Biomedical subjects

W N Konings

Publications and source records attributed to W N Konings.

At least 19 recordsLinked to original sources

PubMed1992

Reconstitution of the leucine transport system of Lactococcus lactis into liposomes composed of membrane-spanning lipids from Sulfolobus acidocaldarius.

The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the bilayer lipid phosphatidylcholine (PC), analyzed on their miscibility, and fused with membrane vesicles from L. lactis. Freeze-fracture electron microscopy demonstrates that the bipolar lipids in the hybrid membranes adopted a monomolecular organization at high S. acidocaldarius lipid content. Leucine transport activity (i.e., delta mu H(+)-driven and counterflow uptake) increased with the content of S. acidocaldarius lipids and was optimal at a one-to-one (w/w) ratio of PC to S. acidocaldarius lipids. Membrane fluidity decreased with increasing S. acidocaldarius lipid content. These data suggest that transport proteins can be functionally reconstituted into membranes composed of membrane-spanning lipids provided that membrane viscosity is restricted.

Biological Transport↗

PubMed1992

What we can learn from the effects of thiol reagents on transport proteins.

Many secondary membrane transport systems contain reactive sulfhydryl groups. In this review the applications of SH reagents for analyzing the role of sulfhydryl groups in membrane transport systems will be discussed. First an overview will be given of the more important reagents, that have been used to study SH-groups in membrane transport systems, and examples will be given of transport proteins in which the role of cysteines have been analyzed. An important application of SH-reagents to label transport proteins using various SH-reagents modified with fluorescent- or spin-label moieties will be discussed. Two general models are shown which have been proposed to explain the role of sulfhydryl groups in some membrane transport systems.

Amino Acid Sequence↗

PubMed1992

Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N.

The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated M(r) of 95,368, which agrees with the apparent M(r) of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis.

Amino Acid Sequence↗

PubMed1992

Effect of the unsaturation of phospholipid acyl chains on leucine transport of Lactococcus lactis and membrane permeability.

The effect of the degree of unsaturation of the phospholipid acyl chains on the branched-chain amino acid transport system of Lactococcus lactis was investigated by the use of a membrane fusion technique. Transport activity was analyzed in hybrid membranes composed of equimolar mixtures of synthetic unsaturated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in which the number of cis double bonds in the 18-carbon acyl chains was varied. The accumulation level and initial rate of both counterflow and protonmotive-force driven transport of leucine decreased with increasing number of double bonds. The reduction in transport activity with increasing number of double bonds correlated with an increase in the passive permeability of the membranes to leucine. The membrane fluidity was hardly affected by the double bond content. It is concluded that the degree of lipid acyl chain unsaturation is a minor determinant of the activity of the branched chain amino acid transport system, but effects strongly the passive permeability of the membrane.

Biological Transport↗

PubMed1992

Nucleotide sequence and functional properties of a sodium-dependent citrate transport system from Klebsiella pneumoniae.

The gene of the sodium-dependent citrate transport system from Klebsiella pneumoniae (citS) is located on plasmid pES3 (Schwarz, E., and Oesterhelt, D. (1985) EMBO J. 4, 1599-1603) and encodes a 446-amino acid protein. Transport of citrate via this citrate transport protein (CitS) is dependent on the presence of sodium ions and is inhibited by magnesium ions. The delta pH (pH gradient across the membrane) is the major driving force for uptake. It is postulated that, in analogy with the proton-dependent citrate carrier (CitH) of K. pneumoniae (van der Rest, M. E., Abee, T., Molenaar, D., and Konings, W. N. (1990) Eur. J. Biochem. 195, 71-77), only one of the protonated species of citrate is recognized by CitS and that citrate is translocated across the membrane in symport with protons and sodium ions. The hydrophobicity profile of CitS suggests that the protein is very hydrophobic and contains 12 membrane-spanning segments. These segments are not centered around a hydrophilic core as has been suggested for other transport proteins, but the protein is asymmetrical with seven transmembrane segments in front of a large hydrophilic loop and five after this loop. The amino acid sequence is highly similar to a citrate transport system of Lactococcus lactis subsp. lactis var. diacetylactis (CitP) (David, S., van der Rest, M. E., Driessen, A. J. M., Simons, G., and de Vos, W. M. (1990) J. Bacteriol. 172, 5789-5794) and less similar to CitH of K. pneumoniae. We conclude that the citS gene of K. pneumoniae encodes a sodium-dependent citrate transport system that belongs to a novel subclass of transport proteins.

Amino Acid Sequence↗

PubMed1992

The stability and functional properties of proteoliposomes mixed with dextran derivatives bearing hydrophobic anchor groups.

Liposomes composed of Escherichia coli phospholipid were coated with polysaccharides bearing hydrophobic palmitoyl anchors. The effect on the stability of liposomes without or with integral membrane proteins was investigated. A high concentration of hydrophobized dextrans protected the liposomes against detergent degradation, decreased the fluidity of the membranes, prevented fusion of the liposomes and enhanced their stability. Proteoliposomes containing beef heart cytochrome-c oxidase and the lactose transport carrier of E. coli were similarly affected by coating with the dextrans. Under these conditions both membrane proteins were still active. Long-term stability of the coated liposomes was obtained only in the absence of the integral membrane proteins.

Animals↗

PubMed1992

Spectral identification of the electrochromically active carotenoids of Rhodobacter sphaeroides in chromatophores and reconstituted liposomes.

Reaction centers with both light harvesting complexes I and II (B875 and B800/850; i.e., RCLHILHII complexes) have been isolated from Rhodobacter sphaeroides. These complexes have been incorporated into liposomes made from lipids purified from Escherichia coli. The electrochromic bandshift of carotenoids, present in these reconstituted complexes, shows shifted minima and maxima with respect to a similar spectrum in chromatophores of Rb. sphaeroides in a potassium diffusion potential induced difference spectrum (see also Crielaard, W., Hellingwerf, K.J. and Konings, W.N. (1989) Biochim. Biophys. Acta 973, 205-211). The absorbance spectrum, at room temperature or at 77 K, of both membrane preparations did not, however, reveal differences in the carotenoid region. The long-wavelength carotenoid peak in both preparations is located at 513 nm (77 K). A small difference could be observed between the 77 K excitation spectra of the B850 fluorescence. Reconstituted complexes show a carotenoid peak at 513 nm, whereas in chromatophores this peak is located at 514.5 nm. When fluorescence was recorded at 805 nm, to detect B800 excitation, there was a marked difference between both preparations. In liposomes the long wavelength B800-associated carotenoid peak is located at 512.5 nm, whereas in chromatophores this peak is located at 516 nm. These results explain the shifted minima and maxima in a potassium diffusion induced difference spectrum in proteoliposomes. The prediction of two carotenoid pools in chromatophores (De Grooth, B.G. and Amesz, J. (1977) Biochim. Biophys. Acta 462, 247-258) is confirmed, and the field sensitive carotenoids are identified as the pool that is associated with the B800 band (Kramer, H.J.M., Van Grondelle, R., Hunter, C.N., Westerhuis, W.H.J. and Amesz, J. (1984) Biochim. Biophys. Acta 765, 156-165).

Bacterial Chromatophores↗

PubMed1992

Acidic phospholipids are required during solubilization of amino acid transport systems of Lactococcus lactis.

The branched-chain amino acid transport system of Lactococcus lactis was solubilized with n-octyl beta-D-gluco-pyranoside and reconstituted into proteoliposomes. Transport activity was recovered only when solubilization was performed in the presence of acidic phospholipids. Omission of acidic phospholipids during solubilization resulted in an inactive transport protein and the activity could not be restored in the reconstitution step. Similar results have been obtained for the arginine/ornithine exchange protein from Pseudomonas aeruginosa and L. lactis. Functional reconstitution of the transport protein requires the presence of aminophospholipids or glycolipids in the liposomes (Driessen, A.J.M., Zheng, T., In't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). We propose that during the detergent solubilization the acidic phospholipids protect the transport systems against denaturation by preventing delipidation.

Amino Acids, Branched-Chain↗

PubMed1992

Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis.

Lactococcus lactis strains produce an extracellular subtilisin-related serine proteinase in which immunologically different components can be distinguished. Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified. By Western-blot analysis it was found that all monoclonal antibodies recognize all denatured proteinase components. The distinction between the different components could be made under native conditions only, indicating that binding regions are masked in the native molecule. In a L. lactis proteinase which was inactivated by the substitution Asp30----Asn under native conditions, only one epitope could be detected. This demonstrates that autoproteolytic activity is required to make specific binding regions accessible for (monoclonal) antibodies.

Amino Acid Sequence↗

PubMed1992

Functional reconstitution of membrane proteins in monolayer liposomes from bipolar lipids of Sulfolobus acidocaldarius.

Membranes of Sulfolobus acidocaldarius, an extreme thermophilic archaebacterium, are composed of unusual bipolar lipids. They consist of macrocyclic tetraethers with two polar heads linked by two hydrophobic C40 phytanyl chains which are thought to be arranged as a monolayer in the cytoplasmic membrane. Fractionation of a total lipid-extract from S. acidocaldarius yielded a lipid fraction which forms closed and stable unilamellar liposomes in aqueous media. Beef heart cytochrome c-oxidase could be functionally reconstituted in these liposomes. In the presence of reduced cytochrome c, a protonmotive force (delta p) across the liposomal membrane was generated of up to -92 mV. Upon fusion of these proteoliposomes with membrane vesicles of Lactococcus lactis, the delta p generated by cytochrome c-oxidase activity was capable to drive uphill transport of leucine. Electron microscopic analysis indicated that the tetraether lipids form a single monolayer liposome. The results demonstrate that tetraether lipids of archaebacteria can form a suitable matrix for the function of exogenous membrane proteins originating from a regular lipid bilayer.

Animals↗

PubMed1992

Energy transduction and transport processes in thermophilic bacteria.

Bacterial growth at the extremes of temperature has remained a fascinating aspect in the study of membrane function and structure. The stability of the integral membrane proteins of thermophiles make them particularly amenable to study. Respiratory enzymes of thermophiles appear to be functionally similar to the mesophilic enzymes but differ in their thermostability and unusual high turnover rates. Energy coupling at extreme temperatures seems inefficient as suggested by the high maintenance coefficients and the high permeability of the cell membrane to protons. Nevertheless, membranes maintain their structure at these extremes through changes in fatty acid acyl chain composition. Archaebacteria synthesize novel membrane-spanning lipids with unique physical characteristics. Thermophiles have adapted to life at extreme temperatures by using sodium ions rather than protons as coupling ions in solute transport. Genetic and biochemical studies of these systems now reveal fundamental principles of such adaptations. The recent development of reconstitution techniques using membrane-spanning lipids allows a rigorous biochemical characterization of membrane proteins of extreme thermophiles in their natural environment.

Archaea↗

PubMed1992

Characterization and functional expression in Escherichia coli of the sodium/proton/glutamate symport proteins of Bacillus stearothermophilus and Bacillus caldotenax.

The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltTBs) and Bacillus caldotenax (gltTBc) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen. The nucleotide sequences of the gltTBs and gltTBc genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltTBs and GltTBc). Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions. These expression signals were functional in E. coli. The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions. The Na+/H+ coupled L-glutamate symport proteins GltTBs and GltTBc are homologous to the strictly H+ coupled L-glutamate transport protein of E. coli K-12 (overall 57.2% identity). Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles. In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons.

Amino Acid Sequence↗

PubMed1992

Can the excretion of metabolites by bacteria be manipulated?

Bacteria can release metabolites into the environment by various mechanisms. Excretion may occur by passive diffusion or by the reversal of the uptake process when the internal concentration of the metabolite exceeds the thermodynamic equilibrium level. In other cases, solutes are excreted against the concentration gradient by special extrusion systems. Their mode of energy coupling is different to that of the well-studied group of uptake systems. A thorough understanding of the transport processes will help to improve the excretion of metabolites of commercial interest, allow a more efficient production of metabolites in bulk quantities, and permit their exploitation to establish new markets.

Bacteria↗

PubMed1992

The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis.

Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.

Adenosine Triphosphate↗

PubMed1992

Mechanism of Na(+)-dependent citrate transport in Klebsiella pneumoniae.

Citrate transport via CitS of Klebsiella pneumoniae has been shown to depend on the presence of Na+. This transport system has been expressed in Escherichia coli, and uptake of citrate in E. coli membrane vesicles via this uptake system was found to be an electrogenic process, although the pH gradient is the main driving force for citrate uptake (M. E. van der Rest, R. M. Siewe, T. Abee, E. Schwartz, D. Oesterhelt, and W. N. Konings, J. Biol. Chem. 267:8971-8976, 1992). Analysis of the affinity constants for the different citrate species at different pH values of the medium indicates that H-citrate2- is the transported species. Since the electrical potential across the membrane is a driving force for citrate transport, this indicates that transport occurs in symport with at least three monovalent cations. Citrate efflux is stimulated by Na+ concentrations of up to 5 mM but inhibited by higher Na+ concentrations. Citrate exchange, however, is stimulated by all Na+ concentrations, indicating sequential events in which Na+ binds before citrate for translocation followed by a release of Na+ after release of citrate. CitS has, at pH 6.0 and in the presence of 5 mM citrate on both sides of the membrane, an apparent affinity (K(app)) for Na+ of 200 microM. The Na+/citrate stoichiometry was found to be 1. It is postulated that H-citrate2- is transported via CitS in symport with one Na+ and at least two H+ ions.

Biological Transport↗

PubMed1992

Isolation and characterization of the high-affinity K(+)-translocating ATPase from Rhodobacter sphaeroides.

Cells of the purple nonsulfur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. A vanadate-sensitive, K(+)-stimulated and Mg(2+)-stimulated ATPase was purified from membranes of these cells by solubilization with decyl-beta-D-maltoside in the presence of Escherichia coli phospholipids followed by triazine-dye affinity chromatography. This primary transport system has a substrate specificity and an inhibitor sensitivity closely similar to those of the Kdp ATPase from E. coli and is composed of three subunits with molecular masses of 70.0, 43.5, and 23.5 kDa.

Adenosine Triphosphatases↗

PubMed1992

arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger.

In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathway, which allows slow growth on rich media. The conversion of arginine to ornithine, CO2, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase pathway are organized in the arcDABC operon. The arcD gene encodes a hydrophobic polytopic membrane protein. Translocation of arginine and ornithine in membrane vesicles derived from an Escherichia coli strain harboring a recombinant plasmid carrying the arcD gene was studied. Arginine and ornithine uptake was coupled to the proton motive force with a bias toward the transmembrane electrical potential. Accumulated ornithine was readily exchangeable for external arginine or lysine. The exchange was several orders of magnitude faster than proton motive force-driven transport. The ArcD protein was reconstituted in proteoliposomes after detergent solubilization of membrane vesicles. These proteoliposomes mediate a stoichiometric exchange between arginine and ornithine. It is concluded that the ArcD protein is a transport system that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway.

Amino Acid Transport Systems↗

PubMed1992

Revised nucleotide sequence of the gltP gene, which encodes the proton-glutamate-aspartate transport protein of Escherichia coli K-12.

The gene encoding the proton-glutamate carrier (GltP) of Escherichia coli K-12 was sequenced, and the primary structure of the protein was analyzed. The nucleotide sequence was found to differ in several aspects from the previously published sequence (B. Wallace, Y. Yang, J. Hong, and D. Lum, J. Bacteriol. 172:3214-3220, 1990). The corrected open reading frame encodes a protein of 437 (instead of 395) amino acids. Hydropathy analysis predicts 12 membrane-spanning alpha-helical regions. The complementary strand does contain an open reading frame possibly encoding a highly hydrophilic polypeptide of 272 amino acids.

Amino Acid Sequence↗