Monoiodotyrosine formation during thyroglobulin processing in Golgi vesicles

A golgi-enriched subfraction was obtained from porcine thyroid glands by differential centrifugation. When incubated in a suitable medium, these vesicles were able to concentrate iodide from the medium and bind it to protein. The iodination process was inhibited by methylmercapto-imidazole and was increased by the addition of an H2O2 generating system to the medium. Analysis of the protein content of the vesicles revealed the presence of 18 and 12-13 S thyroglobulin molecules, lacking mannose residues, and containing only monoiodotyrosine. It is concluded that in vitro, iodination can begin before exocytosis, in the smooth-surfaced vesicles derived from the golgi apparatus, as soon as N-acetylglucosamine is incorporated onto the pre-thyroglobulin molecule.     

Cation transport in vesicles from secreting rabbit stomach

K+ gradient-dependent rubidium flux in vesicles obtained from stimulated rabbit stomach distinguishes two cation pathways. Selective inhibition by vanadate and the (1,2-alpha)-imidazopyridine, SCH 28080 identifies one pathway as H,K-ATPase-mediated passive cation exchange. A second pathway, additive to the first, is inhibited by the protonophore, tetrachlorosalicylanilide and is identified as a K+ conductance pathway present in these vesicles. The conductance was limited to vesicle populations obtained from the stimulated rabbit gastric mucosa and was distributed into both a light microsomal fraction and a heavier membrane fraction. 86Rb+ transport through the cation conductance exhibited a trans-stimulated cation selectivity sequence of K+ greater than Rb+ = Cs+ much greater than Li+. Potential sensitive flux was inhibited by the cyanine dye 3,3'-dipropyl-2,2'-thiodicarbo cyanine iodide, Ba2+, quinine, and the guanidinium compound 1,8-bis-guanidinium-n-octane. The presence of the conductance was correlated with K+-dependent H+ transport which did not require prolonged equilibration in K+ medium for activation. A role for the stimulus-dependent K+ conductance in gastric acid secretion could be its provision of a pathway for net K+ movement to the luminal site of the H,K-ATPase.     

Sulfate transport in porcine thyroid cells. Effects of thyrotropin and iodide

In porcine thyroid cells, thyroglobulin sulfation is controlled by thyrotropin (TSH) and iodide, which contribute to regulating the intracellular sulfate concentration, as we previously established. Here, we studied the transport of sulfate and its regulation by these two effectors. Kinetic studies were performed after [(35)S]sulfate was added to either the basal or apical medium of cell monolayers cultured without any effectors, or with TSH with or without iodide. The basolateral uptake rates were about tenfold higher than the apical uptake rates. TSH increased the basolateral and apical uptake values (by 24 and 9%, respectively, compared with unstimulated cells), and iodide inhibited these effects of TSH. On the basis of results of the pulse-chase experiments, the basolateral and apical effluxes appeared to be well balanced in unstimulated cells and in cells stimulated by both TSH and iodide: approximately 40-50% of the intracellular radioactivity was released into each medium, whereas in the absence of iodide, 70% of the intracellular radioactivity was released on the basolateral side. The rates of transepithelial sulfate transport were increased by TSH compared with unstimulated cells, and these effects decreased in response to iodide. These results suggest that TSH and iodide may each control the sulfate transport process on two sides of the polarized cells, and that the absence of iodide in the TSH-stimulated cells probably results in an unbalanced state of sulfate transport.     

Active transport and accumulation of iodide by newly isolated marine bacteria

Iodide (I(-))-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive (125)I(-). When they were grown in a liquid medium containing 0.1 microM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 microM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 +/- 3.6 (mean +/- standard deviation) pmol of iodide per mg of dry cells and 5.5 x 10(3), respectively. In the presence of much higher concentrations of iodide (1 microM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 muM and 0.55 pmol min(-1) mg of dry cells(-1), respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO(3)(-)), was not transported.     

Trehalose-transporting membrane vesicles from yeasts.

We have isolated and characterized a membrane vesicle fraction from yeasts that is capable trehalose transport. The kinetics of the transport system were similar to those seen in the intact cells. The transport depends on a transmembrane pH gradient. If the gradient is collapsed, trehalose accumulated inside the vesicles is leaked into the medium. After aging for several days the ability of the vesicles to transport was lost. However, transport was partially restored by elevating internal pH in the vesicles.     

Transport of sulphate, thiosulphate and iodide by choroid plexus in vitro

—Isolated choroid plexuses of rabbits and cats were incubated in artificial cerebrospinal fluid medium containing [³⁵S]sulphate, [³⁵S]thiosulphate or [¹²⁵I]iodide and combinations thereof. After 1 hr incubation the mean ratio of tissue concentration to medium concentration was 2·46 for [³⁵S]sulphate, 2·39 for [³⁵S]thiosulphate, and 270 for [¹²⁵I]iodide. Uptake of all three anions was greatly reduced at 0° and by addition of dinitrophenol to the medium. Other inhibitors selectively reduced the uptake of particular anions; non-radioactive sulphate and thiosulphate reduced both [³⁵S]sulphate and [³⁵S]-thiosulphate uptake with much less effect on [¹²⁵I]iodide uptake, while non-radioactive iodide and thiocyanate greatly reduced [¹²⁵]iodide uptake with little or no effect on [³⁵S]sulphate or [³⁵S]thiosulphate uptake. It was concluded: (a) that sulphate and thiosulphate, like iodide, were accumulated by choroid plexus in vitro by active transport; (b) that sulphate and thiosulphate share and compete for a transport mechanism which is separate from the iodide transport mechanism; and (c) that the transport of sulphate out of cerebrospinal fluid demonstrated in vivo could occur at least in part in the choroid plexus.     

Leucine transport coupled to proton movement in membrane vesicles from Chang liver cells

Leucine transport into membrane vesicles obtained from Chang liver cells was stimulated by an inward H+ gradient. The stimulatory effect of the proton gradient on the rate of leucine uptake (1 min) was inhibited by the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. When the vesicles had been preloaded with a high concentration of KCl, addition of valinomycin stimulated leucine uptake by the vesicles, showing that the leucine transport is dependent on potential gradient. Leucine-coupled H+ accumulation inside the vesicles was confirmed by measuring leucine dependent quenching of the fluorescence of 9-aminoacridine added to medium. These results imply that electrochemical gradient of proton can serve as a driving force for leucine transport across the cell membrane and proton movement is coupled to leucine transport.     

Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.     

Modulation of ATP-dependent Ca2+ transport in rat parotid basolateral membrane vesicles by K+ + Cl- flux

In basolateral membrane vesicles (BLMV) isolated from rat parotid glands, the initial rate of ATP-dependent Ca2+ transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N-methyl-D-glucamine (NMDG)-gluconate. Only NH4+, Rb+, or Br- could effectively substitute for K+ or Cl-, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca2+ transport, while equilibration of the vesicles with KCl (greater than 100 mM) increased transport 15-20%. In presence of Cl-, 86Rb+ uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb+ flux by approx. 60% in a Cl- medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca2+ transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 45Ca2+ transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had a higher (approx. 25%) rate of Ca2+ transport than mannitol loaded vesicles. Addition of valinomycin (5 microM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca2+ transport rate. These results suggest that during ATP dependent Ca2+ transport in parotid BLMV, K+ can be recycled by the concerted activities of a K+ and Cl- coupled flux and a K+ conductance.     

Iodide transport in primary cultured thyroid follicle cells: evidence of a TSH-regulated channel mediating iodide efflux selectively across the apical domain of the plasma membrane.

The transport of iodide was studied in porcine thyroid follicle cells cultured in bicameral chambers. The continuous layer of polarized follicle cells, joined by tight junctions, formed a diffusion barrier between the two compartments (apical and basal) of the culture chamber. Uptake and efflux of 125I- at either surface (apical and basolateral) of the cells were thus possible to determine. Protein binding of iodide was inhibited by methimazole (10(-3) M) in all experiments. Radioiodide was taken up by the cells from the basal medium in a thyroid-stimulating hormone (TSH)-dose dependent manner with a maximal cell/medium ratio of 125I- of about 50 in cultures prestimulated with 0.1 to 1 mU/ml for 2 days. This uptake was inhibited by perchlorate and ouabain. In contrast, 125I- was not taken up from the apical medium. In preloaded cells, iodide efflux was rapidly (within 1-2 min) and dose-dependently (0.1-10 mU/ml) stimulated by TSH. Bidirectional measurements revealed that TSH stimulated iodide efflux in apical direction, leaving efflux in basal direction unchanged. In experiments with continuous uptake of label from the basal compartment, the TSH-stimulated efflux in apical direction had a duration of 4 to 6 min and resulted in a reduction in the cellular content of radioiodide by up to 80%. Decreased levels of cellular 125I- remained for at least 15 min after TSH addition. From our observations we conclude that the TSH-regulated uptake and efflux of iodide take place at opposite surfaces of the porcine thyroid follicle cell. Acutely stimulated iodide efflux is not the result of an increased permeability for iodide in the entire plasma membrane but only in the apical domain of this membrane. This implicates the presence of an iodide channel mediating TSH-stimulated efflux across the apical plasma membrane of the follicle cell. The mechanism is suggested to facilitate a vectorial transport of iodide in apical direction, i.e., to the lumen of the intact follicle.     

Phlorizin increases the permeability of intestinal mucosal membrane to sodium

We reported previously that when jejunal transmural glucose transport was inhibited by phlorizin the ratio of Na:glucose transport increased from 2.0:1 (in controls) to 3.3:1. To elucidate the mechanism of this increased ratio of Na:glucose transport, in the present study we have investigated the effect of phlorizin on Na uptake by brush border membrane vesicles and by everted sacs of hamster jejunum. In experiments on membrane vesicles the following observations were made. The time course of Na uptake showed that the control vesicles were in complete equilibrium with a Na-containing (100 mM) medium between 30 and 90 min incubation. In these periods of incubation, the vesicles incubated with phlorizin presumably also equilibrated with the medium, but lost their intravesicular Na during Millipore filtration and washing, and consequently the residual Na content was lower than that of controls. This effect of phlorizin was concentration dependent, and appeared to be unrelated to Na-coupled glucose transport, because it was also observed in the absence of glucose. This loss of Na during Millipore filtration and washing was also observed (i) when vesicles were equilibrated in a Na-containing solution in the absence of phlorizin and then exposed to a similar solution containing phlorizin, or (ii) when vesicles were equilibrated in a Na-containing solution in the presence of phlorizin and then washed repeatedly following Millipore filtration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence measurements of anion transport by the GABAA receptor in reconstituted membrane preparations

A fluorescence assay for measuring the functional properties of the GABAA receptor in reconstituted membrane vesicles is described. This assay is based on a method previously described to measure monovalent cation transport mediated by the nicotinic acetylcholine receptor in membranes from Torpedo electric organ [Moore, H.-P.H., & Raftery, M. A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4509-4513]. The GABAA receptor has been solubilized from bovine brain membranes and reconstituted into phospholipid vesicles. Influx of chloride or iodide into the vesicles has been measured in stopped-flow experiments by monitoring the fluorescence quench of an anion-sensitive fluorophore trapped within the vesicles. Muscimol, a GABAA receptor agonist, stimulated a rapid uptake of either chloride or iodide. Stimulation of chloride influx was dependent on the concentration of muscimol, and the midpoint of the dose-response curve occurred at approximately 0.3 microM. Agonist-stimulated uptake was enhanced by diazepam and blocked by desensitization and by the antagonists bicuculline and picrotoxin. These receptor-mediated effects are shown to be qualitatively similar to measurements of 36Cl- and 125I- efflux using synaptoneurosomes prepared from rat cerebral cortex. The advantages of the fluorescence method in terms of its improved time resolution, sensitivity, and suitability for quantitating GABAA receptor function are discussed.     

Regulation of gastric acid secretion via modulation of a chloride conductance

When gastric microsomes were purified from resting and stimulated rabbit mucosae, they were found to be generally similar in (H+ + K+)-ATPase activity, peptide composition in single-dimension sodium dodecyl sulfate-gel electrophoresis, and in size. In the stimulated vesicles, optimal proton transport activity was found at pH 7.4, 20-50 mM KCl, and 1 mM ATP-Mg. However, in the case of resting vesicles, the presence of valinomycin and an inward Cl-gradient was also necessary for Mg-ATP-dependent proton transport. Measurement of K+ and Cl-diffusion potentials using 3,3-dipropylthiadicarboxocyanine iodide as a potential sensitive dye showed that both resting and stimulated vesicles developed K+ gradient-dependent potentials in the presence of an impermeant anion, but that Cl- gradient-dependent potentials were observed only in the stimulated preparation. 86Rb+ self-exchange was found in both types of vesicles, but Cl- self-exchange was confined to vesicles derived from stimulated mucosae. Putative inhibitors of anion conductance such as furosemide and anthracene 9-carboxylic acid blocked proton transport, Cl- conductance, 36Cl- uptake, and Cl- exchange. The inhibition of proton transport was overcome by valinomycin. ATPase activity in the presence of nigericin, an H+:K+ exchanger, was unaffected by these inhibitors. K+ conductance, Rb+ uptake, and Rb+ exchange were insensitive to these inhibitors. Thus, activation of acid secretion by the stimulated parietal cell appears to involve at least the appearance of a discrete Cl- conductance in the pump-associated membrane.     

Polarization of thyroid cells in culture: evidence for the basolateral localization of the iodide "pump" and of the thyroid-stimulating hormone receptor-adenyl cyclase complex

When cultured in collagen gel-coated dishes, thyroid cells organized into polarized monolayers. The basal poles of the cells were in contact with the collagen gel, whereas the apical surfaces were facing the culture medium. Under these culture conditions, thyroid cells do not concentrate iodide nor respond to acute stimulation by thyroid-stimulating hormone (TSH). To allow the free access of medium components to the basal poles, the gel was detached from the plastic dish and allowed to float in the culture medium. After release of the gel, the iodide concentration and acute response to TSH stimulation were restored. Increased cAMP levels, iodide efflux, and formation of apical pseudopods were observed. When the thyroid cells are cultured on collagen-coated Millipore filters glued to glass rings, the cell layer separates the medium in contact with the apical domain of the plasma membrane (inside the ring) from that bathing the basolateral domain (outside the ring). Iodide present in the basal medium was concentrated in the cells, whereas no transport was observed when iodide was added to the luminal side. Similarly, an acute effect of TSH was observed only when the hormone was added to the basal medium. These results show that the iodide concentration mechanism and the TSH receptor-adenylate cyclase complex are present only on the basolateral domain of thyroid cell plasma membranes.     

Peptide transport in rabbit kidney. Studies with l-carnosine

l-Carnosine was shown to be transported into rabbit renal brush-border membrane vesicles by an Na⁺ - independent mechanism. The transport was competitively inhibited by glycyl-l-proline. Various di- and tripeptides inhibited l-carnosine transport, whereas free amino acids did not. Inhibition studies showed that blocking the free amino and carboxyl groups of the peptide reduced its affinity for the transport carrier. Under the conditions in which there was no detectable hydrolysis of l-carnosine in the medium, intravesicular contents showed a 30% hydrolysis of the peptide within the vesicles. Disruption of membrane vesicles with deoxycholate resulted in a 3-fold increase in l-carnosine hydrolyzing activity over untreated intact vesicles. Based on these observations, a model for peptide transport is proposed in which transport of the intact peptide across the membrane is followed by its partial or complete hydrolysis by a membrane peptidase whose active site is on the cytoplasmic side of the membrane.     

Electrogenic character of the functioning of H+-ATPase of synaptic vesicles in the rat brain

Transmembrane potential (TMP) of synaptic vesicles (SV) from the rat brain was registered by means of potential-sensitive fluorescent probe 3,3' dipropylthiocarbocyanine iodide (dis-C3-(5)). It was found that Mg-ATP generates TMP on the SV membrane suspended in sucrose-buffer medium. TMP did not appear if 0.25 M sucrose medium was replaced by 0.125 M KCl medium or 0.125 NaCl medium, or if the SV membrane was disrupted by triton X-100 or alamethicin, or if there was protonophore m-chlorophenylhydrazone or N-ethylmaleimide influence on SV. The data obtained indicate that functioning of SV proton pump is electrogenic and the level of TMP depends on the presence of anion Cl- in the incubation medium.     

Active Transport and Accumulation of Iodide by Newly Isolated Marine Bacteria

Iodide (I⁻)-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive ¹²⁵I⁻. When they were grown in a liquid medium containing 0.1 μM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 μM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 ± 3.6 (mean ± standard deviation) pmol of iodide per mg of dry cells and 5.5 × 10³, respectively. In the presence of much higher concentrations of iodide (1 μM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 μM and 0.55 pmol min⁻¹ mg of dry cells⁻¹, respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO₃⁻), was not transported.     

Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles.

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.     

Effect of the glycosphingolipid, GM1 on localization of dibucaine in phospholipid vesicles: a fluorescence study

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC) containing different mole percents of monosialoganglioside (GM1) has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in the presence of phospholipid vesicles containing various amounts of GM1 yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more hydrophobic and rigid in membranes that contain GM1 than in membranes without it. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed reduced quenching efficiency with the increase in GM1 content of the vesicles, demonstrating lesser accessibility of the iodide quenchers to dibucaine in the presence of GM1, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of 1 and 2.8–3.1 ns with mean relative contributions of 25 and 75%, respectively. The mean lifetime in vesicles was 20–30% lower than in the aqueous medium and showed a definite increase in presence of GM1 from that in the absence of it. All the spectral properties point that dibucaine encountered regions of membrane containing significant amount of GM1 and penetrated deeper in hydrophobic core of the bilayer.     

A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K+ exhibited high levels of Na+-dependent, alpha-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na+-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by D-glucose, D-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na+-dependent amino acid transport. The use of Na+ in the reconstitution procedure led to formation of liposomes which displayed little Na+-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K+ medium.