Phosphorylation of 3-O-methyl-D-glucose by yeast and beef hexokinase

Beef heart hexokinase and yeast hexokinase both catalyzed the phosphorylation of 3-O-[14C]methyl-D-glucose. The maximal velocity was 3 orders of magnitude lower and the Km for the glucose analogue 40-120-times higher than those observed with D-[U-14C]glucose. Hence, 3-O-methyl-D-glucose should not be considered as a truly nonmetabolized analogue of D-glucose.     

Determination of Glucose Utilization Rates in Cultured Astrocytes and Neurons with [<Superscript>14</Superscript>C]deoxyglucose: Progress,Pitfalls, and Discovery of Intracellular Glucose Compartmentation

2-Deoxy-d-[¹⁴C]glucose ([¹⁴C]DG) is commonly used to determine local glucose utilization rates (CMR_glc) in living brain and to estimate CMR_glc in cultured brain cells as rates of [¹⁴C]DG phosphorylation. Phosphorylation rates of [¹⁴C]DG and its metabolizable fluorescent analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), however, do not take into account differences in the kinetics of transport and metabolism of [¹⁴C]DG or 2-NBDG and glucose in neuronal and astrocytic cells in cultures or in single cells in brain tissue, and conclusions drawn from these data may, therefore, not be correct. As a first step toward the goal of quantitative determination of CMR_glc in astrocytes and neurons in cultures, the steady-state intracellular-to-extracellular concentration ratios (distribution spaces) for glucose and [¹⁴C]DG were determined in cultured striatal neurons and astrocytes as functions of extracellular glucose concentration. Unexpectedly, the glucose distribution spaces rose during extreme hypoglycemia, exceeding 1.0 in astrocytes, whereas the [¹⁴C]DG distribution space fell at the lowest glucose levels. Calculated CMR_glc was greatly overestimated in hypoglycemic and normoglycemic cells because the intracellular glucose concentrations were too high. Determination of the distribution space for [¹⁴C]glucose revealed compartmentation of intracellular glucose in astrocytes, and probably, also in neurons. A smaller metabolic pool is readily accessible to hexokinase and communicates with extracellular glucose, whereas the larger pool is sequestered from hexokinase activity. A new experimental approach using double-labeled assays with DG and glucose is suggested to avoid the limitations imposed by glucose compartmentation on metabolic assays.     

Quantitative estimation of the pathways followed in the conversion to glycogen of glucose administered to the fasted rat

When [6-3H,6-14C]glucose was given in glucose loads to fasted rats, the average 3H/14C ratios in the glycogens deposited in their livers, relative to that in the glucoses administered, were 0.85 and 0.88. When [3-3H,3-14C]lactate was given in trace quantity along with unlabeled glucose loads, the average 3H/14C ratio in the glycogens deposited was 0.08. This indicates that a major fraction of the carbons of the glucose loads was converted to liver glycogen without first being converted to lactate. When [3-3H,6-14C]glucose was given in glucose loads, the 3H/14C ratios in the glycogens deposited averaged 0.44. This indicates that a significant amount of H bound to carbon 3, but not carbon 6, of glucose is removed within liver in the conversion of the carbons of the glucose to glycogen. This can occur in the pentose cycle and by cycling of glucose-6-P via triose phosphates: glucose----glucose-6-P----triose phosphates----glucose-6-P----glycogen. The contributions of these pathways were estimated by giving glucose loads labeled with [1-14C]glucose, [2-14C]glucose, [5-14C]glucose, and [6-14C]glucose and degrading the glucoses obtained by hydrolyzing the glycogens that deposited. Only a few per cent of the glucose carbons deposited in glycogen were deposited in liver via glucose-6-P conversion to triose phosphates. Between 4 and 9% of the glucose utilized by the liver was utilized in the pentose cycle. While these are relatively small percentages, since three NADP3H molecules are formed from each molecule of [3-3H]glucose-6-P utilized in the cycle, a major portion of the difference between the ratios obtained with [3-3H]glucose and with [6-3H]glucose is attributable to metabolism in the pentose cycle. Because 3H of [3-3H]glucose is extensively removed during the conversion of the glucose to glycogen within liver the extent of incorporation of the 3H into liver glycogen is not the measure of glucose's metabolism in other tissues before its carbons are deposited in liver glycogen. The distributions of 14C from the 14C-labeled glucoses into the carbons of the liver glycogens mean that at a minimum about 30% of the carbons of the glucose deposited in the glycogen were first converted to lactate or its metabolic equivalent.     

2-Deoxyglucose Incorporation into Rat Brain Glycogen During Measurement of Local Cerebral Glucose Utilization by the 2-Deoxyglucose Method

The incorporation of 14C into glycogen in rat brain has been measured under the same conditions that exist during the measurement of local cerebral glucose utilization by the autoradiographic 2-[14C]deoxyglucose method. The results demonstrate that approximately 2% of the total 14C in brain 45 min after the pulse of 2-[14C]deoxyglucose is contained in the glycogen portion, and, in fact, incorporated into alpha-1-4 and alpha-1-6 deoxyglucosyl linkages. When the brain is removed by dissection, as is routinely done in the course of the procedure of the 2-[14C]deoxyglucose method to preserve the structure of the brain for autoradiography, the portion of total brain 14C contained in glycogen falls to less than 1%, presumably because of postmortem glycogenolysis which restores much of the label to deoxyglucose-phosphates. In any case, the incorporation of the 14C into glycogen is of no consequence to the validity of the autoradiographic deoxyglucose method, not because of its small magnitude, but because 2-[14C]deoxyglucose is incorporated into glycogen via [14C]deoxyglucose-6-phosphate, and the label in glycogen represents, therefore, an additional "trapped" product of deoxyglucose phosphorylation by hexokinase. With the autoradiographic 2-[14C]deoxyglucose method, in which only total 14C concentration in the brain tissue is measured by quantitative autoradiography, it is essential that all the labeled products derived directly or indirectly from [14C]deoxyglucose phosphorylation by hexokinase be retained in the tissue; their chemical identity is of no significance.     

Incomplete palmitate oxidation in cell-free systems of rat and human muscles

The palmitate oxidation capacity was determined in whole homogenates, postnuclear fractions and mitochondrial fractions of various rat and human muscles and in rat liver, kidney, brain and lung. The oxidation rate (production of 14CO2 and 14C-labeled acid-soluble intermediates) was [1-14C]palmitate greater than [U-14C]palmitate greater than [16-14C]palmitate in all cell-free systems. Oxidation rates were highest in rat heart and liver, intermediate in kidney, diaphragm and m. quadriceps, and low in brain and lung. The capacity of human heart was much lower than that of rat heart and about twice that of human skeletal muscles. Omission of L-carnitine and addition of malonyl-CoA, KCN or antimycin A decreased the oxidation rates in whole homogenates and mitochondrial fractions. Antimycin or KCN increased and malonyl-CoA decreased the ratio of the oxidation rates with [1-14C]- and [16-14C]palmitate. The carnitine concentration had no significant effect on the ratio. 14C-labeled dodecanoic and tetradecanoic acids were identified in homogenates and mitochondrial fractions of m. quadriceps and liver of rat as acid-insoluble intermediates of [16-14C]palmitate oxidation in the presence and absence of antimycin A. Their amounts recovered can account for the differences in oxidation rates found with [1-14C]- and [16-14C]palmitate. The incomplete palmitate oxidation in cell-free systems appears to be mainly caused by an inadequate mitochondrial degradation of peroxisomal oxidation products.     

Correlation of Hexokinase Content and Basal Energy Metabolism in Discrete Regions of Rat Brain

Brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) levels in seven regions of rat brain were estimated by photometric measurement of immunofluorescence in cryostat-cut sections. When compared with basal rates of glucose metabolism in these regions, estimated by the 6-[14C]glucose method, a significant correlation was observed. Thus, hexokinase content reflects metabolic energy demands.     

In vivo protein synthesis from 14C-substrates in porcine tissues during the transition to postnatal development

[2-14C] leucine, [1-14C] alanine, [1-14C] glucose, [1-14C] lactate and [1-14C] pyruvate utilization in the protein synthesis has been studied in vivo at early stages of postnatal development of piglets. It has been established, that during the first 24 hours after birth the protein synthesis intensity, judging by [2-14C] leucine incorporation, in liver, skeletal muscle, duodenal wall and subcutaneous tissue of piglets increases 5, 7, 6.5 and 2.1 times respectively. At the age of 1-2 h the radioactive carbon incorporation from [1-14C] glucose into the brain proteins is more pronounced than into the proteins of liver and skeletal muscle. During the first days of life the intensity of the label incorporation from [1-14C] glucose into liver and skeletal muscle proteins of piglets is enhanced, whereas in brain it remains at the same level. The degree of 14C carbon incorporation from [1-14C]-alanine, [1-14C] pyruvate and [1-14C] lactate into the liver and skeletal muscle proteins of 5-days-old piglets is approximately the same, 14C substrates of protein synthesis in brain and subcutaneous adipose tissue having some peculiarities.     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglycemic (db/db) mice. Effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-3H]glucose, uptake of 3-O-[methyl-3H]glucose (methylglucose) and [2-14C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 +/- 18 and 180 +/- 9 microliter/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V- of methylglucose transport was decreased with no change in Km in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and 3H2O production from [5-3H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Effect of insulin and cortisol on oxidation of (1-14C)glucose, (6-14C)glucose, (1-14C)palmitate and (1-14C)leucine in the tissues of swine during the neonatal period

The intensity of [1-14C]glucose, [6-14C]glucose, [1-14C]palmitate and [1-14C]leucine oxidation and the effect of insulin and hydrocortisone on this process were studied in the brain, duodenum mucosa, liver and skeletal muscle of 1- and 5-day old piglets in vitro. Most of the studied substrates are oxidized in the tissues of 5-day piglets more intensively. Insulin stimulates oxidation of [1-14C]glucose, [6-14C]glucose and [1-14C]leucine in the brain and duodenum mucosa in 1- and 5-day old piglets, while in the liver and skeletal muscle--only in 5-day old piglets. Hydrocortisone administration enhances oxidation of [1-14C]leucine in most of the studied tissues in 1-day piglets and oxidation of [1-14C]glucose and [6-14C]glucose--in 5-day piglets. Both hormones produce no essential influence on the intensity of [1-14C]palmitate oxidation in the studied tissues of piglets or somewhat weaken it.     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues. X. Effect of glucose transport stimuli on the efflux of isotopically labelled calcium and 3-O-methylglucose from soleus muscles and epididymal fat pads of the rat

(1) The relationship between Ca2+ and sugar transport has been studied by comparing the washout of 45Ca and 3-O-[14C]methylglucose from preloaded isolated rat soleus muscles and whole epididymal fat pads. (2) In soleus muscle, nine different agents with well established stimulating effects on glucose transport were all found to produce a marked increase in 3-O-[14C]methylglucose washout, which in each instance was preceded by or coincided with a rise in the washout of 45Ca. (3) Trypsin, 2,4-dinitrophenol, p-chloromercuriphenylsulfonic acid, H2O2 and hyperosmolarity all produced dose-dependent stimulation of the washout of 45Ca and 3-O-[3H]methylglucose. Regression analysis showed a highly significant correlation between the increases in the two parameters (P < 0.001). (4) Depolarization and Na+ influx induced by veratrine were found to be associated with a marked rise in 45Ca release followed by stimulation of 3-O-[14C]methylglucose washout. (5) In epididymal fat pads, six different agents known to stimulate glucose transport were found to produce a highly significant (P < 0.001) increase in the washout of 45Ca and 3-O-[14C]methylglucose. (6) It is concluded that in the major targets for insulin action, activation of the glucose transport system can be elicited by a rise in cytoplasmic Ca2+ concentration brought about by mobilization of Ca2+ from endogenous cellular pools.     

Comparison of Rates of Local Cerebral Glucose Utilization Determined with Deoxy[l-14C]glucose and Deoxy[6-14C]glucose

The activity of the pentose phosphate shunt pathway in brain is thought to be linked to neurotransmitter metabolism, glutathione reduction, and synthetic pathways requiring NADPH. There is currently no method available to assess flux of glucose through the pentose phosphate pathway in localized regions of the brain of conscious animals in vivo. Because metabolites of deoxy[1-14C]glucose are lost from brain when the experimental period of the deoxy[14C]glucose method exceeds 45 min, the possibility was considered that the loss reflected activity of this shunt pathway and that this hexose might be used to assay regional pentose phosphate shunt pathway activity in brain. Decarboxylation of deoxy[1-14C]glucose by brain extracts was detected in vitro, and small quantities of 14C were recovered in the 6-phosphodeoxygluconate fraction when deoxy[14C]glucose metabolites were isolated from freeze-blown brains and separated by HPLC. Local rates of glucose utilization determined with deoxy[1-14C]glucose and deoxy[6-14C]glucose were, however, similar in 20 brain structures at 45, 60, 90, and 120 min after the pulse, indicating that the rate of loss of 14CO2 from deoxy[1-14C]glucose-6-phosphate in normal adult rat brain is too low to permit assay pentose phosphate shunt activity in vivo. Further metabolism of deoxy[1-14]glucose-6-phosphate via this pathway does not interfere during routine use of the deoxyglucose method or explain the progressive decrease in calculated metabolic rate when the experimental period exceeds 45 min.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

Rate-limiting steps of 2-deoxyglucose uptake in rat adipocytes

2-Deoxy[1-¹⁴C]glucose uptake in rat adipocytes was measured as a function of time in the absence and presence of unlabelled glucose or 2-deoxyglucose. Uptake of tracer alone was linear from 2 s to 6 min. At 37°C the rate of uptake in insulin-stimulated cells decreased markedly after a few seconds in the presence of glucose (0.5–10 mM) and after 0.5–2 min in the presence of deoxyglucose (2–10 mM). Similar data were obtained at 22°C. With 10 mM glucose (37°C, 30 s) approx. 80% of the intracellular radioactivity was non-phosphorylated deoxyglucose and with 10 mM deoxyglucose approx. 40% was non-phosphorylated. The results show that deoxy[¹⁴C]glucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose and at least partially in the presence of deoxyglucose. The data suggest caution in using deoxyglucose uptake as a measure of transport, especially in complex kinetic studies.In addition, the initial velocity of tracer ¹$\text{3-O-}\text{methylglucose}$ was found to be approx. 2-fold higher than that of tracer deoxyglucose even though both sugars inhibited the initial velocity of labelled methylglucose half-maximally at a concentration of 5 mM. These data suggest a fundamental difference between deoxyglucose and methylglucose transport.     

Distribution, metabolism and excretion of [1-14C]dolichol injected intravenously into rats.

[1-14C]Dolichol mixed in vitro with rat serum and injected intravenously into rats was rapidly cleared from the circulation in a manner consistent with a two-compartment model. About 80% of the radioactivity recovered from animals killed after 1 day was in the liver, with smaller amounts being found in lung, carcass (internal organs removed), gastrointestinal tract and contents, and spleen. The kidneys, testes and heart contained little radioactivity, and the brain did not appear to take up any [1-14C]dolichol. The half-life for the turnover of radioactivity from [1-14C]dolichol in tissues varied considerably, being 2 days for the lung, 17 for liver and about 50 days for the carcass. After 1 day, and also after 4 and 21 days, most of the radioactivity in all tissues was as [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester, although a small amount of incorporation of [1-14C]dolichol radioactivity into phospholipids was also observed. Faeces collected over the first 4 days after injection contained 13% of the [1-14C]dolichol dose, but urine and expired air contained only small amounts of radioactivity. Radioactivity in faeces was nearly all as unchanged [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester. The [1-14C]dolichol remaining in liver after 21 days appeared to be in a pool (possibly lysosomes) where most of it was not subject to excretion.     

Conversion of arachidonic acid to lipoxygenase products by human fetal tissues

[14C]Arachidonic acid was converted to several lipoxygenase products by homogenates of human fetal tissues as determined by thin-layer chromatography. The net conversions of [14C]arachidonic acid to radiolabeled lipoxygenase products were high (greater than or equal to 5%) in the case of fetal liver and brain, and low (less than or equal to 2%) in the case of fetal adrenal, heart, and kidney.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts

Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.     

Glucose infused through the portal vein enhances liver gluconeogenesis and glycogenesis from [3-14C]pyruvate in the starved rat

After a pulse of [3-14C]pyruvate, 24 hr starved rats were infused through the portal vein with two different doses of glucose (7.8 or 20.8 mg/min) or the medium, and blood was collected from the inferior cava vein at the level of the suprahepatic veins. The highest dose of glucose enhanced the appearance of [14C]glucose in blood from the 2nd to the 20th min after tracer delivery. It also enhanced production of [14C]glycogen and concentration of glycogen in the liver after 5 and 20 min. At 20 min of glucose infusion the appearance of [14C]glyceride glycerol in liver as well as liver lactate concentration and lactate/pyruvate ratio were increased. The low dose of glucose used enhanced liver values of [14C]glycogen, [14C]glycogen specific activity and glycogen concentration. Our results support the hypothesis that in the starved rat glucose is converted into C3 units prior to being deposited as liver glycogen and based on the liver zonation model (Jungermann et al., 1983) it is proposed that glucose stimulated gluconeogenesis by shifting the liver to the cytosolic redox state as a secondary consequence of increased glycolytic activity.     

Inhibition of glucose utilization for acetylcholine synthesis by cerebrospinal fluid.

Abstract— Replacement of bicarbonate-Locke incubation medium with feline CSF reduced [¹⁴C]ACh formation from [U-¹⁴C]glucose by rat brain mince approx 30%. CSF was obtained from a cannula leading to the cisterna magna of freely moving cats. The component of CSF responsible for inhibition was characterized as a dialyzable heat-stable organic anion. Choline acetyltransferase activity was not altered by CSF. [¹⁴C]ACh synthesis and ¹⁴CO₂ production from [U-¹⁴C]glucose but not from [2-¹⁴C]-pyruvate were inhibited by CSF, suggesting inhibition in the metabolism of glucose to pyruvate. The anionic fraction of human CSF was as potent as that from feline CSF in inhibiting ¹⁴CO₂ production from [U-¹⁴C]glucose. Brain hexokinase was inhibited by the anionic fraction of feline CSF. The inhibition was non-competitive with respect to glucose and uncompetitive with respect to ATP. It is suggested that inhibition of hexokinase by CSF was responsible at least in part for the inhibition of glucose metabolism which resulted in decreased [¹⁴C]ACh synthesis and ¹⁴CO₂ production.     

Enzymatic synthesis of UDP-GlcN by a two step hollow fiber enzyme reactor system

UDP-GlcN was synthesized from GlcN and UTP by a two step hollow fiber enzyme reactor method. In step 1, GlcN was converted to GlcN 6-P and then to GlcN 1-P by hexokinase and phosphoglucomutase, respectively, and UTP was used as the phosphate donor. In step 2, GlcN 1-P was converted to UDP-GlcN by UDP glucose pyrophosphorylase. All the enzymes required for the synthesis of UDP-GlcN were enclosed in hollow fiber bundles which allow for the free diffusion of substrates and products across the membranes to and from the enzymes, allow for the reutilization of the enzymes, and simplify the isolation of the product, UDP-GlcN. We show that both UTP and GlcN 6-P are inhibitors of the yeast UDPG pyrophosphorylase and therefore their concentrations must be regulated to obtain maximum yields of UDP-GlcN. The UDP-GlcN produced can be N-acetylated with [14C]acetic anhydride to produce UDP-[14C]GlcNAc. This method can also be used to synthesize [32P]UDP-GlcN and [32P]UDP-GlcNAc from [alpha-32P]UTP and GlcN 1-P.