Transport and phosphorylation of D-galactose in renal cortical cells.

An improved analytical procedure for the extraction and determination of total, free and phosphorylated tissue sugar is described. This method, employing ZnSO4 plus Ba(OH)2 for the precipitation of sugar phosphates, yields values identical with those obtained by the more laborious separation of free and phosphorylated sugar by ion-exchange chromatography. Erroneous values for free sugar due to the action of a Zn2+ -activated phosphatase and/or the lability to acids of some sugar phosphates, are avoided. Using this technique for the sudy of transport and phosphorylation of D-galactose in rabbit renal cortical slices and tissue extracts, it was found: 1. The cellular uptake of D-galactose was associated with the appearance of both free and phosphorylated sugar whether or not external Na+ was present. At 1 mM sugar, galactose was accumulated in the cells against a modest concentration gradient of 1.445 +/- 0.097 (n = 17). Galactose phosphate appeared in the cells considerably faster than free sugar under conditions of net uptake as well as of steady-state exchange (pulse-labelling). 2. Increasing saline pH (6-8) increased the cellular levels of sugar phosphate without affecting the steady-state values of free sugar. With tissue extracts, increasing pH also stimulated the activity of galactokinase and the dephosphorylation of galactose 1-phosphate by a Zn2+ -activated phosphatase. 3. 0.5 mM phlorizin inhibited the tissue uptake of galactose and its subsequent oxidation to CO2 only to a minor degree (30 and 10%, respectively). The absence of external Na+ further depressed the phlorizin effect. Preincubation of the tissue with phlorizin and subsequent washing in part abolished the inhibitory effect. The data suggest that a major portion of the galactose uptake by the tissue proceeds by a mechanism with a low affinity for phlorizin. 4. Efflux studies showed that the wash-out of free galactose from slices was associated with a net decrease of both free and phosphorylated tissue sugar. 5. The above results suggest the possibility that phosphorylation may represent a step in the Na+ -independent, phloretin-sensitive transfer of D-galactose across the antiluminal cell membrane. The participation of intracellular galactokinase and a Zn2+ -activated alkaline phosphatase in the maintenance of the steady state of free and phosphorylated galactose in the cells has been demonstrated.     

Biochimica et biophysica acta: Vol. 647 (1981)

The role of phosphorylation in sugar transport in baker's yeast was studied using 2-deoxy-d-glucose. In wild-type baker's yeast, 2-deoxy-d-glucose is accumulated as a mixture of the free sugar and several derivatives. Pool labeling experiments, designed to determine the temporal order of appearance of labeled 2-deoxy-d-glucose in the intracellular pools, have confirmed previous reports that 2-deoxy-d-glucose first appears in the sugar phosphate pool. Such results are consistent with a transport associated phosphorylation mechanism. Since wild-type yeasts contain three enzymes which could participate in such a process, hexokinase isozymes PI and PII and glucokinase, pool labeling experiments were carried out with single-kinase mutant strains containing only one of these enzymes. Results similar to those for wild-type strains were obtained for all three single-kinase strains, suggesting that if transport associated phosphorylation does occur in baker's yeast, it is not a function of the specific kinase present in the cell. While the results of the pool labeling experiments are consistent with a transport associated phosphorylation mechanism for 2-deoxy-d-glucose, caution is urged in interpreting the results of experiments with whole cells where problems of compartmentation and multiple pools are difficult to assess.     

Asymmetry of glucose transport in the yeast, Kluyveromyces lactis

Uptake and efflux of 6-deoxy-d-[³H]glucose and of 2-deoxy-d-[¹⁴C]glucose by the yeast Kluyveromyces lactis was studied. The tritiated, nonphosphorylatable hexose analogue leaves the cell in the absence and presence of intracellular 2-deoxy-d-glucose 6-phosphate. In energy-rich cells containing pools of hexose 6-phosphate, 2-deoxy-d-glucose is trapped in the cells, for it neither effluxes into glucose-free medium nor exchanges with external, free sugar. In starved, poisoned cells containing negligible amounts of 2-deoxy-d-glucose 6-phosphate, 2-deoxy-d-glucose does leave the cells upon transfer to glucose-free medium. An involvement of analogue structure and availability of metabolites of energy-rich cells in hexose retention is suggested. An internal pool of 6-deoxy-d-glucose does not affect the rate of uptake of 6-deoxy-d-[³H]glucose, nor does internal 2-deoxy-d-[¹⁴C]glucose 6-phosphate influence that rate. Hence, transport of glucose by this yeast is probably not regulated by internal pools of glucose 6-phosphate.     

The involvement of high-energy phosphate in 2-deoxy-d-glucose transport in Kluyveromyces marxianus

Under aerobic conditions 2-deoxy-d-glucose was accumulated in Kluyveromyces marxianus mainly in a phosphorylated form. During sugar uptake both ATP, polyphosphate and orthophosphate levels decreased. Under anaerobic conditions considerably less sugar was taken up. The intracellular free sugar concentration did not exceed the medium concentration, whereas sugar phosphorylation leveled off at about 3 μmol/g yeast. In response to anaerobic 2-deoxy-d-glucose uptake only ATP and polyphosphate appeared to decrease. Within the experimental error sugar phosphorylation was counterbalanced by the polyphosphate decrease. Pulse labeling experiments revealed transport-associated phosphorylation under these anaerobic conditions, Further, kinetic studies on permeabilized cells showed that cytoplasmic ATP could not be the phosphoryl donor in this transport-associated phosphorylation. These results confirm and extend previous observations, indicating that polyphosphate plays a crucial role in 2-deoxy-d-glucose transport in Kluyveromyces marxianus.     

Transport and phosphorylation of 2-deoxy-D-galactase in renal cortical cells.

The transport and phosphorylation of 2-deoxy-D-[3H]galactose in rabbit renal cortical cells was studied. 1. The uptake of 2-deoxy-galactose by cortical slices is associated with an appearance of both free and phosphorylated sugar in the cells. At 1 mM external sugar the cells establish a steady-state gradient of free 2-deoxy-galactose of 3.97 +/- 0.15 (23 animals). 2. The acid-labile sugar phosphate accumulated in the tissue has been identified by a combination of paper and radio-chromatography, as well as on the basis of some of its chemical properties, as 2-deoxy-D-galactose 1-phosphate. Ice-cold trichloroacetic acid produces a decomposition of this compound. 3. Increasing external pH (6-8) brings about a decrease in the steady-state levels of both free and phosphorylated sugar in slices. On the other hand, increasing pH activates the phosphorylation of 2-deoxy-D-galactose by a crude kinase in a tissue extract. 4. Sugar phosphate accumulated in the cells is dephosphorylated by the action of a Zn2+ -activated phosphatase. 5. The efflux of 2-deoxy-D-galactose from the cells is rather slow compared with that found for D-galactose. The efflux is associated with some dephosphorylation of cellular sugar phosphate, and some loss of 2-deoxy-galactose phosphate into the wash-out medium takes place. 6. An inhibition analysis of the uptake of 2-deoxy-D-galactose by the slices indicates that the transport site is shared by D-galactose. The following points of interaction between the sugar molecule and the carrier are identified: C1-OH, C3-OH and C4-OH (both axial) and C6-OH. A (pyranose) ring structure is also essential. A close packing between the substrate and the carrier in the vicinity of C2 is indicated. 7. The data suggest that the above transport system is localized predominantly at the antiluminal (basolateral) face of the renal tubular cells. While the detailed mechanism of the actual transport step (i.e. active transport of the free sugar, or by the action of a phosphotransferase) is still unclear, the data present evidence that both galactokinase and a Zn2+ -activated phosphatase participate in the maintenance of an intracellular steady state of the transported sugar.     

Transport-associated phosphorylation of 2-deoxy-d-glucose in Saccharomyces fragilis

2-Deoxy-d-glucose transport and metabolism was studied in Saccharomyces fragilis. Inside the cells four phosphorylated and three non-phosphorylated derivatives were found and identified. Accumulation of phosphorylated 2-deoxyglucose derivatives was balanced by a concomitant decrease of cellular ATP, orthophosphate and polyphosphates.The free sugar was concentrated against a concentration gradient, contradicting facilitated diffusion. Pulse labeling experiments revealed transport-associated phosphorylation.Theoretical considerations and analysis of the effects of iodoacetate showed that an intracellular hexokinase activity was not involved in 2-deoxyglucose phosphorylation, although this sugar is a good substrate for the enzyme in in vitro experiments.     

Novel phosphoenolpyruvate-dependent futile cycle in Streptococcus lactis: 2-deoxy-D-glucose uncouples energy production from growth.

The addition of 2-deoxy-D-glucose to cultures of Streptococcus lactis 133 that were growing exponentially on sucrose or lactose reduced the growth rate by ca. 95%. Inhibition did not occur with glucose or mannose as the growth sugar. The reduction in growth rate was concomitant with rapid accumulation of the analog in phosphorylated form (2-deoxy-D-glucose 6-phosphate) via the phosphoenolpyruvate-dependent mannose:phosphotransferase system. Within 5 min the intracellular 2-deoxy-D-glucose 6-phosphate concentration reached a steady-state level of greater than 100 mM. After maximum accumulation of the sugar phosphate, the rate of sucrose metabolism (glycolysis) decreased by only 30%, but the cells were depleted of fructose-1,6-diphosphate. The addition of glucose to 2-deoxy-D-glucose 6-phosphate preloaded cells caused expulsion of 2-deoxy-D-glucose and a resumption of normal growth. S. lactis 133 contained an intracellular Mg2+-dependent, fluoride-sensitive phosphatase which hydrolyzed 2-deoxy-D-glucose 6-phosphate (and glucose 6-phosphate) to free sugar and inorganic phosphate. Because of continued dephosphorylation and efflux of the non-metabolizable analog, the maintenance of the intracellular 2-deoxy-D-glucose 6-phosphate pool during growth stasis was dependent upon continued glycolysis. This steady-state condition represented a dynamic equilibrium of: (i) phosphoenolpyruvate-dependent accumulation of 2-deoxy-D-glucose 6-phosphate, (ii) intracellular dephosphorylation, and (iii) efflux of free 2-deoxy-D-glucose. This sequence of events constitutes a futile cycle which promotes the dissipation of phosphoenolpyruvate. We conclude that 2-deoxy-D-glucose functions as an uncoupler by dissociating energy production from growth in S. lactis 133.     

The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The sugar phosphate specificity of the active site of 6-phosphofructo-2-kinase and of the inhibitory site of fructose-2,6-bisphosphatase was investigated. The Michaelis constants and relative Vmax values of the sugar phosphates for the 6-phosphofructo-2-kinase were: D-fructose 6-phosphate, Km = 0.035 mM, Vmax = 1; L-sorbose 6-phosphate, Km = 0.175 mM, Vmax = 1.1; D-tagatose 6-phosphate, Km = 15 mM, Vmax = 0.15; and D-psicose 6-phosphate, Km = 7.4 mM, Vmax = 0.42. The enzyme did not catalyze the phosphorylation of 1-O-methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, 2,5-anhydro-D-mannitol 6-phosphate, D-ribose 5-phosphate, or D-arabinose 5-phosphate. These results indicate that the hydroxyl group at C-3 of the tetrahydrofuran ring must be cis to the beta-anomeric hydroxyl group and that the hydroxyl group at C-4 must be trans. The presence of a hydroxymethyl group at C-2 is required; however, the orientation of the phosphonoxymethyl group at C-5 has little effect on activity. Of all the sugar monophosphates tested, only 2,5-anhydro-D-mannitol 6-phosphate was an effective inhibitor of the kinase with a Ki = 95 microM. The sugar phosphate specificity for the inhibition of the fructose-2,6-bisphosphatase was similar to the substrate specificity for the kinase. The apparent I0.5 values for inhibition were: D-fructose 6-phosphate, 0.01 mM; L-sorbose 6-phosphate, 0.05 mM; D-psicose 6-phosphate, 1 mM; D-tagatose 6-phosphate, greater than 2 mM; 2,5-anhydro-D-mannitol 6-phosphate, 0.5 mM. 1-O-Methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, and D-arabinose 5-phosphate did not inhibit. Treatment of the enzyme with iodoacetamide decreased sugar phosphate affinity in the kinase reaction but had no effect on the sensitivity of fructose-2,6-bisphosphatase to sugar phosphate inhibition. The results suggest a high degree of homology between two separate sugar phosphate binding sites for the bifunctional enzyme.     

The glucose transport system of the hyperthermophilic anaerobic bacterium Thermotoga neapolitana

The glucose transport system of the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DOG). T. neapolitana accumulated 2-DOG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external source of energy, such as pyruvate, and was inhibited by arsenate and gramicidin D. There was no phosphoenolpyruvate-dependent phosphorylation of glucose, 2-DOG, or fructose by cell extracts or toluene-treated cells, indicating the absence of a phosphoenolpyruvate:sugar phosphotransferase system. These data indicate that D-glucose is taken up by T. neapolitana via an active transport system that is energized by an ion gradient generated by ATP, derived from substrate-level phosphorylation.     

Sucrose and starch metabolism in carrot (Daucus carota L.) cell suspensions analysed by 13-labelling: indications for a cytosol and a plastid-localized oxidative pentose phosphate pathway

Cells were grown in batch culture on a mixture of 50 mM glucose and fructose as the carbon source; either the glucose or the fructose was [1-¹³C]-labelled. In order to investigate the uptake and conversion of glucose and fructose during long-term labelling experiments in cell suspensions of Daucus carota L., samples were taken every 2 d during a 2 week culture period and sucrose and starch were assayed by means of HPLC and ¹³C-nuclear magnetic resonance. The fructose moieties of sucrose had a lower labelling percentage than the glucose moieties. Oxidative pentose phosphate pathway activity in the cytosol is suggested to be responsible for this loss of label of especially C-1 carbons. A combination of oxidative pentose phosphate pathway activity, a relatively high activity of pathway to sucrose synthesis and a slow equilibration between glucose-6-phosphate and fructose-6-phosphate could explain these results. Starch contained glucose units with a much lower labelling percentage than glucose moieties of sucrose: it was concluded that a second, plastid-localized, oxidative pentose phosphate pathway was responsible for removal of C-1 carbons of the glucosyl units used for synthesis of starch. Redistribution of label from [1-¹³C]-hexoses to [6-¹³C]-hexoses also occurred: 18-45% of the label was found at the C-6 carbons. This is a consequence of cycling between hexose phosphates and those phosphates in the cytosol catalysed by PFP. The results indicate that independent (oxidative pentose phosphate pathway mediated) sugar converting cycles exist in the cytosol and plastid.Key words: Daucus carotaL., cell suspensions, carbon-13 nuclear magnetic resonance, ¹³C-NMR, carbohydrate cycling, oxidative pentose phosphate pathway, plastid.      

Synthesis, conformation and enzymatic properties of 1-(beta-D-allofuranosyl)uracil and some derivatives.

A new route for the synthesis of 1-(beta-D-allofuranosyl)uracil ("allo-uridine") and the corresponding 6'-deoxy-derivative ("6'-deoxy-allo-uridine") as well as for 1-(beta-D-altrofuranosyl) uracil ("altro-uridine") is described. NMR studies of allo-uridine revealed a preferred conformation with the base in anti-position, C-2'-endo-pucker of the sugar moiety, the 5'-OH-group above the furanose ring and the 5'-CH2OH-group in a gt position with the OH-group in the plane of the furanose ring. The same conformation is found for the 5'- and 6'-phosphate, indicated by the influence of the phosphate group on the H-6 signal. Allo-uridine is phosphorylated by the phosphotransferases from carrot and from malt sprouts only in the 6'-position. The phosphate ester is hydrolysed by unspecific phosphatases but not by 5'-nucleotidase. A (3' leads to 6')-dinucleoside phosphate is formed by pancreatic ribonuclease with 2',3'-cyclic cytidylic acid and allo-uridine. It is split by nuclease S1, but not by snake-venom phosphodiesterase. It has no primer activity for polynucleotide phosphorylase. All-uridine 6'-diphosphate could not be prepared enzymatically by nucleotide kinase or by chemical methods, where 5',6'-cyclic phosphates are formed, which are hydrolysed exclusively to 6'-monophosphates.     

Transport and metabolism of 2-deoxy-D-glucose by Rhodotorula glutinis

2-Deoxy-D-glucose transport by Rhodotorula glutinis is an active process. The intracellular concentration of free deoxyglucose after 15 min incubation of Rhodotorula cells with this sugar was 230 times the extracellular concentration. Although cell extracts at this time contained more 2-deoxy-D-glucose 6-phosphate than deoxyglucose, pulse-labelling experiments demonstrated that deoxyglucose is transported as the free sugar and subsequently phosphorylated. After transport, Rhodotorula cells metabolize deoxyglucose. The major metabolites during 30-90 min incubations were determined to be 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucitol, 2-deoxy-D-gluconate and 2,2'-dideoxy-alpha, alpha'-trehalose. Rhodotorula glutinis also degrades deoxyglucose to CO2. The concentrations of intermediates in this pathway were too low to detect and resolve in extracts of control cells. In 2,4-dinitrophenol-poisoned cells, however, it appears that deoxyglucose degradation is restricted largely to loss of C-1 as CO2 and it was possible to identify 1-deoxy-D-ribulose 5-phosphate as an intermediate presumably arising from metabolism of deoxyglucose by the oxidative portion of the hexose monophosphate pathway.     

Binding of sugar phosphates, inositol phosphates and phosphorylated amino acids to actin.

Binding of biological phosphate compounds to actin was investigated by the effect of these compounds on the critical concentration of the pointed ends of gelsolin-capped actin filaments. According to this assay millimolar concentrations of glucose 6-phosphate and the bisphosphorylated sugars fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, glucose 1,6-bisphosphate, sedoheptulose 1,7-bisphosphate and 2,3-bisphosphoglycerate were found to associate with actin. Glycerophosphoinositol phosphates bound to actin if they were present in millimolar concentrations, and if carbon atom 4 of the inositol ring was phosphorylated and carbon atom 5 was free of phosphate. Also phosphoserine and phosphotyrosine were found to interact with actin. Most of the actin-binding compounds stabilized actin filaments by decreasing the critical concentration suggesting that these compounds had a higher affinity for the subunits along actin filaments than for actin monomers. However, 2,3-bisphosphoglycerate and fructose 2,6-bisphosphate increased the critical concentration probably because these sugar phosphates bound to actin monomers thereby inhibiting actin polymerization.     

Properties of the human erythrocyte glucose transport protein are determined by cellular context

Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific.     

Regulation of the Thiobacillus intermedius glucose uptake system by thiosulfate.

Cells of the mixotrophic chemolithotroph (facultative autotroph) Thiobacillus intermedius which have been grown on a glucose-yeast extract medium, a condition in which glucose is used as a source of energy, accumulate the non-metabolizable analogue 2-deoxy-d-glucose against a concentration gradient in a predominantly unchanged state. On the other hand, cells grown mixotrophically on a thiosulfate-glucose medium, a condition in which glucose provides cell carbon but is not used extensively for energy, and in which enzymes of the Entner-Doudoroff pathway are repressed, do not accumulate 2-deoxy-d-glucose significantly. Similarly, cells grown chemolithotrophically on thiosulfate-carbonate do not take up this sugar. Transfer of thiosulfate-yeast extract-grown cells, which lack the capacity to accumulate 2-deoxy-d-glucose, to a glucose-yeast extract medium results in the induction of the concentrative sugar uptake system. The capacity of induced cells to take up 2-deoxy-d-glucose is inhibited by thiosulfate. Thus, the transport system for glucose appears to be regulated in this organism so that the sugar is accumulated only under conditions where it is utilized as a source of energy, and the presence of the preferred energy source leads to both repression and inhibition of the uptake system.     

A model for accelerated uptake and accumulation of sugars arising from phosphorylation at the inner surface of the cell membrane

A model transport system for cellular accumulation of sugar coupled to phosphorylation is described. Sugar permeates the cell membrane via a passive facilitated transport system. On the inside surface of the membrane the bound sugar is either phosphorylated to form impermeable hexose phosphate, which is released into the intracellular solution, or released directly into the cytosol. Sugar may be regenerated from hexose phosphate in the cytosol via a phosphatase reaction. The reduction of the proportion of sites on the inner membrane surface occupied by permeable sugar, caused by the kinase reaction, increases both net and unidirectional passive inflow and reduces both net and unidirectional exit of sugar, thereby permitting large stationary state gradients of free sugar to be maintained between the cytosol and bathing solution. In cells where there is a high passive membrane permeability to free sugar, steady-state accumulation of free sugar within the cytosol, linked to metabolism is inexplicable in terms of conventional transport kinetics based on equilibrium thermodynamic assumptions. This phenomenon is analysed in terms of non-equilibrium stationary state flows of ligands via a probability network. The effects of metabolism on exchange transport are also examined. The model provides a framework to explain how sugar transport is loosely coupled to phosphorylation in mammalian epithelial cells, adipocytes, yeasts and bacteria, so that a high rate of substrate accumulation is maintained without requiring a reduction in the intracellular concentration of permeable substrate below that in the external solution.     

Glutamine and glutamate nitrogen exchangeable pools in cultured fibroblasts: a stable isotope study

Glutamine's role as an energetic fuel has been extensively studied in the past using ¹⁴C- and ³H-labeled tracers in cultured human cells. Yet another prominent role of glutamine, that of a nitrogen shuttle, cannot be approached without an N-tracer. We therefore used ¹⁵N-labeled glutamine and glutamate to address the following questions: (1) is it possible to study the exchangeable pools of intracellular free glutamine and glutamate nitrogen with stable isotope methods? and (2) to what extent is intracellular glutamine pool regulated by extracellular glutamine? We observed that: (1) intracellular [¹⁵N]-glutamine enrichment reached a plateau at 80% within 20 min of incubation in a buffer containing 0.7 mM pure ¹⁵N-glutamine and no glutamate; in contrast, intracellular ¹⁵N-glutamate enrichment rose only to 40% after 4 hours of incubation in a buffer containing 0.5 mM pure ¹⁵N-glutamate and no glutamine; (2) the cell-free glutamine content was tightly dependent on extracellular glutamine level, while the cell-free glutamate remained steady irrespective of the extracellular glutamate level; (3) the cells took up glutamine and glutamate against a concentration gradient; the rate of glutamine uptake accounted for 90% of the cell glutamine turnover rate; and (4) when cells were confronted with a glutamine-free medium, only one fourth of intracellular glutamine was derived from the exchangeable glutamate. We conclude that: (1) The size and turnover rate of the intracellular pool of free glutamine nitrogen are measurable using stable isotope methodology; (2) glutamine uptake from the extracellular medium accounts for most of glutamine turnover rate in cultured fibroblasts; and (3) intracellular free glutamate is divided up between several pools in cultured human fibroblasts.     

Sugar transformations associated with hexose transport in immature internodal tissue of sugar cane

After a 15 sec incubation in d-glucose-¹⁴C(U), 53–70% of the intracellular radioactivity in immature internodal tissue of sugarcane was in glucose-6-phosphate, and the remainder was in free glucose. Two unmetabolized glucose analogs, 2-deoxy-d-glucosce and 3-O-methyl-d-glucose, were transported at rates comparable to glucose but neither of these analogs was phosphorylated. Doubly-labeled d-glucose-1-¹⁴C-6-phosphate-³²P was dephosphorylated prior to deposition in the inner space, and ¹⁴C was transported into this tissue twice as rapidly as ³²P. It was also shown that ³²P in exogenously supplied glucose-6-³²P was not the source of phosphate for the intracellular synthesis of glucose-6-P. Galactose transport was similar to that of glucose in that the first major product recovered intracellularly was a phosphorylated sugar, i.e. ¹⁴C-galactose-1-P, when the tissue was incubated in d-galactose-¹⁴C(U). Although fructose, glucose, and galactose competed for transport into this tissue, free fructose and glucose predominated in the tissue extract after a 15-sce incubation in d-fructose-¹⁴C(U). This contrasted sharply, with the products of ¹⁴C-glucose transport which were comprised of phosphorylated sugars after 15 sec.     

Redistribution of phosphate pools and the regulation of Escherichia coli adenylate cyclase activity

The enzyme adenylate cyclase plays a key role in mediating the phenomenon of catabolite repression in Escherichia coli. The mechanism by which one sugar prevents the expression of the gene for another catabolite depends on the capacity of the cell to take up the sugar. Sugars that are most effective in the repression mechanism are those that are transported by the phosphoenolpyruvate-energized phosphotransferase system. The hypothesis presented here is that one or more of the proteins associated with this sugar transport system interact with adenylate cyclase and, when they are in their phosphorylated form, activate the enzyme, provided other factors that permit this activation are present. Another essential activator of adenylate cyclase is inorganic orthophosphate. When E. coli are starved for sugars, the pool of total phosphate is accounted for primarily as inorganic orthophosphate, ATP, phosphoenolpyruvate, and transport proteins in their phospho-forms, a condition that promotes activation of adenylate cyclase. When cells are exposed to sugars, the phosphate pool becomes drastically redistributed, such that the level of inorganic orthophosphate and transport phosphoproteins decreases markedly while the pool of sugar phosphate increases. This translation of the extracellular availability of carbon sources into an intracellular phosphate redistribution is the immediate event that is responsible for catabolite repression.