A click chemistry approach to glycomimetics: Michael addition of 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose to 4-deoxy-1,2-O-isopropylidene-L-glycero-pent-4-enopyranos-3-ulose--a convenient route to novel 4-deoxy-(1-->5)-5-C-thiodisaccharides

The base catalyzed conjugate Michael addition of the 1-thiosugar, 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranose, 1, to a new highly reactive enone 4-deoxy-1,2-O-isopropylidene-L-glycero-pent-4-enopyranos-3-ulose, 2, proceeds steroselectively with formation of adduct 3 in 94% yield. Convenient stereoselective reduction of the C-3 keto function of 3 with L-Selectride followed by in situ acetylation produces thiodisaccharide 4 in good 82% yield. Cleavage of the 1,2-O-isopropylidene protecting group with p-toluenesulfonic acid in methanol, followed by de-O-acetylation, produced an inseparable anomeric mixture of methyl 4-deoxy-5-C-(beta-D-glucopyranosyl)-thio-alpha/beta-L-ribo-pyranoside 5 in 72% overall yield. This approach constitutes a new general two-step click chemistry route to the previously unknown class of 4-deoxy-(1-->5)-5-C-thiodisaccharides as stable and biologically important glycomimetics.     

Heterocyclization of 3-deoxy-D-erythro-hexos-2-ulose-1,2-bis(thiosemicarbazone). Crystal structure of the major diastereomer

Both thiosemicarbazone groups of the derivative 1 of 3-deoxy-D-erythro-hexos-2-ulose underwent, on acetylation, a heterocyclization process to give (5R,5'R)-2,2'-diacetamido-4,4'-di-N-acetyl-5'-(1-deoxy-2,3,4-tri-O-acetyl-D-erythritol-1-yl)-5,5'-bis(1,3,4-thiadiazoline) (2) as a major product. The X-ray diffraction data of a single crystal of 2 indicated the R,R configuration for the stereocenters of the thiadiazoline rings (C-5 and C-5'). In the solid state, 2 adopts a sickle conformation (by clockwise rotation of the C-2-C-3 axis of the sugar chain) which has a S//O 1,3-parallel interaction. In solution, as determined by (1)H NMR spectroscopy which included NOE experiments, a similar sickle conformation was observed. From the reaction mixture of acetylation of 1 was isolated the bis(thiadiazoline) 3 as a by-product. The configuration of the C-5 and C-5' stereocenters of 3 were respectively assigned as S,R by comparison of the physical and spectroscopic data of this compound with those of 2.     

Tricyclic furanoid dichloroacetyl orthoesters of D-mannose from 1,2-O-trichloroethylidene-beta-D-mannofuranose

1,2-O-(R)-Trichloroethylidene-beta-D-mannofuranose (1) was obtained from the reaction of D-mannose with chloral. Reaction of 1 with potassium tert-butoxide (3 Mequiv) gave the thermodynamically stable 1,2,5-O-orthodichloroacetyl-beta-D-mannofuranose as the sole product whereas 1.5 Mequiv of reagent gave the kinetically controlled 1,2,3-O-orthodichloroacetyl-beta-D-mannofuranose (10) as the main product. Orthoester 10 gave the 5,6-isopropylidene derivative, which was also obtained from the reaction of 5,6-O-isopropylidene-1,2-O-(R)-trichloroethylidene-beta-D-mannofuranose with potassium tert-butoxide (1.5 Mequiv). These novel orthoesters are expected to prove useful as protecting groups and as building blocks in the formations of new mannofuranisidic units.     

The extended GLUT-family of sugar/polyol transport facilitators: nomenclature,sequence characteristics,and potential function of its novel members

During the last 2 years, several novel genes that encode glucose transporter-like proteins have been identified and characterized. Because of their sequence similarity with GLUT1, these genes appear to belong to the family of solute carriers 2A ( SLC2A, protein symbol GLUT). Sequence comparisons of all 13 family members allow the definition of characteristic sugar/polyol transporter signatures: (1) the presence of 12 membrane-spanning helices, (2) seven conserved glycine residues in the helices, (3) several basic and acidic residues at the intracellular surface of the proteins, (4) two conserved tryptophan residues, and (5) two conserved tyrosine residues. On the basis of sequence similarities and characteristic elements, the extended GLUTfamily can be divided intothree subfamilies, namely class I (the previously known glucose transporters GLUT1-4), class II (the previously known fructose transporter GLUT5, the GLUT7, GLUT9 and GLUT11), and class III (GLUT6, 8, 10, 12, and the myoinositol transporter HMIT1). Functional characteristics have been reported for some of the novel GLUTs. Like GLUT1-4, they exhibit a tissue/cell-specific expression (GLUT6, leukocytes, brain; GLUT8, testis, blastocysts, brain, muscle, adipocytes; GLUT9, liver, kidney; GLUT10, liver, pancreas; GLUT11, heart, skeletal muscle). GLUT6 and GLUT8 appear to be regulated by sub-cellular redistribution, because they are targeted to intracellular compartments by dileucine motifs in a dynamin dependent manner. Sugar transport has been reported for GLUT6, 8, and 11; HMIT1 has been shown to be a H + /myo-inositol co-transporter. Thus, the members of the extended GLUT family exhibit a surprisingly diverse substrate specificity, and the definition of sequence elements determining this substrate specificity will require a full functional characterization of all members.     

Large-scale production of the carbohydrate portion of the sialyl–Tn epitope, α-Neup5Ac-(2→6)-d-GalpNAc, through bacterial coupling

α-Neup5Ac-(2→6)-d-GalpNAc, the carbohydrate portion of sialyl–Tn epitope of the tumor-associated carbohydrate antigen, was prepared by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. Two recombinant E. coli strains overexpressed the CMP-Neup5Ac biosynthetic genes and the α-(2→6)-sialyltransferase gene of Photobacterium damsela. C. ammoniagenes contributed to the production of UTP from orotic acid. α-Neup5Ac-(2→6)-d-GalpNAc was accumulated at 87 mM (45 g/L) after a 25-h reaction starting from orotic acid, N-acetylneuraminic acid, and 2-acetamide-2-deoxy-d-galactose.     

Synthesis of pentopyranosyl-containing thiodisaccharides. Inhibitory activity against β-glycosidases

β-(1→4)-Thiodisaccharides formed by a pentopyranose unit as reducing or non reducing end have been synthesized using a sugar enone derived from a hexose or pentose as Michael acceptor of a 1-thiopentopyranose or 1-thiohexopyranose derivatives. Thus, 2-propyl per-O-acetyl-3-deoxy-4-S-(β-d-Xylp)-4-thiohexopyranosid-2-ulose (3) and benzyl per-O-acetyl-3-deoxy-4-S-(β-d-Galp)-4-thiopentopyranosid-2-ulose (11) were obtained in almost quantitative yields. The carbonyl function of these uloses was reduced with NaBH₄ or K-Selectride, and the stereochemical course of the reduction was highly dependent on the reaction temperature, reducing agent and solvent. Unexpectedly, reduction of 3 with NaBH₄–THF at 0 °C gave a 3-deoxy-4-S-(β-d-Xylp)-4-thio-α-d-ribo-hexopyranoside derivative (6) as major product (74% yield), with isomerization of the sulfur-substituted C-4 stereocenter of the pyranone. Reduction of 11 gave always as major product the benzyl 3-deoxy-4-S-(Galp)-4-thio-β-d-threo-pentopyranoside derivative 14, which was the only product isolated (80% yield) in the reduction with K-Selectride in THF at −78 °C. Deprotection of 14 and its epimer at C-2 (13) afforded, respectively the free thiodisaccharides 19 and 18. They displayed strong inhibitory activity against the β-galactosidase from Escherichia coli. Thus, compound 18 proved to be a non-competitive inhibitor of the enzyme (K_i = 0.80 mM), whereas 19 was a mixed-type inhibitor (K_i = 32 μM).     

Large-scale production of the carbohydrate portion of the sialyl–Tn epitope, α-Neup5Ac-(2→6)- -GalpNAc, through bacterial coupling

α-Neup5Ac-(2→6)- -GalpNAc, the carbohydrate portion of sialyl–Tn epitope of the tumor-associated carbohydrate antigen, was prepared by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. Two recombinant E. coli strains overexpressed the CMP-Neup5Ac biosynthetic genes and the α-(2→6)-sialyltransferase gene of Photobacterium damsela. C. ammoniagenes contributed to the production of UTP from orotic acid. α-Neup5Ac-(2→6)- -GalpNAc was accumulated at 87 mM (45 g/L) after a 25-h reaction starting from orotic acid, N-acetylneuraminic acid, and 2-acetamide-2-deoxy- -galactose.     

Calcium is essential for fructan synthesis induction mediated by sucrose in wheat

The role of Ca²⁺ in the induction of enzymes involved in fructan synthesis (FSS) mediated by sucrose was studied in wheat (Triticum aestivum). Increase of FSS enzyme activity and induction of the expression of their coding genes by sucrose were inhibited in leaf blades treated with chelating agents (EDTA, EGTA and BAPTA). Ca²⁺ channel blockers (lanthanum chloride and ruthenium red) also inhibited the FSS response to sucrose, suggesting the participation of Ca²⁺ from both extra- and intra- cellular stores. Sucrose induced a rapid Ca²⁺ influx into the cytosol in wheat leaf and root tissues, shown with the Ca²⁺ sensitive fluorescent probe Fluo-3/AM ester. Our results support the hypothesis that calcium is a component of the sucrose signaling pathway that leads to the induction of fructan synthesis.     

Direct prediction of bioethanol yield in sugar beet pulp using near infrared spectroscopy

Sugar beets are a raw material for the production of sugar and ethanol. The decision on which end product to pursue could be facilitated by fast and reliable means of predicting the potential ethanol yield from the beets. A Near Infrared (NIR) Spectroscopy-based approach was tested for the direct prediction of the potential bioethanol production from sugar beets. A modified partial least squares (MPLS) regression model was applied to 125 samples, ranging from 21.9 to 31.0 gL(-1) of bioethanol in sugar beet brei. The samples were analyzed in reflectance mode in a Direct Contact Food Analyser (DCFA) FOSS-NIRSystems 6500 monochromator, with standard error of cross validation (SECV), standard error of prediction (SEP), coefficient of determination (r(2)) and coefficient of variation (CV) of 0.51, 0.49, 0.91 and 1.9 gL(-1), respectively. The NIR technique allowed direct prediction of the ethanol yield from sugar beet brei (i.e. the product obtained after sawing beets with a proper machine) in less than 3 min.     

A new method to determine DNA sugar conformation from the joint use of 2D and 3D NMR data

The use of sugar restraints has been proven essential for assessing DNAstructures through molecular modeling studies. We present a new methodcombining 2D (COSY and NOESY) and 3D (NOESY-NOESY) experiments, whereconstraints on either the phase angles or the difference between phase anglesof two residues are obtained from comparison of 2D NOE H1-H4intensities and 3D NOE intensities containing the H1-H4transfer. All experiments lead to restraints that match, proving the validityof the method.     

Synthesis of non-glycosidic 4,6'-thioether-linked disaccharides as hydrolytically stable glycomimetics

Michael addition of 1,2:3,4-di-O-isopropylidene-6-thio-alpha-D-galactose (2) to 2-propyl 6-O-acetyl-3,4-dideoxy-alpha-D-glycero-hex-3-enopyranosid-2-ulose (1) afforded, as the major diastereoisomer, 2-propyl 6-O-acetyl-3-deoxy-4-S-(6-deoxy-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranos-6-yl)-4-thio-alpha-D-threo-hexopyranosid-2-ulose (3, 91% yield). Reduction of the carbonyl group of 3, followed by O-deacetylation gave the two epimers 7 (alpha-D-lyxo) and 8 (alpha-D-xylo) in a 1:2 ratio. On removal of the protecting groups of 8 by acid hydrolysis, formation of an 1,6-anhydro bridge was observed in the 3-deoxy-4-thiohexopyranose unit (10). The free non-glycosidic thioether-linked disaccharide 3-deoxy-4-S-(6-deoxy-alpha,beta-D-galactopyranos-6-yl)-4-thio-alpha,beta-D-xylo-hexopyranose (11) was obtained by acetolysis of 10 followed by O-deacetylation. A similar sequence starting from the enone 1 and methyl 2,3,4-tri-O-benzoyl-6-thio-alpha-D-glucopyranoside (12) led successfully to 2-propyl 3-deoxy-4-S-(methyl 6-deoxy-alpha-D-glucopyranos-6-yl)-4-thio-alpha-D-lyxo-hexopyranoside (17) and its alpha-D-xylo analog (19, major product). In this synthetic route, orthogonal sets of protecting groups were employed to preserve the configuration of both reducing ends and to avoid the formation of the 1,6-anhydro ring.     

乙酸菌糖磷酸化作用的研究

对7种乙酸菌(Acetitomaculum ruminis, Acetobacterium woodii, Eubacterium limosum和分离株A2、A4、A10、H3HH)的葡萄糖和2脱氧葡萄糖磷酸化作用进行了研究。尽管所有机体都存在磷酸化反应,但它们在依赖PEP和ATP的比例上有实质性区别。分离菌株A10具有最高的依赖PEP的葡萄糖磷酸化活力(1162nmol·L-1·mg-1·min-1),A10、H3HH和E.limosum都具有葡萄糖磷酸转移酶系统(phosphotransferase systemPTS)。相反,Ａ.ruminis、A.woodii、A2和A4则不具有PTS活力。这七株菌的葡萄糖依赖ATP的磷酸化活力都高于依赖PEP的磷酸化活力,但其程度有所不同。A10和H3HH的葡萄糖PTS可通过胞外葡萄糖诱导,并且其比活在对数期随培养时间延长而增加。此外,还检测到A10和H3HH对麦芽糖和果糖的依赖ATP和PEP的磷酸化活力。     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle

(1) The effects of vanadate of hexose transport, ⁴⁵Ca-exchange and (Na⁺, K⁺)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[¹⁴C]glucose as well as the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$. (3) Within the same concentration range, vanadate induced an early increase in ⁴⁵Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.98}$). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and this effect was preceded by a rise in the washout of ⁴⁵Ca. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.005}$, $\text{r = 0.98}$). (5) In extensor digitorum longus and soleus muscles, vanadate increased K⁺-contents and decreased Na⁺ contents. (6) The stimulation of ⁴⁵Ca-washout presumably reflects an increase in the cytoplasmic Ca²⁺ level, brought about by an inhibitory effect of vanadate on the Ca²⁺-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca²⁺ level.     

Reaction behavior of molybdocene dichloride towards ribonucleosides and ribonucleoside monophosphates: Rare example of sugar coordination

The coordination behavior of Cp₂Mo²⁺ towards the ribonucleosides and ribonucleoside monophosphates uridine, adenosine, cytidine, guanosine, 5′-UMP, 5′-AMP, 5′-CMP and 5′-GMP has been studied in solution in the range 4 ? pD ? 9 using NMR spectroscopy. The ribonucleosides were found to bind Cp₂Mo²⁺ exclusively through the ribose moiety giving rise to the chelate complexes [Cp₂Mo(urd-O2′,O3′)], [Cp₂Mo(ade-O2′,O3′)], [Cp₂Mo(cyd-O2′,O3′)], and [Cp₂Mo(gua-O2′,O3′)]. The ribonucleotides form three types of complex with Cp₂Mo²⁺ in neutral solution, namely N,PO-macrochelates, PO,O3′-coordinated species as well as O2′,O3′-chelates, while at pD 9 only sugar coordination is observed.     

Transduction pathways mediated by second messengers including cAMP in the sugar receptor cell of the blow fly: study by the whole cell clamp method

The whole cell clamp method was directly applied to the sensory receptor neurons isolated from the adult labellar hair of the blow fly Phormia regina to locate the signal transduction pathways mediated by second messengers. First, the cAMP-mediated transduction pathway was examined to specify its location in the sugar receptor cell. Sugar receptor cell was identified by recording inward current flow under the voltage clamp applying sucrose solution to the surface of the taste neurons. When cyclic nucleotides, such as cGMP and cAMP, were introduced into the sugar receptor cell, inward current was observed (cGMP, 70pA; cAMP, 300pA at 350microM). Inhibitors and activators for the second messengers (GDPbetaS and forskolin) and non-cyclic nucleotides were also examined. Second, non-nucleotide second messengers (IP3 and Ca2+) were examined. The sugar receptor cell was activated when it was injected with IP3 or Ca2+. All the obtained results suggest that the cAMP-mediated signal transduction pathway plays a major role in the sugar receptor cell. The possibility of other transduction pathways mediated by IP3 or Ca2+ was not excluded.     

Sugars proportionately affect artemisinin production

Little is known about the effect of sugars in controlling secondary metabolism. In this study, sugars alone or in combination with their analogs were used to investigate their role in the production of the antimalarial drug, artemisinin, in Artemisia annua L. seedlings. Compared to sucrose, a 200% increase in artemisinin by glucose was observed. Different ratios of fructose to glucose yielded artemisinin levels directly proportional to increases in relative glucose concentration. When the glucose analog, 3-O-methylglucose, was added with glucose, artemisinin production was dramatically decreased, but hexokinase activity was significantly increased compared to glucose alone. In contrast, neither mannose nor mannitol had any significant effect on artemisinin yield. In comparison with 30 g/l sucrose, artemisinin levels were significantly reduced by 80% in the presence of 27 g/l sucrose + 3 g/l palatinose, which cannot be transported into cells through the sucrose transporter. Together these results suggest that both monosaccharide and disaccharide sugars are likely acting not only as carbon sources but also as signals to affect the downstream production of artemisinin, and that the mechanism of these effects appears to be complex.     

Application of the EUF procedure in sugar beet cultivation

Summary The relationship between the EUF-nutrient fractions in the soil on the one hand and the nutrient uptake of sugar beet as well as root yield and quality (polarization, α-amino N etc.) on the other is described on the basis of results obtained over several years in surveys conducted in farmers' fields (5000–6000 fields under sugar beet per year) and in field experiments (25–35 sites per year). Statistically significant close correlations with the respective parameters were found for the following EUF nutrient fractions: EUF-NO₃, EUF-P, EUF-K, EUF-Na, EUF-B and EUF-Mn. Within five years it was possible to determine the EUF-nutrient values which are required for the production of 9 t sugar/ha. These EUF values are the following: Ca: 65–70 mg/100 g at 20°C K: 11–15 mg/100 g at 20°C (depending on the clay content) Mg: 3–5 mg/100 g at 20°C Na: 2–3 mg/100 g at 20°C P: 1.4–1.6 mg/100 g at 20°C For calculation of the N fertilizer requirements of sugar beet it is suggested to use the sum of the EUF-extractable N amounts. It was found in Austria, Yugoslavia and Denmark over a period of 3 years that the EUF-N value of 1 mg/100 g soil determined between June and September was equivalent to 40 kg N/ha. If, for example, the analysed soil contains 3 mg EUF-N/100 g, 3×40=120 kg N/ha will be available to the sugar beet crop in the following year.     

ATP-free biosynthesis of a high-energy phosphate metabolite fructose 1,6-diphosphate by in vitro metabolic engineering

Fructose 1,6-diphosphate (FDP) is a widely used medicine and is also a precursor of two important three-carbon phosphates – glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) for the biosynthesis of numerous fine chemicals. An in vitro synthetic cofactor-free enzymatic pathway comprised of four hyperthermophilic enzymes was designed to produce FDP from starch and pyrophosphate. All of four hyperthermophilic enzymes (i.e., alpha-glucan phosphorylase from Thermotaga maritima, phosphoglucomutase from Thermococcus kodakarensis, glucose 6-phosphate isomerase from Thermus thermophilus, and pyrophosphate phosphofructokinase from T. maritima) were overexpressed in E. coli BL21(DE3) and purified by simple heat precipitation. The optimal pH and temperature of one-pot biosynthesis were 7.2 and 70 °C, respectively. The optimal enzyme ratios of αGP, PGM, PGI and PFK were 2:2:1:2 in terms of units. Via step-wise addition of new substrates, up to 125 ± 4.6 mM FDP was synthesized after 7-h reaction. This de novo ATP-free enzymatic pathway comprised of all hyperthermophilic enzymes could drastically decrease the manufacturing costs of FDP and its derivatives GA3P and DHAP, better than those catalyzed by ATP-regeneration cascade biocatalysis, the use of mesophilic enzymes, whole cell lysates, and microbial cell factories.     

A theoretical study of the conformational behavior of analogues of alpha-L-rhamnose-1-phosphate

The conformational behavior of methyl(2-O-methyl-alpha-L-rhamnopyranosyl)phosphate, together with a group of potentially more stable analogues, was investigated through a DFT approach at the B3LYP/6-31G(d) level; the energy of all the optimized structures was recalculated using a continuum solvent model, C-PCM, choosing water as the solvent. The compounds exhibited several, sometimes tenths of populated conformations so that the overall properties of flexibility and mobility were evaluated. The analogue in which the pyranose oxygen atom is replaced by a methylene group emerges as the best candidate as a mimic of the reference 1-phosphate, in spite of the fact that it lacks the anomeric and exo-anomeric effects. The other analogues result poorer mimics because of a conformational equilibrium at the pyranose ring or of an excessive rigidity of the aglycone moiety.     

Sugar transport in Coprinus cinereus

Two transport systems for glucose were detected: a high affinity system with a K_m of 27 μM, and a low affinity system with a K_m of 3.3 mM. The high affinity system transported glucose, 2-deoxy-d-glucose (K_m = 26 μM), 3-O-methylglucose (K_m = 19 μM), d-glucosamine (K_m = 652 μM), d-fructose (K_m = 2.3 mM) and l-sorbose (K_m = 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°C). The low affinity system transported glucose, 2-deoxy-d-glucose (K_m = 7.5 mM), and 3-O-methylglucose (K_m = 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°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 in sporelings 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 in glucose-free medium. The half-time 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.