The basal Mg2+ ATPase demonstrated Michaelis-Menten kinetics [Km(Mg2+-ATP) = 0.44 +/- 0.01 X 10(-3) M; Vmax = 2.22 +/- 0.01 mumolPi.mg-1.min-1]. The Ca2+-stimulated, Mg2+-ATPase demonstrated apparent substrate inhibition (Ks approximately 10 mM) with no evidence for end-product (ADP) or excess added Ca2+ contributing to this inhibition. Oxalate-supported active calcium uptake velocities also exhibited quantitatively similar substrate inhibition. These results suggest that FSR from vascular smooth muscle contains either two enzymes or one enzyme with two isomeric forms, one of which is associated with the calcium uptake activity of this structure and the other of unknown function.     

Is neurotransmitter histamine predominantly inactivated in astrocytes?

Rat synaptosomes and astroglia cell-enriched fraction were tested for the uptake of histamine (HA) and its precursor histidine, and the activities of the HA-synthesizing enzyme, histidine decarboxylase (HD) and HA-metabolizing enzyme, histamine methyltransferase (HMT). While histidine uptake was more active into synaptosomes than into astrocytes, only astrocytes were capable of a significant HA uptake. Kinetic analysis of the astrocytic HA uptake revealed a high affinity-low capacity system (Km = 5 X 10(-7) M, Vmax = 1.6 X 10(-12) mol.min-1 X mg-1) similar to the astroglial transport systems for other neurotransmitters. HMT was 70% more active in astrocytes than in synaptosomes, whereas HD activity was not different in these two preparations. The results indicate that astrocytes could be the major site of neurotransmitter HA inactivation.     

Uptake and metabolism of [3H]choline mustard by cholinergic nerve terminals from rat brain

The objective of this study was to measure the uptake and metabolism of [3H]choline mustard aziridinium ion in rat brain synaptosomes. In previous investigations, we showed that this compound binds irreversibly to the choline carrier thereby inhibiting choline transport into nerve terminals; it also acts as both a substrate and inhibitor of the acetylcholine biosynthetic enzyme choline acetyltransferase. We now report that [3H]choline mustard aziridinium ion was transported into purified rat brain synaptosomes by a hemicholinium-sensitive mechanism, but at only a fraction of the rate of uptake of [3H]choline. Following 5 min incubation with the nerve terminal preparation, uptake of [3H]choline mustard aziridinium ion was 20% of that of [3H]choline transport, but this fell to 10% of [3H]choline accumulation at 30 min incubation. Apparent Michaelis constants derived from double reciprocal plots of velocity of transport versus substrate concentration revealed that the apparent affinity constants (Km) of the high-affinity choline carrier for [3H]choline mustard aziridinium ion and [3H]choline were not different (1.44 +/- 0.15 and 2.14 +/- 0.80 microM for choline and choline mustard aziridinium ion, respectively). Increasing the incubation time from 5 to 30 min, during which time a proportion of the high-affinity choline carriers were irreversibly inactivated by choline mustard aziridinium ion, did not alter the binding affinity for this compound. The maximum velocity of transport (Vmax) for the two compounds were significantly different with the maximum uptake of [3H]choline mustard aziridinium ion being 19.5% of that for choline at 5 min incubation, and falling to only 10.6% of the maximum rate of choline transport by 30 min incubation. [3H]Choline mustard aziridinium ion transported into synaptosomes on the high-affinity choline carrier was metabolized, with 27% being recovered as [3H]acetylcholine mustard aziridinium ion, 27% as [3H]phosphorylcholine mustard aziridinium ion, 7% as unmetabolized [3H]choline mustard aziridinium ion and 16% recovered as an unidentified metabolite. In parallel samples, [3H]choline taken up into synaptosomes was recovered as [3H]acetylcholine (71%) and unmetabolized [3H]choline (18%) with no net production of [3H]phosphorylcholine. Acetylation of [3H]choline mustard aziridinium ion amounted to only 7.6% of [3H]acetylcholine synthesized under the same conditions. These results show clearly that choline mustard aziridinium ion was accumulated into the cholinergic nerve terminals by the high-affinity choline carrier, but the amount was small relative to the uptake of choline and probably restricted by progressive inactivation of the transporters through covalent bond formation.     

Choline transport by lung epithelium

The uptake of [3H]choline was investigated using isolated perfused rat lungs and primary cultures of granular pneumocytes isolated by tryptic digestion of rat lungs. Metabolic products were separated from free choline by chloroform:methanol extraction and column chromatography. Tissue-associated [3H]choline increased progressively in the perfused lung, and estimated mean intracellular concentration at 2 h was 12 times the extracellular concentration (5 microM). Choline uptake was inhibited by ventilation with CO and by perfusion with the choline analog, hemicholinium-3 (HC-3). Isolated granular pneumocytes also accumulated choline against a concentration gradient by an energy-dependent process. The concentration for half-maximal uptake, after correction for the diffusion component, was estimated at 18 +/- 4 microM (mean +/- SE; n = 3), and the estimated maximal rate of uptake was 213 +/- 44 pmol/min/microliter cell water. HC-3 inhibited uptake by approximately 50% at a concentration of 10(-4) M. There was no effect on uptake when Na+ in the medium was replaced by Li+ or N-methylglucamine+. These results indicate that granular pneumocytes possess a transport system that results in accumulation of choline against a concentration gradient. The characteristics of uptake indicate that this system is similar to the low affinity choline transport system of other organs.     

Type A and B monoamine oxidase activities in the human and rat kidney.

The present work has examined the distribution of the two isoforms of monoamine oxidase (MAO), type MAO-A and MAO-B, in the cortex and medulla of the human and rat kidney. Homogenates of renal cortex and renal medulla were prepared in 67 mmoles l-1 phosphate buffer (pH = 7.2) and MAO activity was determined with [3H]5-hydroxytryptamine ([3H]5HT) and [14C]beta-phenylethylamine ([14C]beta-PEA) as preferential substrates of type A and type B MAO, respectively. Km and Vmax values for the two substrates were also calculated. Both MAO-A and MAO-B are present in the cortex and the medulla of the human and rat kidneys. In the human kidney, MAO-A activity was found to be similar in the cortex (Vmax = 142.70 +/- 45.05 nmoles mg-1 protein h-1) and medulla (Vmax = 133.91 +/- 35.51 nmoles mg-1 protein h-1); MAO-B activity was found to be higher in the cortex (Vmax = 166.19 +/- 19.75 nmoles mg-1 protein h-1) than in the medulla (Vmax = 92.91 +/- 13.22 nmoles mg-1 protein h-1). In the rat kidney, MAO-A was also found to be similar in the cortex (Vmax = 62.35 +/- 1.74 nmoles mg-1 protein h-1) and the medulla (Vmax = 59.42 +/- 0.97 nmoles mg-1 protein h-1) and higher than the activity of MAO-B in the two renal areas (cortex, Vmax = 31.06 +/- 1.09 nmoles mg-1 protein h-1; medulla, Vmax = 14.93 +/- 0.97 nmoles mg-1 protein h-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose transport in Crithidia luciliae.

The glucose analogue, 2-deoxy-D-glucose, was used to characterise the glucose transport system in Crithidia luciliae choanomastigotes. Uptake was temperature dependent with a Q10 of 2, and saturable with a Km of 0.22 mM and Vmax of 5.5 nmol min-1 (mg protein)-1 at 23 degrees C. Preloaded cells showed rapid exchange of intracellular 2-deoxy-D-glucose when incubated with extracellular D-glucose or 2-deoxy-D-glucose but little exchange with L-glucose. The substrate specificity of the uptake was studied using a number of D-glucose analogues. 6-Deoxy-D-glucose, 3-fluoro-3-deoxy-D-glucose and 4-fluoro-4-deoxy-D-glucose all competed for the transporter and had significant inhibitory effects on 2-deoxy-D-glucose transport. In contrast, 1-thio-beta-D-glucose, trehalose, 3-O-methyl-D-glucose, arginine, thymidine, L-sorbose and L-glucose were not inhibitory. The results imply the existence of a glucose transporter. The transport was blocked by a number of inhibitors and ionophores, including fluoride, azide, cyanide, dinitrophenol, valinomycin and nigericin. Overall, the uptake, exchange and efflux of 2-deoxy-D-glucose is consistent with transport via facilitated diffusion.     

Blood-brain barrier choline transport in the senescent rat

Choline is an important membrane phospholipid constituent and a neurotransmitter precursor that is minimally synthesized in brain. The long-term maintenance of brain choline concentration is dependent on uptake from plasma, which occurs via saturable transporter at the blood-brain barrier. Previous studies have suggested that brain choline uptake declined with age. To reevaluate this, brain choline uptake in 3, 12, 24, and 28-month-old Fischer-344 rats was evaluated using the in situ brain perfusion technique. Minimal differences were found with uptake parameters differing by approximately 10% between aged and adult rats for tracer levels while similar trends were observed at higher choline concentrations. Further, estimated Vmax and Km values differed by <30% between the groups. The results suggest that blood-brain barrier choline uptake changes minimally with aging in the rat.     

Secretion of tetraethylammonium by proximal tubules of rabbit kidneys

The secretory transport of tetraethylammonium (TEA) was investigated in perfused and nonperfused isolated S1, S2, and S3 segments of proximal tubules from rabbit kidneys. In the perfused tubules the transepithelial net secretory flux and in nonperfused tubules the TEA cellular uptake were saturable (Km = 67 microM, Vmax = 2,480 fmol X min-1 X mm-1 in perfused S2 segments), energy dependent, and inhibited by mepiperphenidol. The net secretory flux of TEA (J b leads to j TEA) at a bath TEA concentration of 40 microM differed for the three segments and decreased in the order S1 greater than S2 greater than S3. The concentration of TEA in the perfusate leaving the tubule was approximately twice as great and the intracellular TEA concentration approximately 40 times as great as that in the bath. In nonperfused segments (40 microM TEA in the incubation medium) the TEA tissue water-to-medium ratio reached 100. In the three segments the ability to accumulate TEA across the peritubular membrane, thus, was similar, but the transepithelial secretory flux differed significantly. The differences in secretory rate between the three segments presumably result from differences in the luminal membrane permeability.     

Choline transport in collecting duct cells isolated from the rat renal inner medulla

Glycerophosphorylcholine (GPC) plays an important role in the osmoregulation of the renal inner medulla. Under hyperosmotic conditions, a striking increase in cellular GPC content is observed. In order to characterize the cellular events involved in GPC metabolism, we have studied the uptake of choline, a precursor of GPC, by freshly isolated rat inner medullary collecting duct (IMCD) cells at 300 mosmol/l. Choline uptake occurred by a single transport system with an apparent affinity (K _m) of 80 M and a maximal velocity (V _max) of 120 pmol/l cell water/min. Hemicholinium-3, ethanolamine and N,N-dimethylethanolamine were potent inhibitors, but betaine had no effect. Choline uptake was not altered by the replacement of Na⁺ with N-methylglucamine⁺, suggesting a sodium-independent process. Addition of 50 mM KCl to the incubation medium to reduce the cell membrane potential inhibited choline uptake by 19±4% after 10 min. Increasing the extracellular osmolarity to 600 or 900 mosmol/l had no effect on the kinetic parameters of choline uptake. These results suggest that choline uptake into IMCD cells occurs by a sodium-independent transport system driven by the inside negative cell membrane potential. Furthermore, the increase in the GPC content under hyperosmotic conditions is not associated with increased activity of the transport systems of biosynthetic precursors.     

Inhibitors of choline transport in alveolar type II epithelial cells.

Isolated alveolar type II epithelial cells (granular pneumocytes) from rat lung accumulate free choline against a concentration gradient by an energy-dependent saturable transport process with apparent Km approximately 18 microM. In order to evaluate the structural requirements for choline transport by these cells, the inhibition of the initial rate of cellular uptake of [3H]choline (5 microM) by its analogue was measured. There was no significant inhibition of substrate uptake by analogues lacking an amino group while the presence of a quaternary nitrogen was most effective. N,N'-dimethylethanolamine (apparent Ki, 7 microM) and n-decylcholine (apparent Ki, 0.5 microM) were potent competitive inhibitors of choline transport. Substitution of the hydroxyl group in choline greatly diminished the inhibitory effect; fluorocholine, thiocholine, betaine, and betaine aldehyde showed little or no inhibition. This requirement for a hydroxyl group raises the possibility of hydrogen bonding of choline with the transport protein. The choline transport system in granular pneumocytes appears to differ from that in synaptosomes by the lower affinity of the carrier for substrate and for hemicholinium-3 and from that in erythrocytes by the role of the hydroxyl in the substrate molecule. The availability of inhibitory analogues for choline transport will facilitate isolation and study of the granular pneumocyte choline transport protein.     

Transport of adenosine by renal brush border membranes

The transport of adenosine into brush border membrane vesicles from rat kidney is demonstrated. A first, rapid stage of transport is completed by 20 s. It is stimulated by a gradient of sodium. The effect of the concentration of adenosine in the low micromolar range on the initial rate of uptake indicates a saturability. An apparent Km of 1.48 microM and a Vmax of 215 pmol/mg protein/min have been calculated. In the rapid stage of uptake, adenosine is transported into the intravesicular space with no significant binding to the membrane. The following slow uptake is not stimulated by sodium and reaches steady state after about one hour.     

Catecholamine uptake into isolated adrenal chromaffin cells: inhibition of uptake by acetylcholine

We have investigated the process of catecholamine uptake in guinea-pig chromaffin cells. Isolated guinea-pig chromaffin cells accumulate [3H]norepinephrine and [3H]epinephrine by a saturable transport system. Catecholamine uptake is dependent upon temperature, energy, and extracellular Na+. The apparent KmS for norepinephrine and epinephrine transport are approximately 1 and 3.5 microM, respectively; the transport maximum (Vmax) for both compounds is about 100 pmol/min/mg protein. The uptake of norepinephrine into chromaffin cells is inhibited by imipramine (Ki = 50 nM) and by desmethylimipramine (IC50 = 20 nM). In both its substrate specificity and its sensitivity to pharmacological inhibition, the catecholamine uptake system in chromaffin cells is similar to the catecholamine transport system previously described in sympathetic neurons. Decreasing external Na+ from 130 to 19 mM increases the apparent Km for norepinephrine to 2.8 microM. Decreasing external norepinephrine increases the Na+ concentration required for half-maximal transport. Agents that depolarize chromaffin cells, such as acetylcholine and veratridine, significantly inhibit [3H]norepinephrine uptake. This decrease in uptake is due to an increase in the apparent Km for norepinephrine. The inhibition of [3H]norepinephrine uptake by depolarizing agents cannot be accounted for by the preferential release of newly-accumulated [3H]norepinephrine, or by the competitive inhibition of [3H]norepinephrine uptake by secreted catecholamines. The inhibition of catecholamine uptake by depolarizing agents suggests that the transport system may be regulated by the membrane potential. Norepinephrine and epinephrine that are spontaneously released from the adrenal medulla may be recaptured in vivo. The inhibition of transport by acetylcholine may prevent the re-uptake of catecholamine released during the physiological stimulation of secretion.     

Choline uptake in cholinergic nodose cell bodies

Isolated living cell bodies were obtained by mechanical and enzymatic dissociation from adult rabbit nodose ganglion followed by separation of fibres and cells using a Percoll gradient. A purification yield of 45% was measured. Based on previous results obtained in whole ganglion and showing the presence of cholinergic cell bodies among the afferent fibres of the vagus nerve, this preparation was used to study choline uptake by neuron cell somata. Cholinergic cells counted after choline acetyltransferase immunohistological staining showed a stained population of 2.9% among the isolated population. Two [3H]choline uptake mechanisms were detected at the cell body level. The first, with Km1 = 7 microM and Vm1 = 200 pmol/h per ganglion is sodium dependent, related to acetylcholine synthesis (43%) and has an IC50 with hemicholinium-3 equal to 50 microM. The second, with Km2 = 54 microM and Vm2 = 2235 pmol/h per ganglion is sodium independent, poorly associated to acetylcholine synthesis (12%) and exhibits an IC50 of 2 microM with hemicholinium-3. Except for their sensitivity to hemicholinium-3, the high and low affinity choline uptake mechanisms observed at the somatic level have, respectively, the same characteristics as the high and low affinity mechanisms described at the synaptic level. Their physiological role, their opposed sensitivity to hemicholinium-3 compared to the synaptic uptake systems and the relation between the somatic high affinity choline transport and an acetylcholine somatic release are discussed.     

pH regulation in barnacle muscle fibers: dependence on extracellular sodium and bicarbonate

Intracellular pH (pHi) regulation was studied in barnacle muscle fibers with pH-sensitive microelectrodes. The cells were acid loaded, and the subsequent recovery of pHi was monitored. The rate of recovery was reduced by one-third when external Na+ ([Na+]o) was replaced by Li+, but recovery was completely abolished when Na+ was replaced by choline or N-methyl-D-glucamine. In other experiments, varying amounts of Na+ were replaced by choline, and the acid extrusion rate, derived from the recovery rate of pHi, was calculated at a single value of pHi, 6.80. The dependence of the acid extrusion rate on [Na+]o could be described by Michaelis-Menten kinetics; at pHo (extracellular) = 8.0 and [HCO3-]o (extracellular) = 10 mM, the apparent Km and Vmax were 59 mM and 1.3 mmol x l(-1) x min-1. When [HCO3-]o was reduced to 2.5 mM at the same pHo, Km did not change significantly, but Vmax was substantially reduced. On the other hand, when pHo was reduced to 7.4 at constant [HCO3-]o, Vmax changed only slightly, but Km increased substantially. In similar experiments, we examined the dependence of the acid extrusion rate on [HCO3-]o. At pHo = 8.0 and [Na+]o = 440 mM, the apparent Km and Vmax were 4.1 mM and 2.1 mmol x 1-1 x min-1. When pHo was reduced to 7.4, Vmax was not altered, but Km substantially increased. The kinetic data are discussed in terms of the role of pHo, [Na+]o, and [HCO3-]o in the pHi-regulating system.     

Identification and characterization of a ribose transport system in Leishmania donovani promastigotes

A transport system for ribose in Leishmania donovani promastigotes was identified and characterized by measuring the uptake of radioisotope-labeled ribose. The pentoses arabinose, 2-deoxyribose and xylose inhibited ribose uptake, whereas hexoses (glucose, alpha-methylglucoside, thioglucose, galactose, lactose, maltose, mannose), adenosine, and proline did not inhibit uptake, indicating that the transporter exhibited substrate specificity. Intracellular ribose exchanged with 2-deoxyribose. Uptake of ribose showed saturation kinetics with an apparent Km = 2 mM and Vmax = 11 nmol (mg protein)-1 min-1. Both N-ethylmaleimide and p-hydroxymercuribenzoate inhibited ribose uptake which was prevented by dithiothreitol. The uncoupling agents 2,4-dinitrophenol and carbonylcyanide p-(trifluoromethoxy)phenylhydrazone and a variety of inhibitors of energy-driven transport had no significant effect on ribose uptake. Following transport, the intracellular ribose pool contained two-thirds of the sugar in the phosphorylated form and one-third in the neutral form. These cumulative results indicate that a specific carrier mediates ribose uptake via a facilitated diffusion system in L. donovani promastigotes.