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PubMed1992

The electrochemical proton gradient in the bloodstream form of Trypanosoma brucei is dependent on the temperature.

The membrane potential and pH gradient over the plasma membrane of the protozoan parasite Trypanosoma brucei were measured with radioactive indicators in combination with the silicone oil centrifugation technique over a range of temperatures. At 37 degrees C a small membrane potential and pH gradient of similar magnitude, but of opposite polarity, were measured. The resulting electrochemical proton gradient was almost zero. However, when the temperature was lowered from 37 degrees C to 22 degrees C, the internal pH was kept constant independent of the external pH and a membrane potential of between -100 and -150 mV was measured, depending on the external pH. Measurements at various temperatures between 15 degrees C and 37 degrees C revealed that above 26 degrees C the membrane potential collapsed and that this collapse correlated with a sudden increase in membrane fluidity. The uptake of 2-deoxy-D-glucose and of pyruvate, which are both mediated by facilitated diffusion carriers in the plasma membrane of the trypanosome, were also affected by this sudden increase in fluidity of the membrane. The overall rate of the conversion of glucose into its metabolites, which is independent of the plasma membrane, varied only gradually. We conclude (i) that major changes occur in the plasma membrane of T. brucei around 26 degrees C, that affect all membrane related processes; (ii) that the electrochemical proton gradient plays a minor role in the energy metabolism of T. brucei when it resides in the bloodstream of the mammalian host at 37 degrees C; and (iii) that below 26 degrees C an electrochemical proton gradient is maintained over the plasma membrane.

Animals↗

PubMed1992

Glucose transport in Crithidia luciliae.

The glucose analogue, 2-deoxy-D-glucose, was used to characterise the glucose transport system in Crithidia luciliae choanomastigotes. Uptake was temperature dependent with a Q10 of 2, and saturable with a Km of 0.22 mM and Vmax of 5.5 nmol min-1 (mg protein)-1 at 23 degrees C. Preloaded cells showed rapid exchange of intracellular 2-deoxy-D-glucose when incubated with extracellular D-glucose or 2-deoxy-D-glucose but little exchange with L-glucose. The substrate specificity of the uptake was studied using a number of D-glucose analogues. 6-Deoxy-D-glucose, 3-fluoro-3-deoxy-D-glucose and 4-fluoro-4-deoxy-D-glucose all competed for the transporter and had significant inhibitory effects on 2-deoxy-D-glucose transport. In contrast, 1-thio-beta-D-glucose, trehalose, 3-O-methyl-D-glucose, arginine, thymidine, L-sorbose and L-glucose were not inhibitory. The results imply the existence of a glucose transporter. The transport was blocked by a number of inhibitors and ionophores, including fluoride, azide, cyanide, dinitrophenol, valinomycin and nigericin. Overall, the uptake, exchange and efflux of 2-deoxy-D-glucose is consistent with transport via facilitated diffusion.

Animals↗

PubMed1993

Uptake and turnover of glucose in Leishmania donovani.

Glucose uptake and metabolism by Leishmania donovani promastigotes was studied using D-[14C]glucose in combination with the silicone oil centrifugation technique on organisms preadapted to different growth rates and glucose availability in the chemostat. The uptake step was differentiated from the subsequent metabolism by separation in time rather than by using non-metabolisable analogues. The uptake of glucose was measured as a function of time and/or the external glucose concentration on cells grown at high or low growth rate with glucose either as growth rate-limiting substrate, or present in excess. Glucose uptake as a function of its external concentration could be described as consisting of two components (1) a rapid equilibration owing to facilitated diffusion, followed by (2) a much slower uptake that involves an enzymatic component. This slower accumulation of label could be explained as the conversion of glucose into metabolites and a storage carbohydrate. Uptake experiments in the presence of inhibitors indicated that the conversion of glucose was an energy dependent process. These experiments indicate that the active uptake of glucose by L. donovani, as reported by others does not occur across the plasma membrane and should be reinterpreted as the intracellular conversion of glucose into metabolites and storage carbohydrate.

Animals↗

PubMed1985

Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behaviour of these enzymes.

To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), these enzymes were co-immobilized on the surface of a collagen film. The kinetic properties of the immobilized enzymes were compared with those observed with the enzymes in solution. Since the reactions correspond to the cytosolic enzymes, they have been studied in the direction aspartate aminotransferase toward malate dehydrogenase. Coupled enzymes in solution showed classical behaviour. A lag-time was observed before they reached a steady state and this lag-time was dependent on the kinetic properties of the second enzyme, malate dehydrogenase. The same lag-time was observed when malate dehydrogenase in solution was coupled with aspartate aminotransferase bound to the film. When aspartate aminotransferase in solution was coupled with malate dehydrogenase bound to the collagen film, a very long lag-time was observed. Theoretical considerations showed that in the latter case, the lag-time was dependent on the kinetic properties of the second enzyme and the transport coefficient of the intermediate substrate through the boundary layer near the surface of the film. Then both enzymes were co-immobilized on the collagen film. The coupled activity of aspartate aminotransferase and malate dehydrogenase was compared for films with an activity ratio of 5 and 0.8. In both cases, a highly efficient coupling was observed. In the former case, where malate dehydrogenase was rate-limiting, 81% of this limiting activity was observed. In the latter case, aspartate aminotransferase was rate-limiting and 82% of its rate was obtained for the final product formation. The linear increase of product formation with time corresponded fairly well to the theoretical equations developed in the paper. To interpret these rate equations, one should assume that the intermediate substrate oxaloacetate formed by aspartate aminotransferase was used by malate dehydrogenase in the diffusion layer near the film, before diffusing in the bulk solution.

Animals↗

PubMed1994

Creatine-creatine phosphate shuttle modeled as two-compartment system at different levels of creatine kinase activity.

In order to characterize ADP-ATP and creatine-creatine phosphate (Cr-CrP) shuttles a minimal mathematical model with two compartments and cyclic turnover of matter was designed. The 'mitochondrial' compartment contained 'ATP-synthase' and 'mitochondrial creatine kinase' (mitCK). The 'cytoplasmic' compartment consisted of 'ATPase', 'cytoplasmic creatine kinase' (cytCK) and an 'ADP-binding structure'. The exchange of metabolites between these compartments was limited. Different levels of cytCK and mitCK expression as well as different exchange rate constants between the compartments were assigned to obtain several different modes. Every steady state obtained in the presence of low ATPase activity ('resting' conditions) was then disturbed by a steep activation of ATPase ('muscle performance') and afterwards the transition to a new steady state was followed in time. The ATP-buffering capacity of the system initially acquired by cytCK expression significantly increased after additional mitCK supplement. Nevertheless, even the complete Cr-CrP shuttle failed to maintain a high [ATP]/[ADP] ratio during long term 'muscle performance' due to the rate limiting CK-transphosphorylation in the mitochondria. The facilitated diffusion of Cr and CrP was not critical, and the model worked with the same efficiency even at equal permeabilities for nucleotides and guanidines. Under 'resting conditions' the main flux of matter went through the Cr-CrP shuttle, resulting in 'pumping' of CrP. This ensured a 40 s delay in the [ATP] decrease at 'work'. The partial systems without mitCK were not as effective, and this delay was 0-10 s. However, the ADP-ATP shuttle was of more importance at the steady state achieved under 'working' conditions.

Adenosine Triphosphate↗

PubMed1995

Kinetics and mechanism of BAL 31 nuclease action on small substrates and single-stranded DNA.

Kinetic and mechanistic aspects of the action of two forms of the BAL 31 nuclease (EC 3.1.11) from Alteromonas espejiana on model substrates, small oligonucleotides, larger oligonucleotides and poly[d(A)] have been examined. The minimal oligonucleotide substrate is a 5'-phosphorylated dinucleotide and a phosphodiester not containing a nucleotide residue is not cleaved. Both forms act predominantly in an exonucleolytic fashion on single-stranded DNA polymers in a highly processive manner; however, the mechanism becomes distributive for small oligomers (3-4 nucleotide residues). The direction of attack is from the 5' end, in contrast to the mode of digestion of duplex DNA which involves attack at the 3' termini. An endonucleolytic mode of attack also exists, but at a level 2-3% or less of that of the terminally directed cleavage. Apparent values for the catalytic efficiency of the action on long DNA polymers are too large to fit a simple kinetic scheme involving a direct enzyme-substrate encounter and lead to an interpretation in which nuclease molecules are non-productively bound away from the 5' ends and undergo facilitated diffusion to yield productive (terminally bound) enzyme-substrate complexes.

Autoradiography↗

PubMed1986

3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells.

The characteristics and regulatory nature of sugar transport in freshly isolated bovine adrenal chromaffin cells were investigated. Transport was measured by following the cell/medium distribution of non-metabolizable glucose analogue, 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-glucose was was mediated by a saturable transport system with a Km of 8.2 mM and a Vmax of 0.69 nmol/mg protein per min. Basal 3-O-methyl-D-glucose transport was competitively inhibited by D-glucose and a countertransport effect was demonstrated. Cytochalasin B and phloretin, which are specific inhibitors of carrier-mediated glucose transport, significantly decreased basal 3-O-methyl-D-glucose uptake. Basal transport was stimulated by 50 mU/ml insulin, an effect associated with an increase in Vmax. The stimulatory effect of insulin was depressed in medium lacking external Ca2+, or containing the Ca2+-antagonistic ion, La3+, or the Ca2+ channel blocker, methoxyverapamil (D-600). The data suggest that the uptake of 3-O-methyl-D-glucose in freshly isolated bovine adrenal chromaffin cells is mediated by a specific facilitated diffusion mechanism, and is subject to regulation by insulin, thus resembling sugar transport in muscle. In addition, the insulin effect appears to depend on the presence of extracellular Ca2+.

3-O-Methylglucose↗

PubMed1990

Isolation and characterization of Chinese hamster ovary cell mutants defective in glucose transport.

Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.

Animals↗

PubMed1992

Uptake of organic anions by isolated rat hepatocytes. A classification in terms of ATP-dependency.

Uptake of organic anions into isolated rat hepatocytes was studied to examine their ATP dependency. In the presence of rotenone (0.2 microM), the initial velocity of the uptake (Vo) of dibromosulfophthalein (DBSP; 10 microM), 1-anilino-8-naphthalenesulfonate (ANS; 10 microM) and benzylpenicillin (PCG; 0.02 microM) was reduced to 60-70% of the control value, while that of bromosulfophthalein (BSP; 10 microM), rose bengal (RB; 10 microM) and bromophenol blue (BPB; 10 microM) was not affected. Furthermore, we examined the inhibitory effect of rotenone on the uptake at equilibrium of non-metabolizable ligands (DBSP, BPB and RB). The uptake of these ligands reached equilibrium at 30 min with a cel-to-medium concentration ratio (C/M ratio) of 75, 37 and 126, respectively. The C/M ratio at equilibrium of DBSP was reduced by rotenone to approx. 60% of the control value, while that of BPB and RB was not reduced. Other metabolic inhibitors such as sodium azide (10 mM) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 10 microM) also reduced the Vo of DBSP and PCG, while the uptake of BSP and RB was not reduced by these inhibitors. These results indicate that organic anions can be classified into two groups according to whether they are taken up by hepatocytes in an ATP-dependent manner, i.e., via active transport or in an ATP-independent manner, i.e., via facilitated diffusion. DBSP, PCG and ANS belong to the former group, whereas BSP, BPB and RB belong to the latter.

Adenosine Triphosphate↗

PubMed1993

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.

Journal Article↗

PubMedDate not supplied

Inhibition of the serosal sugar carrier in isolated intestinal epithelial cells by saccharin.

Isolated intestinal cells accumulate certain monosaccharides via an Na+-dependent, active transport system localized in the brush-border membrane, and release sugar molecules at the basolateral boundary via a facilitated diffusion, passive system. Work described here indicates that sodium saccharin (25-130 mM) has little if any direct effect on the active transport system, but that the passive transport system is inhibited by saccharin. A short period of exposure (10-60 min) is required for expression of the effect, which is detectable at saccharin concentrations as low as 10 mM. At 100 mM-sodium saccharin, as much as 50% inhibition occurs. Saccharin also appears to act as a weak metabolic inhibitor. The basis of the 'non-specific' effect is not understood, but it can compromise the capacity of the epithelial cells to form sugar gradients. When a sugar is accumulated that satisfies both transport systems (for example 3-O-methylglucose) the effect of saccharin on the passive transport system is the predominant one, and the cells establish a higher sugar gradient than that observed in the absence of saccharin. The 'non-specific' metabolic effect is manifested as an inhibition of sugar gradient formation when sugars that satisfy only the active system (such as alpha-methylglucoside) are accumulated.

Animals↗

PubMed1991

Glucose transporters: structure, function, and regulation.

Glucose is transported into the cell by facilitated diffusion via a family of structurally related proteins, whose expression is tissue-specific. One of these transporters, GLUT4, is expressed specifically in insulin-sensitive tissues. A possible change in the synthesis and/or in the amount of GLUT4 has therefore been studied in situations associated with an increase or a decrease in the effect of insulin on glucose transport. Chronic hyperinsulinemia in rats produces a hyper-response of white adipose tissue to insulin and resistance in skeletal muscle. The hyper-response of white adipose tissue is associated with an increase in GLUT4 mRNA and protein. In contrast, in skeletal muscle, a decrease in GLUT4 mRNA and a decrease (tibialis) or no change (diaphragm) in GLUT4 protein are measured, suggesting a divergent regulation by insulin of glucose transport and transporters in the 2 tissues. In rodents, brown adipose tissue is very sensitive to insulin. The response of this tissue to insulin is decreased in obese insulin-resistant fa/fa rats. Treatment with a beta-adrenergic agonist increases insulin-stimulated glucose transport, GLUT4 protein and mRNA. The data suggest that transporter synthesis can be modulated in vivo by insulin (muscle, white adipose tissue) or by catecholamines (brown adipose tissue).

Adipose Tissue↗

PubMed1984

Digestive and absorptive functions along dog small intestine: comparative distributions in relation to biochemical and morphological parameters.

The digestive (hydrolytic enzymes) and absorptive (sugar and amino acid transport) functions of dog small intestine have been evaluated in different segments and analysed in relation to morphometric and biochemical parameters. The dog small intestine is a cylinder of decreasing diameter in which the underlying mucosa thins down from duodenum to ileum, though maintaining its cellular homogeneity as revealed by measuring the mucosal weight, the total DNA and protein content and the protein content of the brush border membrane. Sucrase, gamma-glutamyltranspeptidase, leucylnaphthylamidase and alkaline phosphatase specific activities, measured both in homogenates of the mucosa and purified brush border membrane fractions, were found distributed along proximo-distal gradients of activity. However, different patterns were obtained which are specific for the enzyme considered. Kinetic parameters, Vmax and Km, were estimated for sucrase and alkaline phosphatase in purified brush border membrane fractions. It appeared that Vmax correlated well with the observed distribution of catalytic sites along the small intestine. Sugar (glucose) and amino acid (alanine and leucine) transport capacities were also distributed according to specific proximo-distal gradients but passive and facilitated diffusions were not affected. Only the active, Na+ -dependent component of transport was sensitive to position along the small intestine and we postulated that this adaptation should involve variations in carrier densities. It is therefore concluded that absorbo-digestive functions are intrinsic characteristics of the brush border membrane which are regulated according to the position along the small intestine.

Animals↗

PubMed1986

Uric acid uptake in erythrocytes of beagle and dalmatian dogs.

Uric acid uptake by erythrocytes of Beagle and Dalmatian dogs has been measured, using (2-14C) uric acid. Uptake was characterized by a fast and a slow component. Urate uptake was inhibited by certain purine and pyrimidine derivatives and by anion transport inhibitors. It was dependent on intraerythrocyte glycolysis. Temperature only influenced uptake by the slow component (Q10 = 2.6). Urate uptake by the slow component is apparently due to the transport into the erythrocytes by facilitated diffusion (Km = 6.6 mmol/l, Vmax = 390 mumol/l/min), whereas the fast component exhibits an adsorption of urate on erythrocyte surface. No difference of urate uptake by erythrocytes of Beagle and Dalmatian dogs has been observed.

Animals↗

PubMed1987

Low-molecular-weight zinc-binding ligand: a regulatory modulator for intestinal zinc transport.

1. In the last two decades, vast numbers of studies on the zinc nutriture of animals and man have been made. However, the biochemical and physiological events in controlling zinc nutrition are still poorly understood. This report concerns the progress made toward understanding the intestinal zinc absorption and secretion mechanisms. 2. Evidence is accumulating that zinc absorption is a facilitated diffusion while zinc secretion is an active transport. 3. It is known that a low molecular weight zinc-binding ligand (LMW-ZBL) is a key regulator of intestinal zinc absorption, possibly a carrier molecule across the intestinal mucosal cells. Some high molecular weight zinc-binding ligands also appear to be involved in regulating intestinal zinc transport. 4. The identity of the LMW-ZBL is a matter of controversy and its specific role in regulating intestinal zinc transport is not well defined. 5. According to the available literature, no systemic investigations have been made to elucidate the intestinal zinc transport mechanisms, and much more information is needed to fully understand them.

Animals↗

PubMed1994

Amino acid transport by human erythrocyte membranes.

The human erythrocyte plasma membrane is permeable to several free amino acids usually present in the bloodstream. Seven distinct routes of entry have been described which represent both secondary active transport and facilitated diffusion (passive transport). Additionally, certain amino acids can enter the cell by simple diffusion, at least to a limited extent. The function of most of these transport systems is unclear, although it has been suggested that the cell can take up certain amino acids and carry them to various parts of the body. In the case of glutamine, cysteine, and glycine, however, it is believed that the biosynthesis of the tripeptide glutathione is the primary reason for their uptake into the cell. Much of the amino acid transport probably has no function in mature red cells, but might be a remnant of the immature cell's needs. This review discusses the various amino acid transport systems known to be present in the red cell plasma membrane.

Amino Acids↗

PubMed1993

Adenosine 5'-triphosphate modulation of nitrobenzylthioinosine binding sites in plasma membranes of bovine chromaffin cells.

Nitrobenzylthioinosine (NBTI) is a high affinity probe for facilitated diffusion nucleoside transporters. Kinetic analysis of the binding of [3H]NBTI to plasma membranes of chromaffin cells was conducted in the presence or absence of adenosine 5'-triphosphate (ATP). Similar curvilinear plots with a Hill number of 1.32 were obtained in both conditions. ATP significantly increased the number of NBTI binding sites in these preparations showing Bmax values of 1.62 +/- 0.20 pmol/mg protein for controls and 3.22 +/- 0.31 pmol/mg protein in the presence of ATP. However, the affinity constant (KD) was not significantly modified. The non-metabolizable ATP analogue, 5'-adenylyl imidodiphosphate (AMP-PNP) and diadenosine tetraphosphate (Ap4A) can mimic the stimulatory ATP effect, but adenosine monophosphate (AMP) has no effect on the NBTI binding to plasma membranes. These results indicate a modulatory role for ATP, non-hydrolysis dependent, on nucleoside transport in chromaffin cells. Therefore, a nucleotide binding site on the nucleoside transporter similar to that described for glucose transporter could be suggested.

Adenosine Triphosphate↗

PubMed1982

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells.

Incubation of L1210 leukemia cells with 10 microM [3H]adenine in the absence of energy substrate results in a very rapid accumulation of 3H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular 3H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4 degree C wash removes the major portion of intracellular adenine and (ii) at 4 degree C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4 degree C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [3H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.

1-Methyl-3-isobutylxanthine↗