Wilhelmus Konings

NarK is a nitrite-extrusion system involved in anaerobic nitrate respiration by Escherichia coli

Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration. A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter. The longest‐lived radioactive isotope of nitrogen, 13N‐nitrate (half‐life = 9.96 min) and the nitrite‐sensitive fluorophore N‐(ethoxycarbonylmethyl)‐6‐methoxyquinolinium bromide have now been used to define the function of NarK. At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange. This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase.

Mutacin II, a Bactericidal Lantibiotic from Streptococcus mutans

Mutacin II is an antibiotic that is produced by group II Streptococcus mutans. It inhibits the growth of other streptococci as well as many other gram-positive microorganisms by a hitherto unknown mechanism. Mutacin II possess bactericidal activity against susceptible cells. It transiently depolarizes the transmembrane electrical potential (delta psi) and the transmembrane pH gradient (delta pH) and partially inhibits amino acid transport. However, it rapidly depletes the intracellular ATP pool in glucose-energized cells and prevents the generation of ATP. It is concluded that mutacin II does not belong to the group of pore-forming antibiotics (type A) or to the type B antibiotics, which inhibit phospholipases or interfere with peptidoglycan biosynthesis. Mutacin II acts by inhibiting essential enzyme functions at the level of metabolic energy generation, an activity that has not yet been classified for antibiotics.

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.

Uptake of Methylamine Via an Inducible, Energy-Dependent Transport System in the Facultative Methylotroph Arthrobacter P1

Cytoplasmic membrane vesicles were prepared by a lysozyme-salt treatment from Arthrobacter P1 grown on methylamine as the carbon and energy source. In the presence of an ascorbate-phenazine methosulphate electron donor system, these vesicles accumulated methylamine in unmodified form by an inducible transport system. This system has a high affinity for methylamine (Kapp=20–25 μM). The effect of the ionophores valinomycin and nigericin combined with membrane potential (Δψ) and pH-gradient (ΔpH) measurements demonstrated that methylamine uptake is electrogenic and driven by the Δψ. Optimal activity is observed at pH 6.5 and 30°C. Methylamine uptake was not affected by the presence of ammonium ions but was inhibited by the primary amines ethylamine (competitively), propylamine, butylamine and benzylamine. In addition, formaldehyde and acetate, at a concentration of 1 mM, inhibited methylamine uptake almost completely. These compounds were shown to be non-competitive inhibitors. A strong inhibition observed in the presence of plumbagin could be relieved by addition of dithiothreitol. This indicates that the oxidation-reduction state of, probably, carrier dithiol-disulfide-groups is an important factor in methylamine translocation in Arthrobacter P1.

Quantitative agreement between the values for the light-induced ΔpH in Rhodopseudomonas sphaeroides measured with automated flow-dialysis and 31P-NMR

Quantitative determination of transmembrane pH and electrical potential gradients is a prerequisite for a further refinement of the concepts of the chemiosmotic hypothesis [ 11. Several methods are available for the measurements of ApH and A

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

(review [21), of which the ‘spectroscopic’ and ‘distribution’ methods are most widely used. The outcome of the various methods, however, does show significant variations and it has been established by stringent comparisons, that the optical methods overestimate the magnitude of the transmembrane gradients [3-61. The distribution methods [2,7] (except flow-dialysis [S]) have the disadvantage that leakage of the probe molecules may occur during the separation step. Furthermore, in any distribution method uncertainties remain concerning: (i) The homogeneity of the internal aqueous phase and the absence of subcellular compartments; (ii) The ‘ideal behaviour’ [2] of the indicator probe; (iii) The activity coefficient of the probe molecules in the internal aqueous phase. With the application of 3’P NMR to biological systems [9-l l] a powerful and independent method has become available for the quantitation of ApH. This technique makes use of the pH dependence of 3’P NMR chemical shifts of phosphate metabolites. It can be used only if calibration curves of the chemical shift

Purification, enzymatic properties, and reconstitution of cytochrome-c oxidase from Bacillus subtilis

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.

Reactive exofacial sulfhydryl-groups on the arginine-ornithine antiporter of Lactococcus lactis

Publisher Summary This chapter describes the methods used for the isolation and purification of cytochrome- c oxidase of B. subtilis W23. The purified enzyme has been biochemically and biophysically characterized and reincorporated into liposomes. The incorporation procedure and the physiological properties of the incorporated enzyme are also described. To determine the energy-transducting capacity of cytochrome- c oxidase, it is essential to reconstitute the purified enzyme into artificial membranes. The efficiency of energy transduction is dependent on the incorporation of the enzyme in the artificial membrane. Several factors influence the incorporation, such as the phospholipid composition of the membrane and the protein-to-lipid ratio used for reconstitution. Reconstitution with the affinity column purified enzyme is not successful until now. The chapter describes the reconstitution using the enzyme prepared by the ammonium sulfate precipitate, after DEAE and gel filtration chromatography.

Protonmotive-force-driven leucine uptake in yeast plasma membrane vesicles

The effect of various sulfhydryl (SH)-specific reagents on arginine-ornithine antiport activity in membrane vesicles of Lactococcus lactis was studied. Little or no inhibition of arginine-ornithine exchange was observed with maleimides and arsenicals. HgCl2 and the organic mercurials p-chloromercuribenzene sulfonate (pCMBS), p-chloromercuribenzoate (pCMB), and O-(3-hydroxymercuri-2-methoxypropyl)carbamylphenoxyacetate completely inactivated the antiporter. This effect could not be attributed to vesicle disruption. Inactivation of arginine-ornithine exchange by pCMBS could be reversed by dithiothreitol. It was reflected by a decreased Vmax with no change in the Kt for arginine uptake, and correlated with a reduction of the number of arginine binding sites. The poorly penetrating pCMBS had at low concentrations only access to the reactive SH-group(s) from the outer surface of the membrane, while the permeant pCMB reacts with SH-group(s) at both membrane surfaces. Arginine and ornithine, if present on the outer surface of the membrane, protected the arginine-ornithine antiporter against pCMBS inactivation. Membrane-impermeable oxidizing agents have no effect on the exchange activity, indicating that the antiporter is not regulated by the redox potential of the environment. It is concluded that the arginine-ornithine antiporter contains one or more reactive exofacial SH-groups which are presumably located in or near the substrate binding site.

Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells.

Plasma membranes prepared from Saccharomyces cerevisiae by the concanavalin A method were fused with liposomes by a freeze‐thaw sonication method. A ΔΨ of −109 mV was generated in the sealed vesicles by a valinomycin‐mediated potassium diffusion potential. This component of protonmotive force was able to drive a transient uptake of leucine.