Transport of hemicellulose monomers in the xylose-fermenting yeastCandida shehatae

Summary Cells ofCandida shehatae repressed by growth in glucose- or D-xylose-medium produced a facilitated diffusion system that transported glucose (K _s±2 mM,V _max±2.3 mmoles g⁻¹ h⁻¹),d-xylose (K _s±125 mM,V _max±22.5 mmoles g⁻¹ h⁻¹) and D-mannose, but neither D-galactose norl-arabinose. Cells derepressed by starvation formed several sugar-proton symports. One proton symport accumulated 3-0-methylglucose about 400-fold and transported glucose (K _s±0.12 mM,V _max ± 3.2 mmoles g⁻¹ h⁻¹) andd-mannose, a second proton symport transportedd-xylose (K _s± 1.0 mM,V _max 1.4 mmoles g⁻¹ h⁻¹) andd-galactose, whilel-arabinose apparently used a third proton symport. The stoicheiometry was one proton for each molecule of glucose or D-xylose transported. Substrates of one sugar proton symport inhibited non-competitively the transport of substrates of the other symports. Starvation, while inducing the sugar-proton symports, silenced the facilitated diffusion system with respect to glucose transport but not with respect to the transport of D-xylose, facilitated diffusion functioning simultaneously with thed-xylose-proton symport.     

Regulation of Sugar Transport Systems in Fusarium oxysporum var. lini

Fusarium oxysporum var. lini (ATCC 10960) formed a facilitated diffusion system for glucose (K_s, about 10 mM) when grown under repressed conditions. Under conditions of derepression, the same system was present together with a high-affinity (K_s, about 40 μM) active system. The maximum velocity of the latter was about 5% of that of the facilitated diffusion system. The high-affinity system was under the control of glucose repression and glucose inactivation. When lactose was the only carbon source in the medium, a facilitated diffusion system for lactose was found (K_s, about 30 mM).     

Substrate and endproduct regulation of cellobiose uptake by Candida wickerhamii

Summary Cellobiose-grown cells of Candida wickerhamii transported cellobiose as glucose by a glucose-proton symport after previous hydrolysis of the disaccharide by an exocellular -glucosidase. Both the symport and the -glucosidase were subject to glucose-induced repression and inactivation while glucose also acted as a competitive inhibitor of the enzyme (K _i 0.3 mM). Under conditions of glucose repression glucose was transported by facilitated diffusion. Cellobiose acted as a competitive inhibitor of the latter (K _i 75 mM) and is possibly a low-affinity substrate, while it inhibited non-competitively the glucoseproton symport (K _i 80 mM). The affinity of cellobiose for the cell-bound -glucosidase was much higher (K _m 4.2 mM) than for the purified enzyme as reported by others (K _m 67–225 mM). Ethanol reversibly inhibited the two glucose transport systems with exponential non-competitive kinetics. The minimum inhibitory concentrations were about 3% and 4% (w/v) for facilitated diffusion and proton symport while the respective exponential inhibition constants were 0.58 l mol^-1 and 1.65 l mol^-1. Ethanol affected the -glucosidase in a complex way, a major effect was deviation from Michaelis-Menten kinetics for ethanol concentrations higher than 4% (w/v), the Hill coefficient increasing up to 1.8 at 6% (w/v) ethanol.     

Facilitated diffusion and active transport systems for glucose in metacercariae of Clinostomum marginatum (Digenea)

Metacercariae of Clinostomum marginatum excysted from yellow perch, Perca flavescens, appear to have two systems for transporting glucose across the tegument, facilitated diffusion and active transport. These systems were distinguished by their differential sensitivities to Na⁺, phlorizin and phloretin. In Ringer's saline for cold-blooded vertebrates, 0.1 mm phlorizin and phloretin were incomplete, but similarly effective inhibitors of glucose uptake in 3 min incubations; worms accumulated in 1 h nonmetabolized 3-O-methylglucose against an apparent concentration difference demonstrating the active transport component. In Na⁺-free saline, phlorizin sensitivity and active transport capacity disappeared, but a phloretin sensitive, Na⁺-independent component remained. The V_max and K₁ of the Na⁺-independent system were 3.0 ± 0.54 μmol/g ethanol-extracted dry wt/h, and 0.8 ± 0.36 mm, respectively. V_max and K₁ of the Na⁺-dependent system, estimated by subtracting the Na⁺-independent values from those obtained in Ringer's saline, were 1.3 ± 0.27 μ mol/g ethanol-extracted dry wt/h, and0.7 ± 0.36mm, respectively.     

Glucose and Galactose Transport in Bifidobacterium bifidum DSM 20082

Sugar uptake was measured with ³H-galactose and ¹⁴C-glucose. Galactose transport system was not modified by inhibitors of known translocases and did not present a saturation kinetic with high concentration of galactose. Glucose incorporation was inhibited by lasalocid (cation symport inhibitor) and increased by KCl. The kinetic parameters K _M and V _max were respectively 9.16 mM and 26.56 nmol/min/mg cell protein. On the basis of this study, galactose crossed through the membrane by diffusion, and glucose was incorporated by a cation symport which is regulated by K⁺ ions. Received: 19 February 1997 / Accepted: 20 March 1997     

Role of de novo protein synthesis in the interconversion of glucose transport systems in the yeast Pichia ohmeri

Glucose-repressed cells of the yeast Pichia ohmeri IGC 2879 transported glucose by facilitated diffusion. Derepression led to the formation of a glucose/proton symport and the simultaneous reduction of the facilitated diffusion capacity by about 70%. Cycloheximide prevented this interconversion indicating its dependence on de novo protein synthesis (proteosynthetic interconversion). In buffer with 2% glucose the glucose/proton symport suffered irreversible inactivation while the facilitated diffusion system was simultaneously restored. This reverse interconversion process did not require de novo protein synthesis as indicated by its lack of sensitivity to cycloheximide (degradative interconversion). Thus the glucose/proton symport system appeared to consist of about 70% of the facilitated diffusion proteins turned silent through association with additional protein(s) the latter being sensitive to glucose-induced repression and glucose-induced inactivation.     

Kinetics of glucose transport in human erythrocytes: zero-trans efflux and infinite-trans efflux at 0°C

The kinetic features of glucose transport in human erythrocytes have been the subject of many studies, but no model is consistent with both the kinetic observations and the characteristics of the purified transporter. In order to reevaluate some of the kinetic features, initial rate measurements were performed at 0°C. The following kinetic parameters were obtained for fresh blood: zero-trans efflux K_m = 3.4 mM, V_max = 5.5 mM/min; infinite-trans efflux K_m = 8.7 mM, V_max = 28 mM/min. For outdated blood, somewhat different parameters were obtained: zero-trans efflux K_m = 2.7 mM, V_max = 2.4 mM/min; infinite-trans efflux K_m = 19 mM, V_max = 23 mM/min. The K_m values for fresh blood differ from the previously reported values of 16 mM and 3.4 mM for zero-trans and infinite-trans efflux, respectively (Baker, G.F. and Naftalin, R.J. (1979) Biochim. Biophys. Acta 550, 474–484). The use of 50 mM galactose rather than 100 mM glucose as the infinite-trans sugar produced no change in the infinite-trans efflux K_m values but somewhat lower V_max values. Simulations indicate that initial rates were closely approximated by the experimental conditions. The observed time courses of efflux are inconsistent with a model involving rate-limiting dissociation of glucose from hemoglobin (Naftalin, R.J., Smith, P.M. and Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235–249). The results presented here support the adequacy of the carrier model to account for the kinetics.     

Transport of lactate and other short-chain monocarboxylates in the yeast Candida utilis

Summary Lactic acid grown cells of the yeast Candida utilis transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoicheiometry of 1:1. The accumulation ratio at pH 5.5 was about twenty. The symport accepted the following monocarboxylates (K _svalues at 25°C, pH 5.5 in brackets): d-lactate (0.06 mM), l-lactate (0.06 mM), pyruvate (0.03 mM), propionate (0.05 mM) and acetate (0.1 mM). The system was inducible and was subject to glucose repression. The affinity of the symport for lactate was not affected by pH over the range 3–6, while the maximum transport velocity was strongly pH dependent, its optimum pH being around pH 5. Undissociated lactic acid entered the cells by simple diffusion. The permeability for the undissociated acid increased exponentially with pH, the diffusion constant increasing 35-fold when the pH was increased from 3 to 5.5.     

Specificity of Sugar Transport in Trypanosoma gambiense*

SYNOPSIS Some carbohydrates inhibited glucose and fructose transport in Trypanosoma gambiense. Glucose transport was inhibited by glycerol, mannose, 2-deoxy-D-glucose, glucosamine and N-acetylglucosamine. Fructose transport was inhibited by glucose, glycerol, mannose, glucosamine and N-acetylglucosamine. Glucosamine transport appeared to be a mediated process and had a K_m of 1.20 mM and a V_max of 28.5 μM glucosamine/g dry wt/2 min. Glucosamine absorption was competitively inhibited by glucose, fructose and N-acetylglycosamine. N-Acetylglucosamine appeared to enter by passive diffusion. Reciprocal inhibition experiments suggested that glucosamine entered entirely via the “fructose site.” Specificity of sugar transport in T. gambiense differs from that of other organisms.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

Inactivation of active glucose transport in Candida wickerhamii is triggered by exocellular glucose

Abstract Under conditions of derepression the yeast Candida wickerhamii formed a high-affinity glucose proton symport. Glucose and glucose analogues induced inactivation of the glucose proton symport and its interconversion into a low-affinity facilitated diffusion system. The specific inactivation rate increased with the concentration of the inactivating sugar and did not obey saturation kinetics. This dependence was still pronounced at sugar concentrations far above saturation of the glucose transport systems. This suggested that the inactivation and interconversion mechanism was triggered by interaction of the inactivating sugar with receptor sites located on the cell surface.     

Glucose transport and metabolism in Gymnodinium breve

Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K_FD of 11 μM and a V_max of 1 × 10^?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (K_i = 40 μM), mannose (K_i = 12O μM), and 2-deoxy-d-glucose (K_i = 190 μM) and non-competitively inhibited by galactose (K_i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.     

Trehalose assimilation and turnover inTorulopsis glabrata

Torulopsis glabrata cells have a specific mechanism for endogenous trehalose mobilization that is associated with the lag phase attending new growth. The degree of internal trehalose depletion depends upon the initial concentration of glucose in the medium. Exogenous trehalose was shown to be assimilated via extra-cytoplasmic hydrolysis prior to, transport of the generated glucose into the cytoplasm. The effect on internal trehalose depletion in this case was commensurate with slower presentation of free glucose and was not indicative of the initial concentration of exterrial glucose residues as trehalose. Kinetic parameters for glucose uptake wereK _m =0.49 mM,V _max =109 μmol/min/g dry weight; those for trehalose assimilation wereK _m =2.5 mM,V _max =17.4μmol/min/g dry weight.     

Hepatic hexose transport and the effect of N2-induced anoxia and KCN on this process

Hepatic hexose transport was characterized using 3-O-methyl-D-glucose, which is not metabolized by the liver. The kinetic parameters determined in the starved state were taken as basal values for the transport system which showed saturation kinetics with high V_max and K_m values of 161 nmol/mg dry wt./rnin and 39 mM respectively. In the fed state, the V_max was found to be increased nearly two-fold; this may be due to a phenomenon known as trans-stirnulation. The effects of N₂-induced anoxia and of KCN were investigated. In the fasted state, anoxia caused the transport characteristics V_max and K_m to decrease nearly two-fold whereas KCN had the opposite effect as the V_max and K_m were increased by three- and two-fold respectively. In the fed state, anoxia and KCN caused a marked decrease in the transport characteristics.     

Glucose Transport in the Malarial (Plasmodium lophurae) Infected Erythrocyte*

Plasmodium lophurae-infected red blood cells utilized considerably greater quantities of glucose than did uninfected duckling red cells. Kinetic analysis of glucose transport showed: (A). Below a concentration of 2 mM in the medium the uptake process followed Michaelis-Menten kinetics (carrier-mediated facilitated diffusion) whereas at concentrations greater than this simple diffusion became the main mode of entry. (B). The apparent transport constants, K_t, for normal and infected cells were similar. However there was an 8-fold increase in the maximal velocity, V_max, for infected cells. (C). “Free” malaria parasites had a significantly lower K_t and a higher V_max than did normal or infected red cells. Entry and exit studies with the nonmetabolizable sugar analog, 3-0-methyl glucose, demonstrated that the enhanced rate of uptake by infected cells involved an increase in the simple diffusion component and the degree of enhancement was correlated with the size of the intracellular parasite. Competition experiments suggested that in the malaria-infected cell one locus is involved in the carrier-mediated transport of glucose, mannose and galactose whereas another locus transports fructose and/or glycerol. These results indicate that the enhanced entry of glucose into the malaria-infected red cell is a consequence of factors other than increased glucose catabolism by the host-parasite complex, and the host cell's capacity to take up greater quantities of sugar directly involves the growing intracellular plasmodium.     

Identification of two forms of the maltose transport system in Saccharomyces cerevisiae and their regulation by catabolite inactivation

The maltose transport system of Saccharomyces cerevisiae exists in two forms with K_m values of approx. 4 mM and 70 mM, respectively. The V_max of the high-K_m form is about 4-fold greater than the V_max of the low one. A rapid and irreversible inactivation of both forms is detected on protein synthesis impairment. This inactivation is stimulated by the catabolism of fermentable sugars and prevented during ethanol catabolism. It is concluded that both forms of the maltose transport system are regulated by catabolite inactivation.     

Isolation and characterization of light- and dithiothreitol-modulatable glucose-6-phosphate dehydrogenase from pea chloroplasts

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) has been purified to electrophoretic homogeneity from pea chloroplasts. The enzyme, which has a Stokes radius of 52 Å, is a tetramer made up of four 56000 Da monomers. The pH optimum is around 8.2. The enzyme is absolutely specific for NADP. The apparent K_m(NADP) is 2.4 ± 0.1 μM. NADPH inhibition of the enzyme is competitive with respect to NADP (mean K_i, 18 ± 5 μM) and is mixed (K_p >K_m, V_max >V_p) with respect to glucose 6-phosphate (mean crossover point, 0.5 ± 0.1 mM). The apparent K_m(glucose 6-phosphate) is 0.37 ± 0.01 mM. The purified enzyme is inactivated in the light in the presence of dilute stroma and washed thylakoids, and by dithiothreitol. Enzyme which has been partially inactivated by treatment with dithiothreitol can be further inactivated in the light in the presence of dilute stroma and washed thylakoids and reactivated in the dark, but only to the extent of the reverse of light inactivation. Dithiothreitol-inactivated enzyme is not reactivated further by addition of crude stroma or oxidized thioredoxin. Dithiothreitol-dependent inactivation of the enzyme follows pseudo-first-order kinetics and shows rate saturation. The enzyme which has been partially inactivated by treatment with dithiothreitol does not differ from the untreated control with respect to thermal and tryptic inactivation. However, enzyme which has been partially light inactivated shows different thermal and tryptic inactivation patterns as compared to the dark control. These observations suggest that the changes in the enzyme brought about by light modulation are not necessarily identical with those brought about by dithiothreitol inactivation.     

Nitrogen and phosphorus uptake by the Brazilian kelp Laminaria abyssalis (Phaeophyta) in culture

The uptake of ammonium, nitrate and phosphate by laboratory-grown young sporophytes of Laminaria abyssalis was measured in a perturbed system (batch mode) at 18 °C and 35 ± 5 µE m^–2 s^–1 photon flux density. Uptake of all appeared to follow saturation-type nutrient uptake kinetics. The NO _inf3 ^sup– (K _s = 14.0 µM, V _max = 5.0 µmol h^–1 g^–1 dry wt) and NH _inf4 ^sup+ (K _s = 4.6 µM, V _max= 2.0 µmol h^–1 g^–1 dry wt) were taken up simultaneously, although NH _inf4 ^sup+ was taken up more rapidly. Values of K ₃ and V _max for phosphate were, respectively, 2.21 µM and 0.83 µmol h^–1 g^–1 dry wt. Nitrate and phosphate were both consumed in similar rates (V _max /Ks 0.37) at low concentrations. NH _inf4 ^sup+ , thus, might be a more efficient form of N fertilizer if artificial enrichment of seawater is used.     

Kinetic Analyses of the Substrate Inhibition of <Emphasis Type="Italic">Paramecium</Emphasis> Arginine Kinase

Paramecium tetraurelia expresses four types of arginine kinase (AK1–AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis–Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3–15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R², AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, V_max^SES?=?30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, V_max^ES?=?241.7. The dissociation constant for the SES complex (K_i^SES: 0.34 mM) was two times smaller than that of the ES complex (K_s^ES: 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower V_max^SES. The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the K_i^SES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, V_max^ES and V_max^SES values for the mutants decreased by one-third, except for the V_max^SES of the S79A mutant, which had a value that was comparable with the value for the wild-type.