Energy coupling in the transport of beta-galactosides by Escherichia coli: effect of proton conductors

Escherichia coli accumulates thiomethyl-beta-d-galactoside against a concentration gradient under anaerobic conditions. The accumulation was abolished by carbonylcyanide m-chlorophenylhydrazone, tetrachlorosalicylanilide, 2,4 dinitrophenol, and other uncouplers of oxidative phosphorylation even though oxidative phosphorylation would not be expected to occur anaerobically. In the presence of the uncouplers, the beta-galactoside carrier remained functional and catalyzed equilibration of thiomethylgalactoside across the membrane. The uncouplers did not inhibit the generation of adenosine triphosphate or protein turnover, or the accumulation of alpha-methylglucoside and glycerol by phosphorylation. We conclude that, at least anaerobically, uncouplers of oxidative phosphorylation do not interfere with energy metabolism in general, but prevent the utilization of metabolic energy for the active transport of galactosides. The uncouplers also facilitate passage of protons across the membrane. Various hypotheses are considered to explain why a proton-impermeable membrane may be required for active transport of galactosides and other substrates.     

Uptake of Branched-Chain Amino Acids by Streptococcus thermophilus

The transport of branched-chain amino acids in Streptococcus thermophilus was energy dependent. The metabolic inhibitors of glycolysis and ATPase enzymes were active, but the proton-conducting uncouplers were not. Transport was optimal at temperatures of between 30 and 45°C and at pH 7.0 for the three amino acids leucine, valine, and isoleucine; a second peak existed at pH 5.0 with valine and isoleucine. By competition and kinetics studies, the branched-chain amino acids were found to share at least a common transport system.     

Branched-chain amino acid transport in Streptococcus agalactiae.

The transport of the branched-chain amino acids in Streptococcus agalactiae was characterized. Glucose-grown cells were able to utilize only glucose as an energy source for transport of L-leucine, whereas lactose-grown cells could utilize both glucose and lactose. It was determined from metabolic inhibitor studies that energy from glycolysis and substrate level phosphorylation was required for active transport. Energy was found to be coupled to transport by the action of adenosine triphosphatase and the generation of a proton motive force. The branched-chain amino acids were found to share a common transport system that may consist of multiple components.     

Branched-chain amino acid transport in Streptococcus agalactiae

The transport of the branched-chain amino acids in Streptococcus agalactiae was characterized. Glucose-grown cells were able to utilize only glucose as an energy source for transport of L-leucine, whereas lactose-grown cells could utilize both glucose and lactose. It was determined from metabolic inhibitor studies that energy from glycolysis and substrate level phosphorylation was required for active transport. Energy was found to be coupled to transport by the action of adenosine triphosphatase and the generation of a proton motive force. The branched-chain amino acids were found to share a common transport system that may consist of multiple components.     

Amino acid transport by the filamentous fungus Arthrobotrys conoides

Uptake of l-valine by germinated spores of Arthrobotrys conoides has all the characteristics of a system of transport that requires an expenditure of energy by the cells. It is dependent on temperature and has an energy of activation of 16,000 cal/mole. Uptake is optimal at pH 5 to 6. l-Valine accumulated against a concentration gradient and is not lost from the cells by leakage or exchange. The process requires energy supplied by the metabolic reactions that are inhibited by catalytic amounts of 2,4-dinitrophenol and azide. The kinetics of the system are consistent with a mechanism of transport that depends on a limited number of sites on the cell surface, and the Michaelis constant for the system is 1.5 x 10(-5) to 7.5 x 10(-5)m. Modification of the amino or carboxyl group abolishes l-valine uptake. The process is competitively inhibited by d-valine, glycine, and other neutral amino acids (K(i) = 1.5 x 10(-5) to 4.0 x 10(-5)m), indicating a lack of stereospecificity, and also indicating that aliphatic side chain is not required for binding with the carrier. The transport system has less affinity for acidic amino acids (glutamic and aspartic acids) than neutral amino acids, and a greater affinity for basic amino acids (histidine, lysine, and arginine). The range of affinity is in the order of 100, as measured in terms of K(i) values for various compounds. The data presented provide suggestive evidence that the uptake by A. conoides of all amino acids except proline is mediated by a single carrier system that possesses an overall negative charge.     

Microbial transport: Adaptations to natural environments

The cytoplasmic membrane of bacteria is the matrix for metabolic energy transducing processes such as proton motive force generation and solute transport. Passive permeation of protons across the cytoplasmic membrane is a crucial determinant in the proton motive generating capacity of the organisms. Adaptations of the membrane composition are needed to restrict the proton permeation rates especially at higher temperatures. Thermophilic bacteria cannot sufficiently restrict this proton permeation at their growth temperature and have to rely on the much␣lower permeation of Na + to generate a sodium motive force for driving metabolic energy-dependent membrane processes. Specific transport systems mediate passage across the membrane at physiological rates of all compounds needed for growth and metabolism and of all end products of metabolism. Some of transport systems, the secondary transporters, transduce one form of electrochemical energy into another form. These transporters can play crucial roles in the generation of metabolic energy. This is especially so in anaerobes such as Lactic Acid Bacteria which live under energy-limited conditions. Several transport systems are specifically aimed at the generation of metabolic energy during periods of energy-limitation. In their natural environment bacteria are also often exposed to cytotoxic compounds, including antibiotics. Many bacteria can respond to this live-threatening condition by overexpressing powerful drug-extruding multidrug resistance systems.     

Co-transport of Potassium and Sugars across the Plasmalemma of Mesophyll Protoplasts

Sugars (sucrose + hexoses) produced photosynthetically by isolated mesophyll protoplasts of wheat and tobacco were effluxed across the plasma membrane (3 to 10 micromoles hexose equivalents per milligram chlorophyll per hour). The efflux was sensitive to uncouplers and oligomycin which indicated a requirement for energy. A proton gradient was probably not coupled directly to the transport because changing the proton gradient across the plasma membrane by varying the pH of the medium or by adding sodium acetate had no significant effect on the rate of sugar release.     

Chloride-dependent restoration of coupling by oligomycin in rat liver mitochondria.

In rat liver mitochondria suspended in KC1 medium, oligomycin interfered with the effect of uncouplers on energy conservation. It antagonized the effect of uncouplers that are weak acids (2,4-dinitrophenol etc.), but enhanced that of the lipid-penetrating cation NN-dimethyl-N'N'-dibenzylammonium. Oligomycin caused none of the above effects when Br- or NO-/3 was substituted for C1- as the major anionic species in the assay medium. The concentration of oligomycin that exerted the above-mentioned effects was lower than that necessary for the inhibition of energy transfer, but was in the range that induced C1- permeation through the cristae membrane. The possible connexion between the effect of oligomycin on C1- permeation and its interference with the action of uncouplers is discussed.     

Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus

The uptake of radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) by protoplasts isolated from leaves of Brassica napus was detected using silicone oil filtration technique. The uptake was pH-dependent with higher uptake rates at acidic pH. Imposition of a pH gradient (internal alkaline) across the plasma membrane resulted in a rapid uptake of p-OHBG, which was inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone, indicating that the uptake is dependent on a proton motive force. Dissipation of the internal positive membrane potential generated a small influx as compared with that seen for pH gradient (DeltapH). Kinetic studies demonstrated the presence of two uptake systems, a saturable and a linear component. The saturable kinetics indicated carrier-mediated translocation with a K(m) of 1.0 mm and a V(max) of 28.7 nmol/microl/h. The linear component had very low substrate affinity. The carrier-mediated transport had a temperature coefficient (Q(10)) of 1.8 +/- 0.2 in the temperature range from 4-30 degrees C. The uptake was against a concentration gradient and was sensitive to protonophores, uncouplers, H(+)-ATPase inhibitors, and the sulfhydryl group modifier p-chloromercuriphenylsulfonic acid. The carrier-mediated uptake system had high specificity for glucosinolates because glucosinolate degradation products, amino acids, sugars, or glutathione conjugates did not compete for p-OHBG uptake. Glucosinolates with different side chains were equally good competitors of p-OHBG uptake, which indicates that the uptake system has low specificity for the glucosinolate side chains. Our data provide the first evidence of an active transport of glucosinolates by a proton-coupled symporter in the plasma membrane of rape leaves.     

An oscillatory mechanism for the gamma-glutamyl transpeptidase-mediated translocation of amino acids across the cell membrane

The reinterpretation of the kinetics of the gamma-glutamyl cycle-mediated uptake of amino acids in the light of the cycle's wave mechanical properties shows that its oscillatory periods are modulated by the chemical nature and the concentrations of amino acids. The periods of the cycle are the half-lives of glutathione whose function is to synchronize the oscillations of the two pathways of the cycle. gamma-glutamyl transpeptidase, an amphipathic membrane protein and the master oscillator of the cycle degrades glutathione and translocates amino acids in a discontinuous manner suggesting that it flip-flops across the membrane, the periods of the flip-flops being the oscillatory periods of the gamma-glutamyl transpeptidase/amino acid complexes. The energies of the flip-flops are quantized and independent of metabolic energy. The principal quantum numbers are dependent on the amino acids being translocated. In their translocation, those amino acids which are good substrates of the gamma-glutamyl transpeptidase possess higher principal quantum numbers than those which are poor substrates, an observation which gives support to the flip-flopping of the gamma-glutamyl transpeptidase/amino acid complexes across the cell membrane.     

Inhibition of amino acid transport in Bacillus subtilis by uncouplers of oxidative phosphorylation.

The effect of three uncouplers of oxidative phosphorylation, trifluoromethoxycarbon-ylcyanidephenylhydrazone (FCCP), 3,3′,4′,5-tetrachlorosalicylanilide (TCSA), and pentachlorophenol (PCP), on transport of glycine and proline by Bacillus subtilis were examined. FCCP inhibited proline uptake uncompetitively, but glycine uptake competitively. TCSA inhibited proline uptake noncompetitively, but glycine uptake competitively. PCP inhibited proline uptake noncompetitively, but glycine uptake uncompetitively. The results indicate that these uncouplers inhibit amino acid transport by interacting at specific sites rather than by reducing any central supply of energy used to fuel metabolic processes.     

Changes of 2-(methylamino) isobutyric acid transport and intracellular pH upon preincubation of rat hepatocyte primary cultures

When rat hepatocyte monolayers were preincubated for 4 h in Hanks' salt solutions at pH 7.0, 7.4 and 8.0, and the Na+-dependent uptake of 2-(methylamino) isobutyric acid (MeAIB) was measured at the same pH values, a stimulation of transport in the order pH 7.0 less than pH 7.4 less than pH 8.0 was observed. Estimations of the intracellular pH from the distribution of DMO revealed a decrease in the internal pH during the preincubation period. The MeAIB transport velocities appear to parallel with the proton gradients across the cell membrane rather than with external (or internal) pH. Analyses of the lactate/pyruvate concentrations in the media indicated that the fall in the intracellular pH is presumably due to an enhanced glycolysis. Suppressive concentrations of system A-reactive amino acids did not prevent the decrease in the internal pH nor did they alter the metabolic data.     

Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system.

The high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.     

Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.     

Glucose and proline transport in kinetoplastids

The parasitic protozoa belonging to the kinetoplastids can use both sugars and amino acids as carbon and energy sources. In this review, Benno ter Kuile discusses nutrient acquisition and utilization and how the metabolic strategies reflect the environment encountered in host and vector. Recent genetic and physiological evidence suggests that facilitated diffusion may be the primary uptake mechanism for glucose, and possibly for proline as well, even though there is biochemical and genetic evidence suggesting that active transport occurs, if not across the plasma membrane, then across the membranes of organelles. Trypanosoma brucei seems to have a metabolic strategy that strives for maximum energy efficiency, making no storage materials and thereby limiting the control over its internal conditions. On the other hand, Leishmania donovani does create a storage buffer, entrapping glucose in the cell. In this manner, it maintains constant internal conditions at the expense of energy, enabling it to survive more adverse conditions in the macrophage and in its vector.     

Evidence for amino-acid: proton cotransport in Ricinus cotyledons

During germination and early growth of the castor-bean (Ricinus communis L.), protein in the endosperm is hydrolyzed and the amino acids are transferred into the cotyledons and then via the translocation stream to the axis of the growing seedling. The cotyledons retain the ability to absorb amino acids after removal of the endosperm and hypocotyl, exhibiting rates of transport up to 70 mol g^-1 h^-1. The transport of L-glutamine was not altered by KCl or NaCl in low concentrations (0–20 mM). High concentrations of KCl (100 mM) inhibited transport, presumably by decreasing the membrane potential. An increase in the pH of the medium bathing the cotyledons was observed for 10 min following addition of L-glutamine but not with D-glutamine, which is not transported. The rate of proton uptake was dependent on the concentration of L-glutamine in the external solution. Inhibitors and uncouplers of respiration (azide, 2, 4-dinitrophenol, carbonyl cyanide phenylhydrazone and N-ethylmaleimide) inhibited both L-glutamine uptake and L-glutamine-induced proton uptake. Amino acids other than L-glutamine also caused a transient pH rise and the rate of proton uptake was proportional to the rate of amino-acid uptake. The stoichiometry was 0.3 protons per amino acid transported. Addition of sucrose also caused proton uptake but the alkalisation by sucrose and by amino acids were not additive. Nevertheless, when sucrose was added 60 min after providing L-glutamine at levels saturating its uptake system, a rise in pH was again observed. The results were consistent with amino-acid transport and sucrose transport in castor-bean cotyledons both occurring by a proton cotransport in the same membrane system but involving separate carriers.     

Inactivation of the intestinal uptake system for beta-lactam antibiotics by diethylpyrocarbonate

The uptake system for beta-lactam antibiotics in the rabbit small intestine was investigated using brush-border membrane vesicles. After treatment of membrane vesicles with the reagent diethylpyrocarbonate (DEP), the uptake of orally active beta-lactam antibiotics with an alpha-amino group in the substituent at position 6 or 7 of the penam or cephem nucleus was significantly inhibited, whereas DEP-treatment had no inhibitory effect on the uptake of beta-lactam antibiotics without an alpha-amino group. The kinetic analysis revealed an apparent competitive inhibition indicating a decreased affinity of the transport system for alpha-amino-beta-lactam antibiotics. Substrates of the intestinal dipeptide transport system - dipeptides and alpha-amino-beta-lactam antibiotics - could protect the transport system from irreversible inhibition by DEP, whereas beta-lactam antibiotics without an alpha-amino group as well as amino acids or bile acids had no effect. Incubation of DEP-treated vesicles with hydroxylamine led to a partial restoration of the transport activity indicating that DEP may have led to a modification of a histidine residue of the transport protein. From the data presented we conclude that a specific interaction of the alpha-amino group in the substituent at position 6 or 7 of the penam or cephem nucleus presumably with a histidine residue of the transport protein is involved in the translocation process of orally active alpha-amino-beta-lactam antibiotics across the intestinal brush-border membrane.     

Energetics of glycylglycine transport in Escherichia coli

The transport system for glycylglycine in Escherichia coli behaves like a shock-sensitive transport system. The initial rate of transport is reduced 85% by subjecting whole cells to osmotic shock, and glycylglycine is not transported by membrane vesicles. The energetics of transport was studied with strain ML 308-225 and its mutant DL-54, which is deficient in Ca(2+)- and Mg(2+)-stimulated adenosine 5'-triphosphatase (EC 3.6.1.3) activity. It is concluded that active transport of glycylglycine, like other shock-sensitive transport systems, has an obligatory requirement for phosphate bond energy, but not for respiration or the energized state of the membrane. The major evidence for this conclusion is as follows. (i) Uptake of glycylglycine is severely inhibited by arsenate. (ii) Oxidizable energy sources such as d-lactate, succinate, and ascorbate, which is mediated by N-methylphenazinium methylsulfate, cannot serve as energy sources for the transport of glycylglycine in DL-54, which lacks oxidative phosphorylation. (iii) When energy is supplied only from adenosine-5'-triphosphate produced by glycolysis (anaerobic transport assays with glucose as the energy source in DL-54), substantial uptake of glycylglycine is observed. (iv) When the Ca(2+)-Mg(2+)-adenosine triphosphatase activity is absent but substrate-level phosphorylations and electron transport are operating (glucose as the energy source in DL-54), transport of glycylglycine shows significant resistance to the uncouplers, dinitrophenol and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone.