Sugar Transport and Sulfite Action in Etioprotoplasts,Semi-Etioprotoplasts and Green Protoplasts of Avena sativa L.

The mechanism of glucose and sucrose transport and the influence of various concentrations of sulfite on its activity was studied in mesophyll protoplasts (etioprotoplasts, semi-etioprotoplasts and green protoplasts) isolated from oat (Avena sativa L.) seedlings. Kinetic analysis of [¹⁴C] glucose loading (in darkness) revealed in each kind of protoplasts the presence of two transport components. At low exogenous glucose concentrations a saturable system was the main mode of transport. At concentrations higher than 20 mM the loading of glucose in all types of protoplasts was dominated by a non-saturable, linear diffusion-like component. The rate of glucose uptake was greatest in etioprotoplasts and lowest in green protoplasts. In contrast to the above we have not found saturable components of sucrose transport in any kind of protoplasts. The rate of its uptake was greatest in semi-etioprotoplasts. Sulfite, at a concentration of < 1.0 mM stimulated and at ≥ 1.0 mM inhibited the uptake of glucose to etioprotoplasts and semi-etioprotoplasts and inhibited that to green protoplasts at any concentration. The transport of sucrose underwent a significant inhibition in the various types of protoplasts only under the influence of 10.0 mM of sulfite ions. Inhibition of glucose uptake by sulfite was of the non-competitive type. Sulfite also affected the level of adenylic nucleotides and lowered the energy charge and ATP/ADP ratio. Intensity of sulfite uptake was significantly higher in green protoplasts than in etioprotoplasts.     

Evidence for functionally distinct glucose transporters in basal and insulin-stimulated adipocytes

The activity and Km of glucose transport of rat adipocytes are quite variable in the basal state. This could be due to differing levels of highly saturable transport against a background of less saturable transport. Such heterogeneity could lead to differing conclusions as to the Km of basal cells compared to insulin-stimulated cells depending on the choice of substrate, the range of concentrations tested, and the rigor of data analysis. In the present work, we used a cell preparation which was stable and partially activated by constant agitation. We used a two-component model to fit the concentration dependence of D-glucose uptake. We defined two parallel pathways of glucose entry, a high-affinity/low-capacity pathway and a low-affinity/high-capacity pathway. Both pathways were stereospecific and were inhibited by cytochalasin B. The low-affinity pathway in basal cells had 97% of the total capacity (Vmax) with a high Km (greater than 50 mM). A second pathway had a very low Km (less than 1 mM) and only 3% of the total capacity, but contributed to 30-60% of glucose uptake at 8 mM glucose. In insulin-stimulated cells, a pathway with a Km of 4-5 mM dominated and contributed 85% of glucose transport. The low-affinity but not the very high affinity pathway persisted in stimulated cells, but its contribution was only 10-15% of transport at 8 mM glucose. These results suggest the presence of at least two functionally distinct transporters whose respective contributions can be characterized by nonlinear regression of data over a wide range of glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The contribution of electrostatic forces to the process of adherence of Candida albicans yeast cells to substrates

Abstract Thermoanaerobacter thermohydrosulfuricus Rt8.B1 catabolized xylose by the pentose phosphate pathway, and xylose isomerase and xylulokinase were inducible. The uptake of xylose was by two low-affinity, inducible systems. Both systems were resistant to the protonophore, tetrachlorosalicylanilide, the F₁F₀-ATPase inhibitor, N , N -dicyclohexylcarboiimide, and the sodium/proton antiporter, monensin. The high capacity system (100 nmol min⁻¹ (mg protein)⁻¹) was only expressed when the bacterium was grown with a high concentration of xylose (50 mM). It took more than 60 mM xylose to saturate the high capacity system. When T. thermohydrosulfuricus was grown with a low concentration of xylose (5 mM), xylose uptake was saturated by as little as 10 mM xylose (18 nmol min⁻¹ (mg protein)⁻¹). Cells grown with 50 mM xylose could not transport glucose, and high capacity xylose transport was not inhibited by glucose or non-metabolizable glucose analogues. Cells grown with 5 mM xylose transported glucose at a rapid rate (30 nmol min⁻¹ (mg protein)⁻¹), and low capacity xylose uptake was competitively inhibited by either glucose or 2-deoxy-glucose. Because the glucose uptake of cells grown on 5 mM xylose was competitively inhibited by xylose, it appeared that the low capacity xylose uptake system was a glucose/xylose carrier.     

Characteristics of glucose uptake by glucose- and NH4-limited grown Penicillium ochrochloron at low, medium and high glucose concentration

Glucose uptake by Penicillium ochrochloron (formerly Penicillium simplicissimum) was studied from 0.01 to 400 mM glucose using chemostat culture and bioreactor batch culture. The characteristics of glucose uptake varied considerably with the conditions of growth, harvest and uptake assay. Glucose-limited grown mycelium showed one saturable transport system [K(S) below 0.01 mM; v(max) 1.1-1.2 mmol (g dry weight)(-1)h(-1)] plus a first order process (permeability P=1.2x10(-7)cm s(-1)). Ammonium-limited grown mycelium showed only one saturable transport system [K(S) 0.3-0.7 mM; v(max) 0.5-0.8 mmol (g dry weight)(-1)h(-1)]. During exponential growth at high glucose concentration (300-400 mM) a first order process was found with a P value of 5.6-9.3x10(-7)cm s(-1). After ammonium exhaustion a second first order phase showed a lower P value (6.1-9.3x10(-8)cm s(-1)). A similar change in permeability was also found after a re-evaluation of published data for Gibberella fujikuroi, Aspergillus niger, Aspergillus awamori and Saccharomycopsis lipolytica. For the first order processes simple diffusion was ruled out as a mechanism for glucose uptake. Glucose uptake by P. ochrochloron was controlled more strongly by metabolism than by transport and was not rate limiting for overflow metabolism.     

Regulation of glucose transport in Candida utilis

The transport systems for glucose present in Candida utilis cells, growing in batch and continuous cultures on several carbon sources, have been studied. Two different systems were found: a proton symport and a facilitated diffusion system. The high-affinity symport (Km for glucose about 15 microM) transported one proton per mole of glucose and was partially constitutive, appearing in cells grown on gluconeogenic substrates such as lactate, ethanol and glycerol. It was also induced by glucose concentrations up to 0.7 mM and repressed by higher ones. The level of repression depended on the external glucose concentration at which cells had grown in a way similar to that shown by the maltose-uptake system, so both systems seem to be under a common glucose control. Initial uptake by facilitated diffusion, the only transport system present in cells growing at glucose concentrations higher than 10 mM, showed a complex kinetic dependence on the extracellular glucose concentration. This could be explained either by the presence of at least two different systems simultaneously active, one with a Km around 2 mM and the other with a Km of about 1 M, or by the allosteric or hysteretic behaviour of a single carrier whose apparent Km would oscillate between 2 and 70 mM.     

Effect of glucocorticoids on the glucose transport system of isolated fat cells.

Glucocorticoids inhibit glucose utilization by fat cells. The possibility that this effect results from altered glucose transport was investigated using an oil-centrifugation technique which allows a rapid (within 45 s) estimation of glucose or 3-O-methylglucose uptake by isolated fat cells. At high concentration (greater than 25 muM), dexamethasone inhibited glucose uptake within 1 min of its addition to fat cells. Efflux of 3-O-methylglucose was also impaired by 0.1 mM dexamethasone. However, diminished glucose uptake was not a specific effect of glucocorticoids; high concentrations (0.1 mM) of 17beta-estradiol, progesterone, and deoxycorticosterone produced a similar response in adipocytes. At a more physiologic steroid concentration (0.1 muM), glucocorticoids inhibited glucose uptake in a time-dependent manner (maximum effect in 1 to 2 hours). This effect was specific for glucocorticoids since, under these conditions, glucose uptake was not changed by the non-glucocorticoid steroids. Lineweaver-Burk analysis showed that 0.1 muM dexamethasone treatment produced a decrease in Vmax for glucose uptake but did not change the Ku. Hexokinase activity and ATP levels were not altered by this treatment, suggesting that processes involved in glucose phosphorylation were not affected. Dexamethasone treatment also caused a reduction in uptake of 3-O-methylglucose when assayed using a low sugar concentration (0.1 mM). At a high concentration (10 mM), uptake of the methyl sugar was only slightly less than normal in treated cells. Stimulation by insulin markedly enhanced uptake of glucose and 3-O-methylglucose by both treated and untreated cells. At a low hexose concentration (0.1 mM) and in the presence of insulin, sugar uptake by dexamethasone-treated cells was slightly less than control cells. Stimulation by insulin did however completely overcome the alteration in hexose uptake when larger concentrations of sugars (greater than 5 mM) were used. There was no detectable change in total protein synthesis during incubation of fat cells with dexamethasone. However, actinomycin C blocked the inhibitory effect of dexamethasone on glucose uptake. Cycloheximide, which caused a small inhibition in glucose uptake, prevented the full expression of the inhibitory effect of dexamethasone on glucose transport. These results indicate that dexamethasone alters the facilitated transport of glucose and, secondly, suggest that synthesis of RNA and protein is needed for glucocorticoid action.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H(+) -dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of D- and L-[U-(14)C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-D-glucose/D-[U-(14)C]glucose and 3-O-methyl-D-glucose/3-O-methyl-D-[U-(14)C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH(4)Cl inhibited neither the linear component of D- and L-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-D-[U-(14)C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol(-1), respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol(-1)). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Kinetics of myo-inositol transport in corneal endothelial cells: diverse effects of sugars and implications in corneal deutergensence [corrected

Kinetics of myo-inositol (MI) uptake into primary cultures of bovine corneal endothelial cells (CEC) were studied. Confluent corneal endothelial cells accumulated 3H-MI in a time dependent and saturable process. At a narrow range of external concentrations of 3H-MI (4-50 microM), the Na(+)-dependent MI uptake followed saturation kinetics. The apparent Km value was 20 microM with a maximum velocity (Vmax) of 16 pmol/20 min/micrograms DNA. At low external 3H-MI concentrations the uptake was dependent on Na ions, but at higher levels the Na(+)-independent fraction of MI uptake significantly increased. The uptake was sensitive to removal of Ca ions and to the presence of inhibitors such as n-ethyl maleimide, phlorizin, ouabain, and amiloride (an inhibitor of Na+/H+ exchanger). The sensitivity of MI uptake toward inhibitors and ionic changes in the bathing media was reduced as external concentrations of 3H-MI increased. Citrate at 0.5 mM increased the uptake, suggesting involvement of mitochondrial oxidative metabolism in the MI uptake. Percent release of radioactivity by 2 min, after an initial 40-min incubation with 20 microM 3H-MI, was 6.6% +/- 0.8 or 35% +/- 4 when release media contained BSS alone or BSS containing 5 mM nonradioactive MI, respectively. Efflux of radioactivity from the cells also was enhanced when release media contained 40 mM glucose. Glucose and galactose as well as nonmetabolizable glucose analogues, such as 3O-methyl glucose or alpha-methyl glucose, at high concentrations (40 mM), acutely (in the incubation media) or chronically (in the growth media) inhibited MI uptake into CEC, and the extent of inhibition was inversely proportional to the external levels of 3H-MI. However, glucose at lower levels (less than or equal to 10 mM) slightly increased MI uptake. These studies indicated that the uptake of MI into corneal endothelial cells was an Na(+)-dependent active process at a narrow range of external radioactive MI concentrations. Higher levels of MI were taken up by the cells via a passive diffusion mechanism, independent of carrier protein(s). Glucose influenced the uptake of MI in a complex manner. The increased MI efflux by glucose or by MI was perhaps due to the limited capacity of CEC for accumulation or compartmentalization of this or other solutes/osmolytes, a phenomenon that may be related to the role of CEC in maintenance of corneal deutergence. High glucose-induced inhibition of Na(+)-dependent MI uptake may be in part due to glucose regulation of Na+ fluxes and cell volume.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of kinase-dependent glucose uptake in Saccharomyces cerevisiae

There are both low- and high-affinity mechanisms for uptake of glucose in Saccharomyces cerevisiae; high-affinity uptake somehow depends on the presence of hexose kinases (L. F. Bisson and D. G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983; L. F. Bisson and D. G. Fraenkel, J. Bacteriol. 155:995-1000, 1983). We report here on the effect of culture conditions on the level of high-affinity uptake. The high-affinity component was low during growth in high concentrations of glucose (100 mM), increased as glucose was exhausted from the medium, and decreased again during prolonged incubation in the stationary phase. The higher level of uptake was found in growth on low concentrations of glucose (0.5 mM) and in growth on normal concentrations of galactose, lactate plus glycerol, or ethanol. These results suggest that some component of high-affinity uptake is repressible by glucose. A shift from medium with 100 mM glucose to medium with 5 mM glucose resulted in up to a 10-fold increase in the level of high-affinity uptake within 90 min; the increase did not occur in the presence of cycloheximide or 2,4-dinitrophenol or in buffer alone with low glucose, suggesting that protein synthesis or energy metabolism (or both) was required. Reimposition of the high glucose concentration caused loss of high-affinity uptake, a process not prevented by cycloheximide. The use of hexokinase single-gene mutants showed that the derepression of high-affinity uptake was not clearly correlated with changes in levels of the kinases themselves. These results place the phenomenon of high- and low-affinity uptake in a physiological context, in that high-affinity uptake seems to be expressed best in conditions where it might be needed. Apparent similarities between glucose uptake in yeast and animal cells are noted.     

Interactions between nitrogen fixation and osmoregulation in the methanogenic archaeon methanosarcina barkeri 227

The nitrogenase enzyme complex of Methanosarcina barkeri 227 was found to be more sensitive to NaCl than previously studied molybdenum nitrogenases are, with total inhibition of activity occurring at 190 mM NaCl, compared with >600 mM NaCl for Azotobacter vinelandii and Clostridium pasteurianum nitrogenases. Na+ and K+ had equivalent effects, whereas Mg2+ was more inhibitory than either monovalent cation, even on a per-charge basis. The anion Cl- was more inhibitory than acetate was. Because M. barkeri 227 is a facultative halophile, we examined the effects of external salt on growth and diazotrophy and found that inhibition of growth was not greater with N2 than with NH4+. Cells grown with N2 and cells grown with NH4+ produced equal concentrations of alpha-glutamate at low salt concentrations and equal concentrations of Nepsilon-acetyl-beta-lysine at NaCl concentrations greater than 500 mM. Despite the high energetic cost of fixing nitrogen for these osmolytes, we obtained no evidence that there is a shift towards nonnitrogenous osmolytes during diazotrophic growth. In vitro nitrogenase enzyme assays showed that at a low concentration (approximately 100 mM) potassium glutamate enhanced activity but at higher concentrations this compound inhibited activity; 50% inhibition occurred at a potassium glutamate concentration of approximately 400 mM.     

Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells

Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 microM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.     

Effects of pH and type of sugar in the medium on tyrosinase activity in cultured melanoma cells.

The tyrosinase (EC 1.14.18.1) activity of cultured mouse melanoma cells B16 in the stationary phase of growth, depends greatly on the pH of the medium and the kind of sugar present. The enzyme activity of a homogenate of cells grown at pH 7.2 in Eagles's MEM supplemented with 10% new born calf serum and con taining galactose in place of glucose, was about ten times that of a homogenate of cells cultured at pH 6.3 in the same medium. The tyrosinase activity changed reversibly on changing the pH of the culture medium. When cultured at a constant pH of 7.2, cells grown with 1 mM galactose had about five times higher tyrosinase activity than cells grown with 1 mM glucose. Only a small amount of lactate accumulated in cultures with glucose and it had little effect on the enzyme activity. These two findings explain the very low tyrosinase activity of cells cultured in medium with 5 mM glucose: the low activity is due to the presence of glucose and to the low pH resulting from conversion of glucose to lactic acid.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.     

Interaction between facilitated diffusion of glucose across the plasma membrane and its metabolism in Trichomonas vaginalis

Abstract The parasitic protist Trichomonas vaginalis transport glucose across the plasma membrane by facilitated diffusion. The K _m of the transporter for glucose was 1.6 mM. The uptake of labelled glucose in a minimal medium not allowing growth reached saturation only after 2.5 h, indicating the turnover of storage carbohydrate. Organisms grown on glucose showed higher activities both of the transporter and of the subsequent metabolic pathway than organisms grown on maltose. At low external glucose concentrations the transport step was rate limiting, at higher levels a subsequent enzymatic step. The uptake mechanism for glucose of T. vaginalis resembled that of parasitic kinetoplastid protist and Entamoeba histolytica .     

Heme Iron Uptake by Caco-2 Cells is a Saturable,Temperature Sensitive and Modulated by Extracellular pH and Potassium

It is known that heme iron and inorganic iron are absorbed differently. Heme iron is found in the diet mainly in the form of hemoglobin and myoglobin. The mechanism of iron absorption remains uncertain. This study focused on the heme iron uptake by Caco-2 cells from a hemoglobin digest and its response to different iron concentrations. We studied the intracellular Fe concentration and the effect of time, K⁺ depletion, and cytosol acidification on apical uptake and transepithelial transport in cells incubated with different heme Fe concentrations. Cells incubated with hemoglobin-digest showed a lower intracellular Fe concentration than cells grown with inorganic Fe. However, uptake and transepithelial transport of Fe was higher in cells incubated with heme Fe. Heme Fe uptake had a low V _max and K _m as compared to inorganic Fe uptake and did not compete with non-heme Fe uptake. Heme Fe uptake was inhibited in cells exposed to K⁺ depletion or cytosol acidification. Heme oxygenase 1 expression increased and DMT1 expression decreased with higher heme Fe concentrations in the media. The uptake of heme iron is a saturable and temperature-dependent process and, therefore, could occur through a mechanism involving both a receptor and the endocytic pathway.     

Developmental and other characteristics of lysine uptake by rat brain synaptosomes.

Synaptosomes isolated from adult or newborn rat cerebrum take up L-lysine by two saturable systems, one with a high affinity low capacity and the other with a low affinity high capacity. Initial rate of uptake for low lysine concentrations is mort tissue. Analysis of kinetic data indicates that synaptosomes of the newborn have a higher Vmax than those of the adult for high affinity system but adult for high affinity system but adult synaptosomes have a higher Vmax than newborn for low affinity system. At a physiological lysine concentration of 0.5 mM, the calculated contributions of two systems indicate that the adult uptake occurs for about 71% by low affinity system but the newborn utilizes both systems to the same extent. The uptake is sodium independent but pH dependent. Lysine uptake is inhibited by other dibasic amino acids, arginine and ornithine but not cystine. Kinetic analysis indicates that arginine specifically inhibits the high affinity, low Km system for lysine uptake.     

Increased glucose permeability in Babesia bovis-infected erythrocytes

Glucose influx into bovine erythrocytes was found to be significantly increased upon infection with the parasite, Babesia bovis. The influx of glucose into the infected cells over 4 min was not saturable at high concentrations of glucose (240 mM), nor was it affected by established inhibitors of mammalian glucose transport, such as cytochalasin B and phloretin (0.1-100 microM). Glucose uptake into the parasitized cells was, however, inhibited by phloridzin (phloretin-2-beta-glucoside) at concentrations over the range of 10-500 microM. Further inhibition of glucose uptake by adenosine (2.5-15 mM) was found to occur in B. bovis-infected bovine erythrocytes, suggesting an interaction of adenosine with the new or altered component of glucose transport in the parasitized cells.     

Adaptive control at low glucose concentration of HEK-293 cell serum-free cultures.

Fed-batch cultures were implemented to study the metabolism of HEK-293 cells. Glucose, measured every 30 min by a FIA biosensor system, was maintained at 1 mM throughout the culture using an adaptive nonlinear controller based on minimal process modeling. The controller performed satisfactorily at both low and high cell concentrations without the need for retuning between different culture phases. Overall, lactate production was significantly reduced by maintaining a low glucose concentration, thus decreasing the rate of glycolysis. The rates of glucose and glutamine uptake as well as the lactate and ammonia production were compared to those obtained in batch mode with an initial glucose concentration of 21 mM. Basically, three phases were observed in both culture modes. The metabolic shift from the first to the second phase was characterized by a significant reduction in glucose consumption and lactate production while maximum growth rate was maintained. The specific respiration rate appeared unchanged during the first two phases, suggesting that no change occurred in the oxidative pathway capacity. In the third phase, cell growth became slower very likely due to glutamine limitation.     

Cation transport in Escherichia coli. VIII. Potassium transport mutants

Analysis of K transport mutants indicates the existence of four separate K uptake systems in Escherichia coli K-12. A high affinity system called Kdp has a Km of 2 muM, and Vmax at 37 degrees C of 150 mumol/g min. This system is repressed by growth in high concentrations of K. Two constitutive systems, TrkA and TrkD, have Km's of 1.5 and 0.5 mM and Vmax's of 550 and 40 at 37 and 30 degrees C, respectively. Mutants lacking all three of these saturable systems take up K slowly by a process, called TrkF, whose rate of transport is linearly dependent on K concentration up to 105 mM. On the whole, each of these systems appears to function as an independent path for K uptake since the kinetics of uptake when two are present is the sum of each operating alone. This is not true for strains having both the TrkD and Kdp systems, where presence of the latter results in K uptake which saturates at a K concentration well below 0.1 mM. This result indicates some interaction between these systems so that uptake now has the affinity characteristic of the Kdp system. All transport systems are able to extrude Na during K uptake. The measurements of cell Na suggest that growing cells of E. coli have very low concentrations of Na, considerably lower than indicated by earlier studies.     

Studies on choline permeation through the plasma membrane and its incorporation into phosphatidyl choline of Ehrlich-Lettré-ascites tumor cells in vitro.

The initial rate of incorporation of 14C or 3H-labeled choline into Ehrlich-Lettre ascites cells of the glycogen-free strain seven days after inoculation was investigated in vitro. 1. At choline concentrations in the medium between 6 to 30 muM and 100 to 500 muM the choline uptake by the cells followed Michaelis-Menton Kinetics with V values between 31 to 100 and 59 to 500 pmol per minute at a given cell density, and average Q10-values of 2.1 at the high and of 2.4 at the low choline molarity. The K-m-values increased from 27 muM to 58.8 muM at low and from 0.11 mM to 0.22 mM at high choline concentrations over a temperature range between 15 degrees C and 37 degrees C. Arrhenius plot of the V values gave two lines, one with a transition temperature at 25 degrees C at low and one straight line at high choline concentrations, from which the energy of activation for choline uptake was determined to be 16 kcal/mol. 2. It is assumed that two systems exist for the choline uptake by the ascites cells. One, operative at low substrate concentrations, which is saturable and probably is to be classified as a carrier-mediated facilitated diffusion process, can be strongly inhibited by deoxyglucose or 2,4-dinitrophenol and also by substrate analogues such as chlorocholine or benzoylcholine. Ouabain affects this system to a lesser extent. The other system functioning at high choline concentrations may be a simple diffusion process, which is little inhibited by substrate analogues, ouabain and deoxyglucose; however, it is also inhibited by 2,4-dinitrophenol and p-chloromercuribenzoate. 3. Choline incorporation into the acid-insoluble material (lecithin) gave linear Michaelis-Menton kinetics at the low and the high substrate concentration respectively. K-m-values decreased with an increase in temperature at low and increased with rising temperature at high substrate concentrations thus reflecting a close relationship between choline uptake and its metabolism. Labeling of lecithin choline in the various subcellular fractions under the conditions of the functioning of a carrier-mediated process was in the order: mitochondria (50%) greater than plasma membranes (25%) greater nuclei (14%) greater than microsomes (9%) greater than supernatant (1.5%). 4. Treatment of the cells with p-chloromercuribenzoate or heat shock at 50 degrees C markedly reduced the cholinee uptake and concomitantly its conversion into lecithin. Kinetic analysis revealed that the inhibitory effect of p-chloromercuribenzoate was competitive and that of the heat shock non-competitive in nature. Further the choline uptake by the cells was found to be the rate-limiting step, since the rate of choline phosphorylation was determined by the extracellular choline concentration. Pulse chase experiments showed a rapid turnover of the choline moiety with a concomitant increase in activity of the lecithin fraction and little change within the choline phosphate pool.