Search PubMed⌕ Search

Biomedical subjects

B Poolman

Publications and source records attributed to B Poolman.

117 records · Page 7Linked to original sources
PubMed1987

Regulation of the glutamate-glutamine transport system by intracellular pH in Streptococcus lactis.

Various methods of manipulation of the intracellular pH in Streptococcus lactis result in a unique relationship between the rate of glutamate and glutamine transport and the cytoplasmic pH. The initial rate of glutamate uptake by S. lactis cells increases more than 30-fold when the intracellular pH is raised from 6.0 to 7.4. A further increase of the cytoplasmic pH to 8.0 was without effect on transport. The different levels of inhibition of glutamate and glutamine transport at various external pH values by uncouplers and ionophores, which dissipate the proton motive force, can be explained by the effects exerted on the intracellular pH. The dependence of glutamate transport on the accumulation of potassium ions in potassium-filled and -depleted cells is caused by the regulation of intracellular pH by potassium movement.

Biological Transport, Active↗
PubMed1987

Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris.

In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 +/- 0.3 microM, independent of the extracellular pH. The KTS for glutamate uptake are 3.5, 11.2, 77, and 1200 microM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTS of 1.8 +/- 0.5 microM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 X 10(3) for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and greater than 30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the the proton motive force.

Adenosine Triphosphate↗
PubMed1983

The location of redox-sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli.

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane. Treatment of right-side-out membrane vesicles from E. coli ML 308-225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the delta approximately mu H+. Subsequent addition of reducing agents restored proline transport activity. The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide. Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a delta approximately mu H+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group. Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a delta approximately mu H+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.

Arsenicals↗
PubMedDate not supplied

Peptides and ATP binding cassette peptide transporters.

In this review our knowledge of ATP binding cassette (ABC) transporters specific for peptides is discussed. Besides serving a role in nutrition of the cell, the systems participate in various signaling processes that allow (micro)organisms to monitor the local environment. In bacteria, these include regulation of gene expression, competence development, sporulation, DNA transfer by conjugation, chemotaxis, and virulence development, and the role of ABC transporters in each of these processes is discussed. Particular attention is paid to the specificity determinants of peptide receptors and transporters in relation to their structure and to the mechanisms of peptide binding.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis.

The di-tripeptide transport system (DtpT) of Lactococcus lactis was purified to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C10E8), followed by solubilization with n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin. The DtpT protein was reconstituted into detergent-destabilized preformed liposomes prepared from E. coli phospholipid/phosphatidylcholine. A variety of detergents were tested for their ability to mediate the membrane reconstitution of DtpT and their effectiveness to yield proteoliposomes with a high transport activity. The highest activities were obtained with TX100, C12E8 and DM, whereas DDM yielded relatively poor activities, in particular when this detergent was used at concentrations beyond the onset of solubilization of the preformed liposomes. Parallel with the low activity, significant losses of lipid were observed when the reconstitution was performed at high DDM concentrations. This explained at least part of the reduced transport activity as the DtpT protein was highly dependent on the final lipid-to-protein ratios in the proteoliposomes. Consistent with the difference in mechanism of DDM- and TX100-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when TX100 was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.

Bacterial Proteins↗
PubMedDate not supplied

The application of pH-sensitive fluorescent dyes in lactic acid bacteria reveals distinct extrusion systems for unmodified and conjugated dyes.

Intracellular pH in bacteria can be measured efficiently between internal pH values of 6.5 and 8.5 with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and-6]-carboxyfluorescein (BCECF). A new fluorescent pH probe with a lower pKa(app) than BCECF was synthesized from fluorescein isothiocyanate and glutamate. The new probe, N-(fluorescein thio-ureanyl)-glutamate (FTUG), was much less sensitive to changes in concentrations of KCl than was BCECF. Similar to BCECF, an efflux of FTUG independent of the proton motive force, but dependent on ATP, was observed both in Lactobacillus plantarum and Lactococcus lactis. Corrections for probe efflux allowed accurate measurements of the pHin. Similar intracellular pH values were determined with FTUG and BCECF, in the range where both probes can be applied, and the pH values correlated well with those estimated from the distribution of radio-labelled benzoic acid. Since FITC can easily be coupled to substrates containing an amino group, it is possible to develop other FITC derivatives as well. The mechanisms of probe excretion and the nature of the excreted product(s) were studied in further detail for BCECF and FTUG. BCECF was excreted from wild-type L. lactis in an unmodified form as was determined by chromatographic and mass spectrometry analysis. In the case of FTUG, the excreted product was a conjugated derivative. Unmodified FTUG was not excreted, although it was present in cellular extracts from L. lactis. Exit of BCECF was completely inhibited in a BCECF efflux mutant (Bef-) of L. lactis, whereas FTUG-conjugate efflux in this mutant was similar to the wild-type. Addition of indomethacin, a known inhibitor of BCECF efflux in human epithelial cells, resulted in complete inhibition of BCECF efflux in wild-type L. lactis, whereas FTUG-conjugate exit was only slightly affected. The results of the mutant and inhibitor studies suggest that FTUG-conjugate and BCECF efflux in L. lactis are mediated by different ATP-driven extrusion systems for organic anions.

Fluorescein-5-isothiocyanate↗