Anion Transport Systems: Continuous Monitoring of Transport by Fluorescence (CMTF) in Cells and Vesicles

1991 ◽  
pp. 341-367
Author(s):  
Z. Ioav Cabantchik ◽  
Ofer Eidelman
Keyword(s):  
Continuous Monitoring ◽  
Anion Transport ◽  
Transport Systems ◽  
In Cells

scholarly journals BetS Is a Major Glycine Betaine/Proline Betaine Transporter Required for Early Osmotic Adjustment in Sinorhizobium meliloti

2002 ◽  
Vol 184 (10) ◽  
pp. 2654-2663 ◽  
Author(s):  
Alexandre Boscari ◽  
Karine Mandon ◽  
Laurence Dupont ◽  
Marie-Christine Poggi ◽  
Daniel Le Rudulier
Keyword(s):  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Transport Systems ◽  
High Osmolarity ◽  
Carnitine Transporter ◽  
Rapid Acquisition ◽  
Common Structure ◽  
Pcr Product ◽  
Transport Kinetic ◽  
In Cells

ABSTRACT Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with Km s of 16 ± 2 μM and 56 ± 6 μM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na+ driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. β-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.

Transport of L-tyrosine by B16/F10 melanoma cells: the effect of the intracellular content of other amino acids

1990 ◽  
Vol 97 (3) ◽  
pp. 479-485
Author(s):  
J.R. Jara ◽  
J.H. Martinez-Liarte ◽  
F. Solano ◽  
R. Penafiel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Initial Rate ◽  
Aromatic Amino Acids ◽  
Melanoma Cells ◽  
Transport Systems ◽  
Specific Amino Acid ◽  
Intracellular Content ◽  
Tyrosine Hydroxylation ◽  
In Cells

The uptake of L-Tyr by B16/F10 malignant melanocytes in culture has been studied. These melanoma cells can either be depleted of amino acids by 1 h preincubation in Hanks' isotonic medium or preloaded with a specific amino acid by 1 h preincubation in the same solution containing 2 mM of the amino acid to be preloaded. By means of these pretreatments, it is shown that the rate of L-Tyr uptake is greatly dependent on the content of other amino acids inside the cells. The L-Tyr uptake is higher in cells preloaded with amino acids transported by the L and ASC systems than in cells depleted of amino acids or preloaded with amino acids transported by the A system. It is concluded that L-Tyr is mainly taken up by an exchange mechanism with other amino acids mediated by the L1 system, although the ASC system can also participate in the process. In agreement with that, the homo-exchange performed by cells preloaded with unlabelled L-Tyr is more efficient than any other hetero-exchange, although L-Dopa, the product of tyrosine hydroxylation in melanin synthesis, is almost as efficient as L-Tyr. Apart from aromatic amino acids, melanoma cells preloaded with L-Met and L-His also yield a high initial rate of L-Tyr uptake. The results herein suggest that melanoma cells do not have transport systems specific for L-Tyr, even if this amino acid is needed to carry out the differential pathway of this type of cells, melanosynthesis.

Active HCO3− transport in cyanobacteria

1991 ◽  
Vol 69 (5) ◽  
pp. 936-944 ◽  
Author(s):  
George S. Espie ◽  
Anthony G. Miller ◽  
Ramani A. Kandasamy ◽  
David T. Canvin
Keyword(s):  
Inorganic Carbon ◽  
Dissolved Inorganic Carbon ◽  
Reaction Medium ◽  
Transport Systems ◽  
Bicarbonate Transport ◽  
Key Words Cyanobacteria ◽  
Sodium Dependent ◽  
Rate Of Photosynthesis ◽  
Low Levels ◽  
In Cells

Cyanobacteria possess systems for the active transport of both CO2 and HCO3−. While the active CO2 transport system seems to be present in cells grown on all levels of CO2 or dissolved inorganic carbon, the bicarbonate transport systems are only present in cells grown on low levels of CO2 or dissolved inorganic carbon (air levels or lower). Active bicarbonate transport can be shown to occur when the rate of photosynthesis exceeds that which could be sustained by the production of CO2 from the dehydration of bicarbonate or when CO2 transport is inhibited with carbon oxysulfide or hydrogen sulfide. Two systems for active bicarbonate transport have been identified: one is dependent on the presence of millimolar concentrations of sodium, and the other is independent of the sodium requirement. Cells grown with air bubbling normally possess the first whereas cells grown in standing culture normally possess the second. The sodium-dependent bicarbonate transport can be inhibited by omitting sodium from the reaction medium or competitively with lithium when sodium is present. Monensin and amiloride also inhibit sodium-dependent bicarbonate transport. It does not appear to be inhibited by ethoxyzolamide. The inhibition of sodium-independent bicarbonate transport is not yet established. Bicarbonate transport appears to have no effect on CO2 transport and CO2 transport appears to have no effect on bicarbonate transport. Hence, the transport systems seems to be independent. Although a number of mechanisms have been proposed for bicarbonate transport, the experimental data are not sufficient to clearly distinguish between them. Key words: cyanobacteria, active CO2 transport, active HCO3− transport, photosynthesis, sodium.

scholarly journals Calpastatin in erythrocytes of young and old individuals

1994 ◽  
Vol 304 (2) ◽  
pp. 365-370 ◽  
Author(s):  
N Schwarz-Benmeir ◽  
T Glaser ◽  
S Barnoy ◽  
N S Kosower
Keyword(s):  
Molecular Mass Distribution ◽  
Intact Cell ◽  
Specific Inhibitor ◽  
Anion Transport ◽  
Erythrocyte Membranes ◽  
Specific Antibodies ◽  
Induced Changes ◽  
Initial Binding ◽  
Enhanced Degradation ◽  
In Cells

To gain knowledge about the behaviour of calpastatin (the specific inhibitor of the Ca(2+)-dependent thiol protease calpain) in the intact cell, we analysed the inhibitor by specific antibodies and determined its activity in erythrocytes from individuals 20-34 years old (young) and 70-93 years old (old). Differences between old and young in the behaviour of erythrocyte calpastatin were observed. Erythrocytes of old individuals had lower amounts of calpastatin and less calpastatin activity than those of young ones. A difference between old and young was also found in the molecular-mass distribution of calpastatin subunits. Increasing the erythrocyte Ca2+ induced changes in calpastatin in young individuals, rendering it similar to calpastatin in cells of old individuals. When calpastatin (isolated from erythrocytes of a young individual) was added to erythrocyte membranes, the initial binding and subsequent association of calpastatin with the membrane were lower in old than in young individuals. We had previously found that calpain binding and activation were enhanced in erythrocyte membranes from old individuals, along with enhanced degradation of band 3 (a major erythrocyte transmembrane anion-transport protein). The overall results indicate an interaction of calpain with calpastatin in the intact cell. Enhanced activation of erythrocyte calpain and degradation of calpastatin occur under conditions of increased cellular Ca2+ and in cells of the aged.

Two pathways for choline transport in eel erythrocytes: a saturable carrier and a volume-activated channel

1994 ◽  
Vol 267 (3) ◽  
pp. R773-R779 ◽  
Author(s):  
S. E. Joyner ◽  
K. Kirk
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Anguilla Anguilla ◽  
Anion Transport ◽  
Response Curves ◽  
Choline Transport ◽  
Human Red Blood Cells ◽  
Selective Channel ◽  
Transport Inhibitors ◽  
In Cells

Choline transport in eel (Anguilla anguilla) erythrocytes was investigated in cells suspended in isotonic and hypotonic media. In cells in isosmotic solution choline transport was mediated by a saturable system with a Michaelis constant (Km; 62 +/- 6 microM) similar to that of the choline carrier of human erythrocytes but a maximal transport rate (Vmax; 4.5 +/- 0.4 mmol.1 red blood cells-1.h-1) almost two orders of magnitude higher than that in human red blood cells. This pathway was inhibited by hemicholinium-3 and dodecyltrimethylammonium, but not by any of a range of anion transport inhibitors tested. Swelling the cells by suspending them in hyposmotic media activated a second choline transport component that was kinetically and pharmacologically distinct from the saturable system. The volume-activated component was nonsaturable (up to 50 mM choline). It was not inhibited by hemicholinium-3 or dodecyltrimethylammonium but was inhibited by anion transport inhibitors, the most potent of which was 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; half-maximal inhibitory concentration = 14 microM). Dose-response curves for the effect of NPPB on swelling-activated choline transport and the swelling-activated transport of taurine, a sulfonic amino acid, were superimposable. It is postulated that the transport of choline and taurine (as well as that of other small organic solutes) in osmotically swollen fish erythrocytes is mediated by a volume-activated, anion-selective channel.

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