Hexose transport in L6 rat myoblasts. I. Rate-limiting step, kinetic properties, and evidence for two systems

The hexose transport system of undifferentiated L6 rat myoblasts was investigated. 2-Deoxy-D-glucose (2-DOG) and 2-deoxy-2-fluoro-D-glucose (2FG) were used as analogues to investigate the rate-limiting step of hexose uptake into the cell. Virtually all of the 2-DOG or 2FG taken up into the cell was found to be in the phosphorylated form. No significant pool of intracellular free sugar could be detected. This demonstrates that hexose transport, not phosphorylation, is the rate-limiting step. The inhibitory effect of various glucose analogues on 2-DOG and 3-O-methyl-D-glucose (3-OMG) uptake revealed that these two sugars may be taken up into the cell by different carriers. In addition, kinetics analysis of the transport of both sugars also indicates that two hexose transport systems may be present in L6 cells. 2-DOG is transported by high and low affinity transport systems (Km 0.6 mM and 2.9 mM, respectively), whereas 3-OMG is transported by a low affinity system (Km 3.5 mM). Treatment of cells with ionophores or energy uncouplers results in inactivation of the high affinity system, but not the low affinity system.     

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts

Hexose transport in plasma membrane vesicles prepared from L6 rat myoblasts was shown to be stereospecific, activated by glucose starvation and occurred by both high and low affinity systems. Transport by the high affinity system was shown to occur by an active transport process. Furthermore, the high affinity system was shown to be defective in vesicles prepared from F72 cells (hexose transport mutant). These results indicate that the high affinity hexose transport system is retained in the plasma membrane vesicles. Thus plasma membrane vesicles could be of value in further characterization of the L6 high affinity hexose transport system, without interference from the various metabolic events occurring in whole cells.     

Hexose transport in L6 rat myoblasts. II. The effects of sulfhydryl reagents

The importance of sulfhydryl groups for hexose transport in undifferentiated L6 rat myoblasts was investigated. N-ethylmaleimide (NEM) and p-chloromer-curibenzenesulfonic acid (pCMBS) inhibited 2-deoxy-D-glucose (2-DOG) transport in a time and concentration-dependent manner. The inhibition produced by both reagents was virtually complete within 5 min, although neither reagent inhibited transport more than 70–80% regardless of the concentrations or incubation times used. Furthermore, the inhibition of 2-DOG transport by pCMBS or NEM could not be prevented by simultaneous preincubation of cells with 20 mM D-glucose or 20 mM 2-DOG. This suggests that sulfhydryl groups required for transport are separate from the hexose binding and transport site. By comparing the effects of the membrane impermeant pCMBS to those of the membrane permeant NEM, cell surface sulfhydryl groups were shown to be essential for hexose binding and transport. In contrast to the inhibition of 2-DOG transport, pCMBS and NEM had much less of an effect on 3-O-methyl-D-glucose (3-OMG) transport. For example, 1 mM NEM inhibited 2-DOG transport by 66%, whereas 3-OMG transport was inhibited by only 7%. This supports the suggestion that these hexose analogues may be transported by different carriers. Kinetic analysis of transport shows that treatment of cells with 1 mM NEM or 1 pCMBS results in inactivation of the high affinity 2-DOG transport system, whereas the low affinity transport system is unaffected. 3-OMG is preferentially transported by the low affinity system.     

Hexose transport in human myoblasts.

The present investigation reports on the hexose transport properties of human myoblasts isolated from normal subjects and from patients with Duchenne muscular dystrophy (DMD). Similar to rat myoblast L6, normal human myoblasts possess a high- (HAHT) and a low- (LAHT) affinity hexose transport system. The non-metabolizable hexose analogue, 2-deoxyglucose, is preferentially taken up by HAHT. The transport of this analogue is the rate-limiting step in the uptake process. This human myoblast HAHT is also similar to that of the rat myoblast in its substrate specificity and in response to the energy uncouplers, cytochalasin B and phloretin. The human myoblast LAHT resembles that of rat myoblast in its insensitivity to energy uncouplers, and in its transport affinity and capacity for 3-O-methyl-D-glucose. Although DMD myoblasts resemble their normal counterpart in their ability to differentiate, they differ significantly in their hexose transport properties. In addition to HAHT and LAHT present in normal human myoblast, DMD myoblasts contain a super-high-affinity hexose transport system (SHAHT). SHAHT can be detected only at very low substrate concentrations. It differs from HAHT not only in its much higher transport affinity, but also in its response to the traditional hexose transport inhibitors. For example, SHAHT can be activated by cytochalasin B and phlorizin, whereas it is more sensitive to inhibition by phloretin. Unlike HAHT, energy uncouplers are found to be ineffective in inhibiting SHAHT. It should be mentioned that SHAHT cannot be detected in myoblasts isolated from patients with other types of myopathy. The present study serves to demonstrate that more than one hexose transport system is operating in human skeletal muscle cells, as found in other cell types.     

Cytochalasin B as a probe for the two hexose-transport systems in rat L6 myoblasts.

We have recently demonstrated that two hexose-transport systems are present in undifferentiated rat L6 myoblasts: D-glucose and 2-deoxy-D-glucose are preferentially transported by the high-affinity system, whereas 3-O-methyl-D-glucose is transported primarily by the low-affinity system. Mutant D23 is found to be defective only in the high-affinity hexose-transport system. The low-affinity transport system is much more sensitive to inhibition by cytochalasin B (CB). The present study examines the identity, properties and regulation of the CB-binding sites by measuring CB binding to both whole cells and plasma membrane. Scatchard analysis of the binding data revealed the presence of two CB-binding sites, namely CBH and CBL. These two sites differ not only in their affinity for CB, but their levels can also be differentially altered by various biochemical, physiological and genetic manipulations. CBL resembles the high-affinity hexose-transport system in that it is absent in mutant D23 and is present in larger quantities in glucose-starved cells. Moreover, CB binding to this site is inhibited by D-glucose and 2-deoxy-D-glucose, the preferred substrates of the high-affinity hexose-transport system. On the other hand, CBH is found to be unaltered in mutant D23, which also retains the normal low-affinity hexose-transport system. CBH also resembles the low-affinity transport system in that it is not elevated in glucose-starved cells. Furthermore, binding of CB to this site can be inhibited by 3-O-methyl-D-glucose, the preferred substrate of the low-affinity transport system. It should be noted that 2-deoxy-D-glucose does not have much effect on CBH, and vice versa. Studies with purified membrane preparations indicate that both CB-binding sites are present in similar ratios in the plasma membrane and the low-density microsomal fraction. Plasma-membrane studies also reveal that D-glucose 6-phosphate, but not 2-deoxy-D-glucose 6-phosphate, is very effective in activating CB binding. Data presented suggest that CB binding may be regulated by sugar analogues in an allosteric manner.     

Regulation of hexose transport in rat myoblasts during growth and differentiation

We report here the effects of growth conditions and myogenic differentiation on rat myoblast hexose transport activities. We have previously shown that in undifferentiated myoblasts the preferred substrates for the high (HAHT)- and low (LAHT)-affinity hexose transport systems are 2-deoxyglucose (2-DG) and 3-O-methyl-D-glucose (3-OMG), respectively. The present study shows that at cell density higher than 4.4 x 10(4) cells/cm2, the activities of both transport processes decrease with increasing cell densities of the undifferentiated myoblasts. Since the transport affinities are not altered, the observed decrease is compatible with the notion that the number of functional hexose transporters may be decreased in the plasma membrane. Myogenic differentiation is found to alter the 2-DG, but not the 3-OMG, transport affinity. The Km values of 2-DG uptake are elevated upon the onset of fusion and are directly proportional to the extent of fusion. This relationship between myogenesis and hexose transport is further explored by using cultures impaired in myogenesis. Treatment of cells with 5-bromo-2'-deoxyuridine abolishes not only myogenesis but also the myogenesis-induced change in 2-DG transport affinity. Similarly, alteration in 2-DG transport affinity cannot be observed in a myogenesis-defective mutant, D1. However, under myogenesis-permissive condition, the myogenesis of this mutant is also accompanied by changes in its 2-DG transport affinity. The myotube 2-DG transport system also differs from its myoblast counterpart in its response to sulfhydryl reagents and in its turnover rate. It may be surmised from the above observations that myogenesis results in the alteration of the turnover rate or in the modification of the 2-DG transport system. Although glucose starvation has no effect on myogenesis, it is found to alter the substrate specificity and transport capacity of HAHT. In conclusion, the present study shows that hexose transport in rat myoblasts is very sensitive to the growth conditions and the stages of differentiation of the cultures. This may explain why different hexose transport properties have been observed with myoblasts grown under different conditions.     

Isolation and characterization of hexose transport mutants in L6 rat myoblasts

A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2-deoxy-D-glucose (2-DOG)-and 2-deoxy-2-fluoro-D-glucose (2FG)-resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2-DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2-DOG, 2FG, or 3-O-methyl-D-glucose (3-OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four- to five-fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for alpha-aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2-DOG and 3-OMG transport in mutant F72 demonstrated that the Vmax for 2-DOG uptake was significantly reduced, whereas the Vmax for 3-OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high-affinity hexose transport system.     

Involvement of hexose transport in myogenic differentiation

A high (HAHT) and a low (LAHT) affinity hexose transport system are present in undifferentiated rat L6 myoblasts; however, only the latter can be detected in multinucleated myotubes. This suggests that HAHT is either down-regulated or modified as a result of myogenesis. The present investigation examined the relationship between HAHT and myogenic differentiation. While myogenesis could be inhibited by the potent hexose transport inhibitor phloretin, it was not affected by phlorizin which had no effect on hexose transport. This relationship was further explored using six different HAHT-defective mutants. All six mutants, altered in either the HAHT transport affinity (Type I mutants) or capacity (Type II mutants), were impaired in myogenesis. Since these mutants were selected from both mutagenized and non-mutagenized cells with different reagents, or with different concentrations of the same reagent, the deficiency in myogenesis was likely due to changes in HAHT properties. This notion was confirmed by the observation that growth of Type I mutants in high D-glucose concentrations could rectify the defect in myogenesis. D-glucose was unlikely to rectify the defect in myogenesis, if this defect was due to a second unrelated mutation that may have arisen during isolation of the mutants. Since both types of mutants were not altered in LAHT, D-glucose should still be taken up into the cells. The fact that the glucose-mediated increase in fusion could not be observed in Type II mutants (deficient in the HAHT transporter) suggested that myogenesis was dependent on the presence of D-glucose or its metabolites in specific HAHT-accessible compartments. It is tempting to speculate that trans-acting regulators involved in myogenesis may be synthesized from the glucose metabolites in these specialized HAHT-accessible compartments.     

Sugar transport in Coprinus cinereus.

Two transport systems for glucose were detected: a high affinity system with a Km of 27 muM, and a low affinity system with a Km of 3.3 mM. The high affinity system transported glucose, 2-deoxy-D-glucose (Km = 26 muM), 3-O-methylglucose (Km = 19 muM), D-glucosamine (Km = 652 muM), D-fructose (Km = 2.3 mM) and L-sorbose (Km = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45 degrees C). The low affinity system transported glucose, 2-deoxy-D-glucose (Km = 7.5 mM), and 3-O-methylglucose (Km = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30--50 degrees C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-D-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present insporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation inglucose-free medium. The half-ti me for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5--7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-D-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity. Analysis of transport defective mutants revealed defects in both transport systems although the mutants used were alleles of a single gene. It is concluded that this gene (the ftr cistron) is the structural gene for an allosteric molecule which serves both transport systems.     

Hexose transport properties of myoblasts isolated from a patient with suspected muscle carnitine deficiency

The human primary carnitine deficiency syndromes are potentially fatal disorders affecting children and adults. The molecular etiologies of these syndromes have not been fully determined. Muscle carnitine deficiency syndrome is characterized by mild to severe muscle weakness, lipid accumulation in muscle, and reduced muscle carnitine concentration. In the present investigation, the hexose transport properties of muscle cells isolated from a patient with suspected muscle carnitine deficiency (MCD) were examined. We have previously shown that myoblasts from normal human subjects possessed at least two hexose transport systems, the low (LAHT) and the high (HAHT) affinity hexose transport systems. Their preferred substrates were 3-O-methyl-D-glucose and 2-deoxyglucose (dGlc), respectively; HAHT, but not LAHT, was sensitive to inhibition by carbonyl cyanide m-chlorophenylhydrazone (CCCP). Here we show that the kinetic properties of HAHT in the MCD myoblasts differ significantly from those of normal myoblasts and that the rates of dGlc transport by MCD myoblasts are restored to normal by growth in 40 microM L-carnitine. We also demonstrate that the kinetic properties of LAHT are quite similar in both normal and MCD myoblasts. It can be inferred from these findings that HAHT and LAHT may be coded or regulated by different genes. Based on the finding that the dGlc transport system in L-carnitine grown cells is no longer sensitive to inhibition by CCCP, it is thought that L-carnitine may play a regulatory role in HAHT, viz., by maintaining the HAHT transporter in a functional state, even in energy-uncoupled cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutated PtsG, the glucose transporter, allows uptake of D-ribose.

Mutations arose from an Escherichia coli strain defective in the high (Rbs/ribose) and low (Als/allose and Xyl/xylose) affinity D-ribose transporters, which allow cells to grow on D-ribose. Genetic tagging and mapping of the mutations revealed that two loci in the E. coli linkage map are involved in creating a novel ribose transport mechanism. One mutation was found in ptsG, the glucose-specific transporter of phosphoenolpyruvate:carbohydrate phosphotransferase system and the other in mlc, recently reported to be involved in the regulation of ptsG. Five different mutations in ptsG were characterized, whose growth on D-ribose medium was about 80% that of the high affinity system (Rbs+). Two of them were found in the predicted periplasmic loops, whereas three others are in the transmembrane region. Ribose uptakes in the mutants, competitively inhibited by D-glucose, D-xylose, or D-allose, were much lower than that of the high affinity transporter but higher than those of the Als and Xyl systems. Further analyses of the mutants revealed that the rbsK (ribokinase) and rbsD (function unknown) genes are involved in the ribose transport through PtsG, indicating that the phosphorylation of ribose is not mediated by PtsG and that some unknown metabolic function mediated by RbsD is required. It was also found that D-xylose, another sugar not involved in phosphorylation, was efficiently transported through the wild-type or mutant PtsG in mlc-negative background. The efficiencies of xylose and glucose transports are variable in the PtsG mutants, depending on their locations, either in the periplasm or in the membrane. In an extreme case of the transmembrane change (I283T), xylose transport is virtually abolished, indicating that the residue is directly involved in determining sugar specificity. We propose that there are at least two domains for substrate specificity in PtsG with slightly altered recognition properties.     

Citrate transport in Salmonella typhimurium.

Citrate was rapidly metabolized in wild-type cells of Salmonella typhimurium but actively accumulated in both aconitase mutants and fluorocitrate-poisoned cells. In aconitase mutants citrate was transported by a single high affinity system (K_m 23 μm, V_max 27.2 nmol min^?1 mg^?1), characterized by a single pH optimum of 7.0 and a Q₁₀ of 3.0, and was stimulated by Na⁺. cis-Aconitate, tricarballylate, trans-aconitate, and dl-fluorocitrate were weak competitive inhibitors of citrate transport whereas various other tricarboxylic acid cycle intermediates and carboxylates were ineffective. Spontaneous citrate transport mutants were unable to oxidize citrate, cis-aconitate, or tricarballylate. Such mutants were specific for citrate and transported dicarboxylates normally whereas dicarboxylate transport mutants transported and oxidized citrate normally. In whole cells of an aconitase mutant citrate transport was strongly dependent on an energy source. d(?)-Lactate dehydrogenase mutants were singularly defective in energization by d(?)-lactate. Membrane vesicles of wild-type cells were capable of energized transport by d(?)-lactate or ascorbate-phenyl-methyl sulfonate. Citrate transport in whole cells was primarily energized aerobically, and ATPase deficient mutants were still able to transport citrate in whole cells.     

Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. strain 6803

The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp. strain 6803 was demonstrated by following the initial rates of uptake with 14C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids. One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport. The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in one of two transport systems operating on glutamine. The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate. The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine. Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system. Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent Km ranging from 6 to 60 microM) and of Vmax.     

Inorganic cation transport and the effects on C4 dicarboxylate transport in Bacillus subtilis

The complex interrelationships between the transport of inorganic cations and C4 dicarboxylate were examined using mutants defective in potassium transport and retention, divalent cation transport, or phosphate transport. The potassium transport system, studied using 86Rb+ as a K+ analogue, kinetically appeared as a single system (Km 200 microM for Rb+, Ki 50 microM for K+), the activity of which was only slightly reduced in K+ retention mutants. Divalent cation transport, studied using 54Mn2+, 60Co2+, and 45Ca2+, was more complex being represented by at least two systems, one with a high affinity for Mn2+ (Km 2.5 microM) and a more general one of low affinity (Km 1.3-10 mM) for Mg2+, Mn2+, Ca/2+, and Co2+. Divalent cation transport was repressed by Mg2+, derepressed in K+ retention mutants, and defective in Co2+-resistant mutants. Phosphate was required for both divalent cation and succinate transport, and phosphate transport mutants (arsenate resistant) were found to be defective in both divalent cation and succinate transport. Divalent cations, especially Mg2+ and Co2+, decreased Km for succinate transport approximately 20-fold over that achieved with K+; neither cation was required stoichiometrically for succinate transport. The loss of divalent cation transport in cobalt-resistant mutants has been correlated with the loss of a 55,000 molecular weight membrane protein. Similarly, the loss of phosphate transport in arsenate-resistant mutants has been correlated with the loss of a 35,000 molecular weight membrane component.     

Properties of hexose-transport regulatory mutants isolated from L6 rat myoblasts.

A hexose-transport regulatory mutant (D1/S4) was isolated from L6 rat myoblasts on the basis of its resistance to detachment and cell lysis in the presence of antibody and complement. Growth studies indicated that D1/S4 cells had a slower doubling time (29 h) compared with the parental L6 cells (22 h). Furthermore, after 9 days growth, less than 1% cell fusion was observed with D1/S4 cells, whereas 95% cell fusion was observed with the L6 cells. When the parental L6 cells were starved of glucose or treated with anti-L6 antibody, a significant increase in the Vmax, of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose (MeGlc) transport was observed. Although glucose-grown D1/S4 cells possessed normal hexose-transport activity, the above treatments had no effect on dGlc and MeGlc transport in these cells. Electrophoresis and immunoblotting studies revealed that D1/S4 cells possessed decreased amounts of a 112 kDa plasma-membrane protein. It is conceivable that this protein may play a role in triggering the antibody- and glucose-starvation-mediated activation of hexose transport and in myogenic differentiation. Unlike D1/S4, mutant F72, a mutant defective in the high-affinity hexose-transport system, was found to possess normal amounts of the 112 kDa protein. Although glucose starvation has no effect on the hexose-transport activity in this mutant, its hexose transport activity can be increased by antibody treatment. These studies with mutants suggest the involvement of regulatory components in the activation of hexose transport.     

The deoxyribonucleoside transport systems of cultured Novikoff rat hepatoma cells

The transport of various deoxyribonucleosides by cultured Novikoff rat hepatoma cells (subline N1S1-67) follows normal Michaelis-Menten kinetics. The transport reactions are competitively inhibited by most heterologous deoxy- and ribonucleosides and by Persantin and Cytochalasin B. Comparisons of the transport kinetics of the various deoxyribonucleosides (K_m and V_max ) and of the K_m/K_i ratios for the inhibitions indicate that deoxythymidine, deoxyuridine and 5-fluordeoxyuridine are transported by a single system, whereas deoxycytidine and the purine deoxyribonucleosides are transported by other systems. The data suggest that deoxyadenosine, deoxyguanosine and deoxyinosine, are not transported by a single system, but the number of transport systems involved could not be established unequivocally. Similar comparisons also suggest that the deoxyribonucleosides are transported by different systems than the ribonucleosides. All deoxyribonucleoside transport systems are inhibited to about the same extent by Persantin (K_i = 1–2 μM) and Cytochalasin B (K_i = 4–12 μM). The inhibitions of deoxynucleoside transport resulted in corresponding apparent competitive inhibitions of their incorporation into nucleic acids.     

Succinate transport in Bacillus subtilis. Dependence on inorganic anions

Cations were generally ineffective in stimulating succinate transport in a succinate dehydrogenase mutant of Bacillus subtilis unless accompanied by polyvalent anions; phosphate and sulfate being particularly active. The K_m values for the phosphate or sulfate requirement were approx. 3 mM.Biphasic kinetics were characteristic of both the succinate (K_m values 0.1 and 1 mM), and inorganic phosphate (K_m values 0.1 and 3 mM) transport system(s). The phosphate transport system(s) was repressed by high inorganic phosphate and a coordinate increase in the transport of phosphate, arsenate, and phosphate-stimulated succinate transport accompanied growth in low phosphate media.A class of arsenate resistant mutants were simultaneously defective in the transport of arsenate, phosphate and succinate when cells were repressed for phosphate transport, however, the transport of these ions was regained in these mutants when grown in low phosphate media. Organic phosphate esters did not stimulate succinate transport in arsenate resistant mutants but were effective after growth in low phosphate media. Growth under phosphate limitation permitted the simultaneous regain of both phosphate and sulfate dependent succinate transport activities whereas sulfate limitation alone was ineffective.Succinate was not transported by an anion exchange diffusion mechanism since phosphate efflux was low or absent during succinate transport.The transport of C₄-dicarboxylates in B. subtilis is strongly stimulated by intracellular polyvalent anions. The absence of an anion permeability mechanism precludes succinate transport but partial escape from this restriction is mediated by the derepression of a phosphate transport system.     

GLYCINE UPTAKE IN RAT CENTRAL NERVOUS SYSTEM SLICES AND HOMOGENATES: EVIDENCE FOR DIFFERENT UPTAKE SYSTEMS IN SPINAL CORD AND CEREBRAL CORTEX

Abstract— Evidence is presented that glycine is taken up by two different transport systems in rat CNS tissue slices; one system has relatively low affinity for glycine (K_m = 300 μ m ) and predominates in cerebral cortex, cerebellum and mid-brain, the other has a higher affinity for glycine (K_m = 40 μ m ) and is detectable only in spinal cord, medulla and pons. The low affinity transport system appears to be shared by other small neutral amino acids, whereas the high affinity system is very specific for glycine. Both transport systems were shown to be present in particles in homogenates of CNS tissue by incubation with glycine in vitro , and subcellular fractionation studies suggested that synaptosomes were partly responsible for such uptake. Various substances were tested as inhibitors of the high affinity uptake system for glycine in spinal cord slices; the most potent inhibitors were p -chloro-mercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine, imipramine, desipramine, hydrazinoacetic acid and haloperidol. No competitive inhibitors of the high affinity glycine uptake were found. It is suggested that the high affinity transport system is associated with inhibitory synapses where glycine is a transmitter.     

Uridine and cytidine transport in Escherichia coli B and transport-deficient mutants.

Three mutants of Escherichia coli B which are defective in components of the transport system for uridine and uracil were isolated and utilized to study the mechanism of uridine transport. Mutant U- was isolated from a culture resistant to 77 micronM 5-fluorouracil. Mutant U-UR-, isolated from a culture of mutant U-, is resistant to 770 micronM 5-fluorouracil and 750 micronM adenosine. Mutant NUC- is resistant to 80 micronM showdomycin and has been reported previously. The characteristics of uridine transport by E. coli B and the mutants provide data supporting the following conclusions. The transport of adenosine, deoxyadenosine, guanosine, deoxyguanosine, adenine, or guanine by mutant U- and mutant U-UR- is identical with that in the parental strain. Uridine is transported by E. coli B as intact uridine. In addition, extracellular uridine is also rapidly cleaved to uracil and the ribose moiety. The latter is transported into the cells, whereas uracil appears in the medium and is transported by a separate uracil transport system. The entry of the ribose moiety of uridine is fast relative to the uracil and uridine transport processes. The Km values and the inhibitory effects of heterologous nucleosides for the transport of uridine and the ribose moiety of uridine are similar. Studies of cytidine uptake in the parental and mutant strains provide evidence that cytidine is transported by two independent systems, one of which is the same as that involved in the transport of intact uridine. Uridine inhibits but is not transported by the other system for cytidine transport. Evidence for the above conclusions was based on comparisons of the characteristics of [2-14C]uridine, [U-14C]uridine, and [2-14C]cytidine transport using E. coli B and the three transport mutants under conditions which measure initial rates. The nature of the inhibitory effects of heterologous nucleosides on the uridine transport processes and identification of extracellular components from radioactive uridine provides supportive data for the conclusions.     

Stimulation of Unbalanced Ribonucleic Acid Synthesis in Escherichia coli by Methanol

Data are presented which support the view that l-lysine is transported by two systems in Streptococcus faecalis. The system with the higher affinity for l-lysine appears to be specific for l-lysine among the common amino acids and to require an energy source. The second system transports both l-lysine and l-arginine and does not appear to require an energy source. Both of these systems will accept hydroxy-l-lysine as a substrate as shown by the energy requirement for hydroxy-l-lysine transport and by the inhibition of uptake by l-arginine as well as by l-lysine. The affinity of both systems appears to be considerably lower for hydroxy-l-lysine than for l-lysine. A mutant of S. faecalis which is resistant to the growth inhibitory action of hydroxy-l-lysine appears to differ from the parent strain by having a defective l-lysine-specific transport system. In this mutant, hydroxy-l-lysine is not readily transported via the l-lysine-specific system because of the mutation or via the second system because of the high concentration of l-arginine present in the growth medium. This overall lack of transport prevents hydroxy-l-lysine from reaching inhibitory levels within the cell.