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PubMed1977

Selectivity of the 2-deoxyglucose transport system in human and guinea pig polymorphonuclear leukocytes.

To determine whether the deleterious action of D-galactose upon phagocyte function could be related to inhibition of glucose uptake, the properties of glucose transport were investigated by following the incorporation of [G-3H]2-deoxyglucose into human and guinea pig polymorphonuclear leukocytes (PMN). Uptake of [G-3H]2-deoxyglucose by guinea pig PMN proceeded in vitro with a Km of 1.8 mM and Vmax of 0.67 nmol/min per 10(6) cells. This system was competitively inhibited by glucose and mannose but was not significantly affected by galactose, fructose, or 3-0-methylglucose. Maximal uptake of 2-deoxyglucose occurred at 41 degrees C, and phosphorylation was necessary for its intracellular concentration. Transport of 2-deoxyglucose, although not altered by uncouplers of oxidative phosphorylation, was sensitive to inhibitors of glycolysis. Preincubation of cells with 2 mM iodoacetate for 30 min significantly reduced the uptake of 2-deoxyglucose and the intracellular levels of adenosine-5'-triphosphate without decreasing cell viability. These results indicated that uptake of 2-deoxyglucose in guinea pig PMN occurred by facilitated diffusion with subsequent phosphorylation. Similar results were obtained with PMN isolated from human peripheral blood.

Adenosine Triphosphate↗
PubMed1970

Extrusion of sodium and hydrogen ions as the primary process in potassium ion accumulation by Streptococcus faecalis.

Glycolyzing cells of Streptococcus faecalis accumulate K(+) with concurrent extrusion of equivalent amounts of H(+) and Na(+). An attempt was made to clarify the retionship between the movements of Na(+) and K(+). Sodium was displaced from cells glycolyzing in the presence of ammonia, diethylamine, tris(hydroxymethyl)aminomethane, and other nitrogenous cations; by contrast, K(+) was completely retained. Accumulation of K(+) by heterologous exchange for Na(+) was not inhibited by antibiotics which facilitate diffusion of K(+) across the membrane, but was blocked by proton conductors. The results indicate that extrusion of Na(+) and H(+) from the cells is a primary, energy-linked process which generates an electrical potential (interior negative); K(+) accumulation occurs in response to this potential. Two mutants deficient in K(+) accumulation and retention were examined in terms of this model. One mutant is apparently defective in exchange of K(+) for H(+). In the other mutant, exchange of K(+) for Na(+) is impaired.

Amines↗
PubMed1970

Galactose transport in Saccharomyces cerevisiae. 3. Characteristics of galactose uptake in transferaseless cells: evidence against transport-associated phosphorylation.

The characteristics of the inducible galactose transport system in bakers' yeast were studied in uridine diphosphate, galactose-1-phosphate uridylyl-transferaseless cells. Transferaseless cells transport galactose at the same initial rate as wild-type cells and accumulate a mixture of free galactose and galactose-1-phosphate. The addition of (14)C-labeled galactose to cells preloaded with unlabeled galactose and galactose-1-phosphate results in a higher rate of labeling of the free-sugar pool than of the galactose-1-phosphate pool. These results support other evidence that galactose uptake in bakers' yeast is a carrier-mediated, facilitated diffusion and that phosphorylation is an intracellular event after uptake of the free sugar.

Biological Transport↗
PubMed1971

Transport of glycerol by Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the transport of glycerol was shown to be genetically controlled and to be dependent on induction by glycerol. Accumulation of (14)C-glycerol was almost completely absent in uninduced cells and in a transport-negative mutant. Kinetic studies with induced cells suggested that glycerol may be transported by two systems with different affinities for glycerol. Osmotically shocked cells did not transport glycerol, and the supernatant fluid from shocked cells contained glycerol-binding activity demonstrable by equilibrium dialysis. The binding protein was not glycerol kinase. Binding activity was absent in shock fluids from the transport-negative mutant and from uninduced cells. The glycerol-binding protein was partially purified by precipitation with ammonium sulfate. Mild heat treatment completely eliminated the binding activity of shock fluid and of the partially purified protein. Sodium azide and N-ethylmaleimide inhibited both transport by whole cells and binding of glycerol by shock fluid. It is concluded that transport of glycerol by P. aeruginosa involves a binding protein responsible for recognition of glycerol and may occur by facilitated diffusion or active transport. A requirement for energy has not been demonstrated.

Ammonium Sulfate↗
PubMed1975

Urea transport in Saccharomyces cerevisiae.

Urea transport in Saccharomyces cerevisiae occurs by two pathways. The first mode of uptake is via an active transport system which: (i) has an apparent Km value of 14 muM, (ii) is absolutely dependent upon energy metabolism, (iii) requires pre-growth of the cultures in the presence of oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, and (iv) is sensitive to nitrogen repression. The second mode of uptake which occurs at external urea concentrations in excess of 0.5 mM is via either passive or facilitated diffusion.

Ammonia↗
PubMed1975

Characterization of the glucose transport systems in Neurospora crassa sl.

Neurospora crassa sl, a mutant that lacks a rigid cell wall, exhibits transport systems for glucose similar to those of wild-type strain 1A. When the orgnism is grown in a medium containing 50 mM glucose as the carbon source, glucose is transported primarily by a glucose-facilitated diffusion system (GluI). When it is grown in a medium with little or no glucose present, a glucose active transport system (Glu II) is expressed. Both of these systems are similar kinetically to those in the wild type. Significant differences do exist between strains sl and 1A with respect to genetic regulation of the glucose active transport system.

Biological Transport, Active↗
PubMed1975

Glucose transport and its inhibition by short-chain n-alkanes in Cladosporium resinae.

Glucose transport in Cladosporium resinae was studies with the aid of the non-metabolizable glucose analogue 3-O-methyl-D-glucose (3-O-MG). 3-O-MG, transported as a free sugar without phosphorylation, was found to inhibit glucose uptake competitively. Conversely, glucose was a competitive inhibitor of 3-O-MG uptake. Moreover, both glucose and 3-O-MG were able to bring about rapid counterflow intracellular 3-O-MG. Thus, glucose and 3-O-MG share the same entry and exit systems. The transport of 3-O-MG is carrier mediated and energy dependent as shown by saturation kinetics, strong temperature dependence, accumulation of unaltered 3-O-MG against a concentration gradient, and inhibition of uptake by NaN3, NaCN, and 2,4-dinitrophenol. The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, mannose, xylose, or glucose. There was no derepressible low-Km glucose transport system in C. resinae. n-Hexane and n-heptane were found to inhibit 3-O-MG uptake rapidly at temperatures above 20 C. Over 50% inhibition of the uptake rate occurred after only 10 min of incubation with n-hexane at 30 C. The percentage of inhibition in the presence of n-hexane, compared to controls in the absence of n-hexane, was found to increase with increasing temperature. Longer-chain n-alkanes (C8 to C18) had no significant effect on uptake. The efflux of intracellular 3-O-MG, which appeared to occur by facilitated diffusion, was not affected by any of the n-alkanes tested including n-hexane.

Alkanes↗
PubMed1976

Urea transport-defective strains of Saccharomyces cerevisiae.

Experiments characterizing the urea active transport system in Saccharomyces cerevisiae indicate that (i) formamide and acetamide are strong competitive inhibitors of urea accumulation, (ii) uptake is maximal at pH 3.3 and is 80% inhibited at pH 6.0, and (iii) adenosine 5'-triphosphate generated by glycolysis in conjunction with formation of an ion gradient is likely the driving force behind urea transport. Mutant strains were isolated that are unable to accumulate urea at external concentrations of 0.25 mM. These strains also exhibit a depressed growth rate on 10 mM urea, indicating existence of a relationship between the active transport and facilitated diffusion modes of urea uptake.

Acetamides↗
PubMed1976

Energy cost of galactoside transport to Escherichia coli.

Energy reserves of Escherichia coli can be depleted by our previously reported procedure to a level such that even the "downhill" transport of o-nitrophenyl-beta-D-galactopyranoside (ONPG) is completely dependent upon the exogenous energy supply. The ONPG concentration is high externally to the cells and is low intracellular because of the action of cytoplasmic beta-galactosidase. In the present work, depleted cell suspensions have been infused at low, steady rates with glucose and other energy sources while measurements of transport were being made. Comparing the rate of ONPG transport with the rate of introduction of glucose under conditions where the chosen glucose infusion rate limits transport, we find that 89 molecules of ONPG are transported per molecule of fully oxidized glucose. This transport yield is constant over a 6.5-fold range in rate of glucose addition. This constancy over a range of infusion rates implies that transport is the major cellular function under these special conditions. The yield value if 89 is in the agreement with the predicitions of 76 from Mitchell's chemiosmotic theory and constitutes an independent proff of its validity, since all the other proposed mechanisms of engery coupling predict much smaller yields. The lag from the start of glucose infusion into the reaction cuvette, to the extrapolated time at which a steady rate of transport and concomitant hydrolysis are achieved, is short (approximately 1 min). Similarly, the time after the infusion is stopped until the rate of transport returns to the background rate is also short. The latter implies that the energy metabolism is directed almost entirely to transport and/or other ongoing cellular processes and not to repair or renewal of an energy-independent, facilitated diffusion system.

Biological Transport, Active↗
PubMed1977

Isolation, genetic analysis, and characterization of Escherichia coli mutants with defects in the lacY gene.

Five hundred thirty-five lacY mutants were isolated from an Escherichia coli strain carrying the lactose operon on an F' factor, either without mutagenesis or after mutagenesis with 2-aminopurine or N-methyl-N'-nitro-N-nitrosoguanidine. Crosses against 48 independently isolated deletions ending in the lacY gene divided the gene into 36 deletion groups. Suppressibility studies with 7 nonsense suppressor strains classified 276 mutants as nonsense mutants and 78 as missense (or nonsuppressible) mutants. One hundred seventy-nine mutants were "leaky" and could not be so allocated, and two were found to have small internal deletions. Nonsense mutants could in many cases be subdivided even within deletion groups on the basis of their suppressibility pattern, giving a total of 70 groups of nonsense mutants. Studies of these mutants allow the following conclusions: lactose and melibiose most probably do not have separate binding sites on the permease; the lacY region most likely consists of one cistron, and so both active transport and facilitated diffusion are functions of one protein; and finally, there is probably no small defined region of the permease responsible for energy coupling of transport. Furthermore, the strains and the analysis form the basis for a future functional study of the permease by biochemical techniques.

2-Aminopurine↗
PubMed1978

Binding and transport of thiamine by Lactobacillus casei.

The relationship between thiamine transport and a membrane-associated thiamine-binding activity has been investigated in Lactobacillus casei. Thiamine transport proceeds via a system whose general properties are typical of active uptake processes; entry of the vitamin into the cells requires energy, is temperature dependent, exhibits saturation kinetics, and is inhibited by substrate analogs. A considerable concentration gradient of unchanged thiamine can be achieved by the system, although the vitamin is slowly metabolized to thiamine pyrophosphate. Consistent with these results, L. casei also contains a high-affinity, thiamine-binding component which could be measured by incubation of intact cells with labeled substrate at 4 degrees C (conditions under which transport is negligible). Binding was insensitive to iodoacetate, occurred at a level (0.5 nmol per 10(10) cells) nearly 20-fold higher than could be accounted for by facilitated diffusion, and was found to reside in a component of the cell membrane. Participation of this binder in thiamine transport is supported by the observations that the processes of binding and transport showed similarities in their (i) regulation by the concentration of thiamine in the growth medium, (ii) binding affinities for thiamine, and (iii) susceptibility to inhibition by thiamine analogs.

Binding, Competitive↗
PubMed1978

Evolution of propanediol utilization in Escherichia coli: mutant with improved substrate-scavenging power.

Wild-type strains of Escherichia coli are unable to use L-1,2-propanediol as a carbon and energy source. A series of mutants, able to grow on this compound at progressively faster rates, had been isolated by repeated transfers to a medium containing 20 mM L-1,2-propanediol. These strains synthesize at high constitutive levels a propanediolmicotinamide adenine dinucleotide oxidoreductase, an enzyme serving as a lactaldehyde during L-fucose fermentation by wild type cells. In this study, a mutant that can grow rapidly on the novel carbon source was subjected to further selection in a medium containing L-1,2-propanediol never exceeding 0.5 mM to obtain a derivative that has an increased power to extract the substrate from the medium. The emerging mutant exhibited four changes at the enzymatic level: (i) fuculose 1-phosphate aldolase activity is lost; (ii) the constitutive propanediol oxidoreductase activity is increased in its level; (iii) lactaldehyde dehydrogenase becomes constitutive and shows an elevated specific activity in crude extracts; and (iv) at low concentrations of propanediol, the facilitated diffusion across the cell membrane is enhanced. Changes two to four seem to act in concert in the trapping of propanediol by hastening its rate of entry and conversion to an ionized metabolite, lactate.

Alcohol Oxidoreductases↗
PubMed1979

Transport of cyclic adenosine 3',5'-monophosphate across Escherichia coli vesicle membranes.

The uptake and efflux of cyclic adenosine 3',5'-monophosphate (3',5'-cAMP) by Escherichia coli membrane vesicles were studied. Metabolic energy was not required for the uptake process and was found to actually decrease the amount of 3',5'-cAMP found in the vesicles. 3',5'-cAMP uptake exhibits saturation kinetics (Km = 10 mM, Vmax = 2.8 nmol/mg of protein per min) and was competitively inhibited by a number of 3',5'-cAMP analogs. The uptake of 3',5'-cAMP was found to be sharply affected by a membrane phase transition. The excretion of 3',5'-cAMP was studied by using everted membrane vesicles. Efflux in this system was dependent upon metabolic energy and was reduced or abolished by uncouplers. Different energy sources powered efflux at different rates, showing a relationship between the degree of membrane energization and rate of excretion of 3',5'-cAMP. The efflux process also displayed saturation kinetics (Km = 10.0 mM, Vmax = 0.98 nmol/mg of protein per min) and was competitively inhibited by the same 3',5'-cAMP analogs and to the same degree as was the uptake process. 3',5'-cAMP was found to be chemically unaltered by both the uptake and excretion processes. These data are interpreted as showing that the uptake and excretion of 3',5'-cAMP in E. coli membrane vesicles are carrier-mediated phenomena, possibly employing the same carrier system. Uptake is by facilitated diffusion whereas efflux is via an energy-dependent, active transport process. Evidence is presented showing that cells can regulate the number of 3',5'-cAMP transport carriers. The rate of 3',5'-cAMP excretion is possibly regulated by both the degree of membrane energization and the number of carriers present per cells.

Biological Transport↗
PubMed1980

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.

Biological Transport, Active↗
PubMed1985

Maltose-binding protein does not modulate the activity of maltoporin as a general porin in Escherichia coli.

Maltoporin (lambda receptor) is part of the maltose transport system in Escherichia coli and is necessary for the facilitated diffusion of maltose and maltodextrins across the outer membrane. Maltoporin also allows the diffusion of nonmaltodextrin substrates, albeit with less efficiency. The preference of maltoporin for maltodextrins in vivo is thought to be the result of an interaction of maltoporin with the maltose-binding protein, the malE gene product. In a recent report Heuzenroeder and Reeves (J. Bacteriol. 144:431-435, 1980) suggested that this interaction establishes a gating mechanism which inhibits the diffusion of nonmaltodextrin substrates, such as lactose. To reinvestigate this important conclusion, we constructed ompR malTc strains carrying either the malE+ gene, the nonpolar malE444 deletion, or the malE254 allele, which specifies an interaction-deficient maltose-binding protein. Lactose uptake was measured at different concentrations below the Km of this transport system and under conditions where transport was limited by the diffusion through maltoporin. We found no difference in the kinetics of lactose uptake irrespective of the malE allele. We conclude that the maltose-binding protein does not modulate the activity of maltoporin as a general outer membrane porin.

ATP-Binding Cassette Transporters↗
PubMed1986

Uptake of benzoate by Rhodopseudomonas palustris grown anaerobically in light.

The uptake and anaerobic metabolism of benzoate were studied in short-term experiments with phototrophic cells of Rhodopseudomonas palustris. Cells that were preincubated and assayed anaerobically in the presence of 1 mM dithiothreitol accumulated [7-14C]benzoate at a rate of at least 0.5 nmol . min-1 . mg-1 of protein. Cells that were preincubated aerobically, or anaerobically in the absence of a reducing agent or an electron donor such as succinate, took up benzoate at reduced rates. Benzoate was removed from the external medium with remarkably high efficiency; initial uptake rates were independent of substrate concentration, and uptake remained linear down to concentrations of less than 1 microM. Uptake rates were not sensitive to external pH in the range of 6.5 to 8.1, and very little free benzoate was found associated with the cells. By contrast, benzoyl coenzyme A (CoA) was formed rapidly in cells exposed to labeled benzoate. Its appearance in such cells, together with the more gradual accumulation of other compounds tentatively identified as reduction products, is consistent with the identification of benzoyl CoA as an intermediate in the anaerobic reductive metabolism of benzoate. The very effective uptake of external benzoate can be explained by its conversion to benzoyl CoA immediately after its passage across the cell membrane by simple or facilitated diffusion. Such a chemical conversion would serve to maintain a downhill concentration gradient between the cell cytoplasm and the cell surroundings, even at very low external benzoate concentrations.

Acyl Coenzyme A↗
PubMed1987

Transport of branched-chain amino acids in membrane vesicles of Streptococcus cremoris.

The kinetics, specificity, and mechanism of branched-chain amino acid transport in Streptococcus cremoris were studied in a membrane system of S. cremoris in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (delta p)-generating system. Influx of L-leucine, L-isoleucine, and L-valine can occur via a common transport system which is highly selective for the L-isomers of branched chain amino acids and analogs. The pH dependency of the kinetic constants of delta p-driven L-leucine transport and exchange (counterflow) was determined. The maximal rate of delta p-driven transport of L-leucine (Vmax) increased with increasing internal pH, whereas the affinity constant increased with increasing external pH. The affinity constant for exchange (counterflow) varied in a similar fashion with pH, whereas Vmax was pH independent. Further analysis of the pH dependency of various modes of facilitated diffusion, i.e., efflux, exchange, influx, and counterflow, suggests that H+ and L-leucine binding and release to and from the carrier proceed by an ordered mechanism. A kinetic scheme of the translocation cycle of H+-L-leucine cotransport is suggested.

Amino Acids, Branched-Chain↗
PubMed1987

Regulation of beta-galactoside transport and accumulation in heterofermentative lactic acid bacteria.

Galactose-grown cells of the heterofermentative lactic acid bacteria Lactobacillus brevis and Lactobacillus buchneri transported methyl-beta-D-thiogalactopyranoside (TMG) by an active transport mechanism and accumulated intracellular free TMG when provided with an exogenous source of energy, such as arginine. The intracellular concentration of TMG resultant under these conditions was approximately 20-fold higher than that in the medium. In contrast, the provision of energy by metabolism of glucose, gluconate, or glucosamine promoted a rapid but transient uptake of TMG followed by efflux that established a low cellular concentration of the galactoside, i.e., only two- to fourfold higher than that in the medium. Furthermore, the addition of glucose to cells preloaded with TMG in the presence of arginine elicited a rapid efflux of the intracellular galactoside. The extent of cellular TMG displacement and the duration of the transient effect of glucose on TMG transport were related to the initial concentration of glucose in the medium. Exhaustion of glucose from the medium restored uptake and accumulation of TMG, providing arginine was available for ATP generation. The nonmetabolizable sugar 2-deoxyglucose elicited efflux of TMG from preloaded cells of L. buchneri but not from those of L. brevis. Phosphorylation of this glucose analog was catalyzed by cell extracts of L. buchneri but not by those of L. brevis. Iodoacetate, at a concentration that inhibits growth and ATP production from glucose, did not prevent efflux of cellular TMG elicited by glucose. The results suggested that a phosphorylated metabolite(s) at or above the level of glyceraldehyde-3-phosphate was required to evoke displacement of intracellular TMG from the cells. Counterflow experiments suggested that glucose converted the active uptake of TMG in L. brevis to a facilitated diffusion mechanism that allowed equilibrium of TMG between the extra- and intracellular milieux. The means by which glucose metabolites elicited this vectorial regulation is not known, but similarities to the inducer expulsion that has been described for homofermentative Streptococcus and Lactobacillus species suggested the involvement of HPr, a protein that functions as a phosphocarrier protein in the phosphotransferase system, as well as a presumptive regulator of sugar transport. Indeed, complementation assays wit extracts of Staphylococcus aureus ptsH mutant revealed the presence of HPr in L. brevis, although this lactobacillus lacked a functional phaosphoenolpyruvate-dependent phosphortransferase system for glucose, 2-deoxyglucose, or TMG.

Arginine↗