Sodium-dependent uptake of [3H]scyllo-inositol by Tetrahymena: incorporation into phosphatidylinositol, phosphatidylinositol-linked glycans, and polyphosphoinositols.

[3H]Scyllo-inositol was taken up by Tetrahymena cells through a sodium-dependent pathway wherein unlabeled scyllo- and myo-inositol competed for uptake. d-Glucose was a competitor of [3H]myo-inositol uptake, but did not appear to compete for [3H]scyllo-inositol uptake. Transport of [3H]scyllo- and [3H]myo-inositol was inhibited when sodium was removed from the labeling buffer and by phlorizin, an inhibitor of sodium-dependent transporters. Cytochalasin B, an inhibitor of facilitated glucose transporters, had no significant effect on inositol transport. Internalized [3H]scyllo-inositol was readily incorporated intact into phosphatidylinositol, phosphatidylinositol-linked glycans, and polyphosphoinositols. Distribution of [3H]scyllo- and [3H]myo-inositol radioactivity into individual polyphosphoinositols was found to differ.     

Putative cocaine receptor in striatum is a glycoprotein with active thiol function

Dopamine transporters of bovine and rat striata were identified by their specific [3H]cocaine binding and cocaine-sensitive [3H]dopamine [( 3H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes. Inorganic and organic mercury reagents inhibited both [3H]DA uptake and [3H]cocaine binding, though they were all more potent inhibitors of the former. n-Ethylmaleimide inhibited [3H]DA uptake totally but [3H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [3H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [3H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (less than 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (greater than 10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation.     

Identification and characterization of GABA, proline and quaternary ammonium compound transporters from Arabidopsis thaliana.

Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants. Heterologous complementation of a GABA uptake-deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9 +/- 1.7 and 1.7 +/- 0.3 mM at pH 4, respectively. The simultaneous transport of [1-14C]GABA and [2,3-3H]proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition. ProT2-mediated [1-14C]GABA transport was inhibited by proline and quaternary ammonium compounds.     

Ubiquitin-Directed Antibodies Inhibit Neuronal Transporters in Rat Brain Synaptosomes

Affinity-purified antibodies specific for ubiquitin were found to inhibit the sodium-dependent uptake of [3H]choline, gamma-[3H]aminobutyric acid [( 3H]GABA), [3H]glutamate, [3H]norepinephrine, [3H]aspartate, and [3H]serotonin in rat cerebral cortical synaptosomes at a low concentration (10 micrograms/ml). These antibodies (termed anti-Ub) had no effect on the sodium-independent uptake of these substances or their calcium-dependent efflux. Synaptosomal [3H]deoxyglucose uptake was not affected in normal Krebs Ringer buffer containing 10 mM glucose, but was inhibited in glucose-free medium. Other nonneuronal sodium-dependent transport processes were found to be unaffected by 10 micrograms/ml anti-Ub, suggesting that anti-Ub does not bind indiscriminantly to sodium-binding sites on sodium-dependent organic solute transporters. Finally, anti-Ub inhibited sodium-dependent [3H]GABA and [3H]glutamate uptake in plasma membrane ghosts, devoid of membrane potential, which were derived from rat cerebral cortical synaptosomes. These results suggest that neuronal transporters or sites proximal to them may be ubiquitinylated on the plasma membrane surface.     

Effects of Heavy Metal Cations and Other Sulfhydryl Reagents on Brain Dopamine D1 Receptors: Evidence for Involvement of a Thiol Group in the Conformation of the Active Site

To investigate aspects of the biochemical nature of membrane-bound dopamine D1 receptors, rat striatal homogenates were pretreated with heavy metal cations and some other chemical agents, and their effects on D1 receptors were subsequently determined using a standard [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-N-3- benzazepine([3H]SCH 23390) binding assay. Incubation of striatal membranes with as little as 1 microM Hg2+, 10 microM Cu2+, and 10 microM Cd2+ completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 microM, was noncompetitive in nature, whereas 3-5 microM Cu2+ afforded mixed-type inhibition. The inhibitory effect of Cu2+ was fully reversed by dithiothreitol (0.1-1 mM). Cu2+ (2 microM) did not affect the affinity of cis-flupenthixol or clozapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+ (30 microM), Ni2+ (30 microM), Mn2+ (1 mM), Ca2+ (25 mM), and Ba2+ (20 mM), inhibited specific [3H]SCH 23390 binding by 50% at the concentrations indicated. The thiol alkylating reagent N-ethylmaleimide (NEM) (0.2 mM) reduced specific binding by 70%. The effect of NEM was completely prevented by coincubation with a D1 receptor saturating concentration of SCH 23390 (20 nM) or dopamine (10 microM). The results indicated that the dopamine D1 receptor is a thiol protein and that a thiol group is essential for the ligand binding.     

Characterization of alpha 2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[3H]Yohimbine, a potent alpha 2-adrenergic antagonist, was used to label the alpha-adrenergic receptors in membranes isolated from human platelets. Binding of [3H]yohimbine to platelet membranes appears to have all the characteristics of binding to alpha-adrenergic receptors. Binding reached a steady state in 2-3 min at 37 degrees C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10(-5) M; t1/2 = 2.37 min). [3H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [3H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (-) isomer was 11-times more potent that the (+) isomer. Catecholamine agonists competed for the occupancy of the [3H]yohimbine-binding sites with an order of potency: clonidine greater than (-)-epinephrine greater than (-)-norepinephrine much greater than (-)-isoproterenol. The potent alpha-adrenergic antagonist, phentolamine, competed for the sites whereas the beta-antagonist, (+/-)-propranolol, was very weak inhibitor. 0.1 mM GTP reduced the binding affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonin competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [3H]yohimbine binding. These results suggest that [3H]yohimbine binding to hunan platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label alpha 2-adrenergic receptors.     

Transport of Leucine-Enkephalin Across the Blood-Brain Barrier in the Perfused Guinea Pig Brain

Transport of [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) [( 3H]Leu-enkephalin) across the blood-brain barrier was studied in the adult guinea pig, by means of vascular perfusion of the head in vivo. The unidirectional transfer constant (Kin) estimated from the multiple-time uptake data for [3H]Leu-enkephalin ranged from 3.62 X 10(-3) to 3.63 X 10(-3) ml min-1 g-1 in the parietal cortex, caudate nucleus, and hippocampus. Transport of [3H]Leu-enkephalin was not inhibited by unlabelled L-tyrosine (the N-terminal amino acid) at a concentration as high as 5 mM, or by the inhibitor of aminopeptidase activity bacitracin (2 mM), suggesting that there was no enzymatic degradation of peptide at the blood-brain barrier. By contrast, 2 mM unlabelled Leu-enkephalin strongly inhibited the unidirectional blood-to-brain transport of [3H]Leu-enkephalin by 74-78% in the parietal cortex, caudate nucleus, and hippocampus. The tetrapeptide tyrosyl-glycyl-glycyl-phenylalanine (without the C-terminal leucine of Leu-enkephalin), at a concentration of 5 mM, caused a moderate inhibition ranging from 15 to 29% in the brain regions studied, whereas the tetrapeptide glycyl-glycyl-phenylalanyl-leucine (without the N-terminal tyrosine) at 5 mM was without effect on Leu-enkephalin transport. Unidirectional brain uptake of Leu-enkephalin was not altered in the presence of naloxone at a concentration as high as 3 mM (1 mg/ml), suggesting that there is no binding of Leu-enkephalin to opioid receptors at the blood-brain barrier. It is concluded that there is a specific transport mechanism for Leu-enkephalin at the blood-brain barrier in the guinea pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes. Direct evidence that latent transporters become exposed to the extracellular space in response to insulin

Irradiation of intact rat adipocytes with high intensity ultraviolet light in the presence of 0.5 microM [3H] cytochalasin B results in the labeling of Mr 43,000 and 46,000 proteins that reside in the plasma membrane fraction. In contrast to the Mr 46,000 protein, the Mr 43,000 component is not observed in the microsome fraction and exhibits lower affinity for [3H]cytochalasin B. Photolabeling of the Mr 43,000 protein is inhibited by cytochalasin D, indicating it is not a hexose transporter component. The Mr 46,000 protein exhibits characteristics expected for the glucose transporter such that D-glucose or 3-O-methylglucose but not cytochalasin D inhibits its photolabeling with [3H] cytochalasin B. Furthermore, insulin addition to intact cells either prior to or after photoaffinity labeling of the Mr 46,000 protein causes a redistribution of this component from the low density microsomes to the plasma membrane fraction, as expected for the hexose transporter. Photolabeling of transporters in both the low density microsome and plasma membrane fractions is inhibited when intact cells are equilibrated with 50 mM ethylidene glucose prior to irradiation with [3H]cytochalasin B. Incubation of intact cells with 50 mM ethylidene glucose for 1 min at 15 degrees C leads to an intracellular concentration of only 2 mM. Under these conditions, the photoaffinity labeling in intact cells of hexose transporters that fractionate with the low density microsomes is unaffected, indicating these transporters are not exposed to the extracellular medium. In contrast, photolabeling in intact insulin-treated cells of hexose transporters that fractionate with the plasma membrane is inhibited under these incubation conditions. The results demonstrate that insulin action results in the exposure to the extracellular medium of previously sequestered hexose transporters.     

Myo-inositol transport through the blood-brain barrier

The unidirectional transport of [³H]myo-inositol across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured using an in situ rat brain perfusion technique. Myo-inositol was transported across the blood-brain barrier by a low capacity, saturable system with a one-half saturation concentration of 0.1 mM. The permeability surface-area product was 6.2×10^–5S^–1 with a myo-inositol concentration of 0.02 mM in the perfusate. The myo-inositol stereoisomer scyllo-inositol but not (+)-chiro-inositol (both 1 mM) inhibited myo-inositol transfer through the blood-brain barrier. These observations provide evidence that myo-inositol is transferred through the blood-brain barrier by simple diffusion and a stereospecific, saturable transport system.     

Sugar transport in rat liver lysosomes. Direct demonstration by using labelled sugars.

Purified rat liver lysosomes ('tritosomes') were prepared from rats injected with Triton WR-1339. 2. The water space of tritosomes, measured by using [3H]water and [14C]sucrose, was 2.15 +/- 0.72 microliter/mg of protein (mean +/- S.E.M., n = 12). 3. Tritosomes, when compared with a crude preparation of normal lysosomes by an indirect method of study, showed sugar specificity but decreased stereospecificity of sugar uptake. 4. At 125 mM the relative rates of net uptake of D-[14C]ribose, D-[14C]- or D-[3H]glucose and 2-deoxy-D-[3H]glucose were the same as that inferred from the indirect study. 5. The entry of D-[3H]glucose into tritosomes showed concentration-dependence suggestive of saturation, with a Km of 48 +/- 18 mM (4). 6. D- and L-glucose, D-ribose, 2-deoxy-D-glucose and D-mannose competed with D-[14C]glucose or D-[14C]ribose for uptake. 7. Cytochalasin B inhibited D-[3H]glucose uptake. 8. Uptake of 1 mM-L-[14C]glucose was slower than for 1 mM-D-[14C]glucose. 9. It is concluded that a facilitated-diffusion transport system is present in purified rat liver lysosomes.     

Involvement of noradrenaline transporters in S-nitrosocysteine-stimulated noradrenaline release from rat brain slices: existence of functional Na(+)-independent transporter activity

Noradrenaline (NA) can be released by both exocytosis and by the membrane transporter responsible for transmitter uptake. Previously, we reported that S-nitrosocysteine (SNC), an S-nitrosothiol, stimulated [3H]NA release from the rat hippocampus. In this study, we investigated the involvement of the NA transport system in SNC-stimulated NA release from rat brain (cerebral cortex and hippocampus) slices. [3H]NA release by SNC in normal Na(+) (148 mM)-containing buffer from both slices was slightly, but significantly, inhibited by 1 microM desipramine, an NA transporter inhibitor. [3H]NA release in low Na(+) (under 14 mM)-containing buffer was inhibited by over 50% by desipramine. [3H]NA release by tyramine from both slices in normal and low Na(+) buffer was almost completely inhibited by desipramine. [3H]NA uptake into cerebral cortical slices was observed in low Na(+) buffer at 20-30% of normal Na(+) buffer levels. [3H]NA uptake in both normal and low Na(+) buffers was inhibited by desipramine and by SNC. Although [3H]NA uptake in normal Na(+) buffer was almost completely inhibited by 500 microM ouabain, the uptake in low Na(+) buffer was resistant to ouabain. These findings suggest the existence of a functional Na(+)-independent NA transport system and that SNC stimulates NA release at least partially via this system in brain slices.     

A Conserved Salt Bridge between Transmembrane Segments 1 and 10 Constitutes an Extracellular Gate in the Dopamine Transporter

Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the “thin gate” formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT), the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [³H]CFT. Also, the coordination of Zn²⁺ between introduced histidines (R85H/D476H) caused a ∼2.5-fold increase in [³H]CFT binding (B_max). Importantly, Zn²⁺ also inhibited [³H]dopamine transport in R85H/D476H, suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally, we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [³H](S)-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport.     

A purine-selective nucleobase/nucleoside transporter in PK15NTD cells

Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purine-selective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with K(m) values of 2.5 +/- 0.7 microM for [(3)H]adenine, 6.4 +/- 0.5 microM for [(3)H]guanine, 1.1 +/- 0.1 mM for [(3)H]guanosine, and 4.2 +/- 0.5 mM [(3)H]adenosine. The uptake of [(3)H]guanine (0.05 microM) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5-1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine > thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2' and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [(3)H]guanine: 2-chloro-2'-deoxyadenosine > 2-chloroadenosine > 2'3'-dideoxyadenosine = 2'-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2'-deoxyadenosine and 2-chloroadenosine inhibited [(3)H]guanine uptake with IC(50) values of 68 +/- 5 and 99 +/- 10 microM, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-beta-D-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 (IC(50) of approximately 1 microM), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells.     

Phosphatidylinositol synthase of Tetrahymena: inositol isomers as substrates in phosphatidylinositol biosynthesis and headgroup exchange reactions

Phosphatidylinositol (PtdIns) synthase in microsomal fractions derived from Tetrahymena vorax was studied to determine its activity requirements. The suitability of inositol isomers as substrates for the synthase and in headgroup exchange reactions also was investigated. Tetrahymena PtdIn synthase activity was optimum in the presence of 2 mM MgCl2 plus 2 mM MnCl2, a pH of 7.8, and a temperature of 30 degrees C. The enzyme retained approximately 80% of its activity after incubation at 70 degrees C for 10 min. PtdIns headgroup exchange activity was maximal in the presence of cytidine monophosphate. By following either the accumulation of radiolabeled reaction products or the loss of radiolabel from precursors, each of the inositol isomers tested appeared to serve as substrates for both the PtdIns synthase and PtdIns:inositol phosphatidyl transferase activities. In each case, myo-inositol and scyllo-inositol were the preferred substrates. The data suggest two routes for the formation of phosphatidyl-non-myo-inositols in Tetrahymena and the potential for the production of novel, non-myo-inositol-containing second messengers.     

[3H]dipyridamole binding to nucleoside transporters from guinea-pig and rat lung.

Membranes from guinea-pig lung exhibited high-affinity binding of [3H]dipyridamole, a potent inhibitor of nucleoside transport. Binding (apparent KD 2 nM) was inhibited by the nucleoside-transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and lidoflazine and by the transported nucleosides uridine and adenosine. In contrast, there was no detectable high-affinity binding of [3H]dipyridamole to lung membranes from the rat, a species whose nucleoside transporters exhibit a low sensitivity to dipyridamole inhibition. Bmax. values for high-affinity binding of [3H]dipyridamole and [3H]NBMPR to guinea-pig membranes were similar, suggesting that these structurally unrelated ligands bind to the NBMPR-sensitive nucleoside transporter with the same stoichiometry.     

Identification and characterization of GABA, proline and quaternary ammonium compound transporters from Arabidopsis thaliana

Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants. Heterologous complementation of a GABA uptake-deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9±1.7 and 1.7±0.3 mM at pH 4, respectively. The simultaneous transport of [1-¹⁴C]GABA and [2,3-³H]proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition. ProT2-mediated [1-¹⁴C]GABA transport was inhibited by proline and quaternary ammonium compounds.     

Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity

Although high inorganic phosphate (Pi) concentration in culture media directly inhibits generation of new osteoclasts and also inhibits bone resorption by mature osteoclasts, its precise mechanism and the physiological role have not been elucidated. The present study was performed to investigate these issues. Increase in extracellular Pi concentration ([Pi](e)) (2.5-4 mM) concentration dependently inhibited 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] or parathyroid hormone (PTH)-(1-34)-induced osteoclast-like cell formation from unfractionated bone cells in the presence of stromal cells. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited 1,25(OH)(2)D(3)-, PTH-(1-34)-, or receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells. Increase in [Pi](e) (2.5-4 mM) dose dependently stimulated the expression of osteoprotegerin (OPG) mRNA and increased the expression of OPG mRNA suppressed by PTH-(1-34) or 1,25(OH)(2)D(3) in unfractionated bone cells, while it did not affect RANKL mRNA. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited the bone-resorbing activity of isolated rabbit osteoclasts. Increase in [Pi](e) (4 mM) induced the apoptosis of isolated rabbit osteoclasts while it did not affect the apoptosis of osteoclast precursor cells and mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of RANKL and M-CSF. These results indicate that increase in [Pi](e) inhibits osteoclast differentiation both by up-regulating OPG expression and by direct action on osteoclast precursor cells. It is also indicated that increase in [Pi](e) inhibits osteoclastic activity at least in part by the direct induction of apoptosis of osteoclasts.     

The Transport Systems for Selenomethionine/Methionine and Selenocystine/Cystine in Escherichia Coli K-12 Appear to be Cooperative

Dopamine transporters of bovine and rat striata were identified by their specific [³H]cocaine binding and cocaine-sensitive [³H]dopamine ([³H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes

Inorganic and organic mercury reagents inhibited both [³H]DA uptake and [³H]cocaine binding, though they were all more potent inhibitors of the former, n- Ethylmaleimide inhibited [³H]DA uptake totally but [³H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [³H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [³H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (< 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (>10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation     

Characteristics of Glucose Transport in Neuronal Cells and Astrocytes from Rat Brain in Primary Culture

Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2-deoxy-D-[3H]glucose ([3H]2-DG) into the cells. Various sugars inhibited 2-DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Km values for 2-DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2-DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2-DG uptake competitively in both neuronal cells and astrocytes. The uptake by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a probe.     

Mechanisms of DNA Utilization by Estuarine Microbial Populations

The mechanisms of utilization of DNA by estuarine microbial populations were investigated by competition experiments and DNA uptake studies. Deoxyribonucleoside monophosphates, thymidine, thymine, and RNA all competed with the uptake of radioactivity from [³H]DNA in 4-h incubations. In 15-min incubations, deoxyribonucleoside monophosphates had no effect or stimulated [³H]DNA binding, depending on the concentration. The uptake of radioactivity from [³H]DNA resulted in little accumulation of trichloroacetic acid-soluble intracellular radioactivity and was inhibited by the DNA synthesis inhibitor novobiocin. Molecular fractionation studies indicated that some radioactivity from [³H]DNA appeared in the RNA (10 and 30% at 4 and 24 h, respectively) and protein (approximately 3%) fractions. The ability of estuarine microbial assemblages to transport gene sequences was investigated by plasmid uptake studies, followed by molecular probing. Although plasmid DNA was detected on filters after filtration of plasmid-amended incubations, DNase treatment of filters removed this DNA, indicating that there was little transport of intact gene sequences. These observations led to the following model for DNA utilization by estuarine microbial populations. (i) DNA is rapidly bound to the cell surface and (ii) hydrolyzed by cell-associated and extracellular nonspecific nucleases. (iii) DNA hydrolysis products are transported, and (iv) the products are rapidly salvaged into nucleic acids, with little accumulation into intracellular nucleotide pools.