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PubMed1977

Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate.

Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which completes with UDP-glucose for uridylylating the enzyme. The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains more than one active site per molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical. The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-ens of the uridylyl-enzyme under the latter conditions is [l]ump. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies on the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue. The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0 degrees C and reactivated by NH2OH. UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate while 70 mM galactose-1-P has only a slight protective effect. Uridylyl-enzyme in inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme. The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose. It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates.

Binding Sites↗
PubMed1985

Cerebrospinal fluid glucose: turnover and metabolism.

The turnover of cerebrospinal fluid (CSF) glucose was studied in cats during steady-state perfusion. In all experiments, the perfusion fluid contained either tracer [14C]glucose alone or tracer glucose along with 4.45 mM unlabeled glucose. In some studies, serum glucose was lowered with insulin. The concentration of glucose and [14C]glucose in the effluent fluid was measured, and the distribution of 14C between glucose and lactate was determined by chromatography. From these values, the extraction of glucose and the metabolism of glucose to lactate were calculated. From the decrease in the specific activity of glucose in the perfusion fluid, the influx of glucose from serum was also determined. During steady-state perfusion, 71% of the radioactivity was recovered in the effluent fluid: 50% in the form of glucose, 6% in the form of lactate, and 15% in forms that were not identified. Thus, 50% of the perfusion fluid glucose was cleared, of which 29% was extracted and 21% metabolized. The influx of glucose was proportional to the serum glucose when the latter was about 2.5 mM or 10.0 mM. During perfusion with tracer glucose only, the concentration of glucose in the effluent fluid was 25% that of serum. The transport of glucose from serum was independent of the glucose concentration gradient between serum and perfusion fluid. However, when perfusion fluid glucose concentration was greater than that of serum, transport was inhibited. These studies suggest that in maintaining CSF glucose at a lower concentration than serum glucose, with equal amounts of glucose entering and leaving the CSF, 50% of CSF glucose concentration cleared is replaced by 25% of serum glucose concentration.

Animals↗
PubMed1979

Anabolism versus catabolism of [5-3H]uridine and its relationship to ribonucleic acid labelling in mouse liver after partial hepatectomy.

The balance between anabolism and catabolism of [5-(3)H]uridine was studied in the mouse after partial hepatectomy. Labelling of RNA and UDP-glucose was determined and evaluated in relation to changes in the specific radioactivity of UTP. The amounts of labelled catabolic products of uridine were increased several-fold in liver and blood after partial hepatectomy. The specific radioactivity of RNA decreased to about 60% of the control value at 6h and was in the same range as that of control liver at 24h after operation. Decreased labelling of RNA and UDP-glucose was attributable to decreased specific radioactivity of UTP. No changes in the size of the UTP pool or in the balance between uridine anabolism and catabolism were found that could explain the decreased specific radioactivity of UTP. Rather, the alterations in the labelling of this metabolite induced by the partial hepatectomy may be related to decreased phosphorylating capacity in the liver cells and/or dilution of the labelled precursor in an expanded uridine pool. The enhanced amounts of uridine catabolic products in liver and blood were probably a consequence of accumulation and altered incorporation of the metabolites from the blood into the liver cells. Despite the increased amounts of labelled catabolic products and the decreased labelling of RNA, the results reported here actually suggest decreased uridine catabolism and slightly increased RNA synthesis in mouse liver after partial hepatectomy. The results stress the importance of proper controls in determination of nucleic acid synthesis and in metabolic studies by use of labelled precursors.

Animals↗
PubMed1989

F-18 fluorodeoxyglucose: its potential in differentiating between stress fracture and neoplasia.

F-18 fluorodeoxyglucose (FDG) accumulates into regions of enhanced glucose uptake and metabolism such as the brain, heart, and malignant tumors. The clinical usefulness of this positron-emitting radiopharmaceutical is illustrated in a case where the clinical picture and CT indicated a malignant bone lesion in the clavicle. Histologically a stress fracture was found secondary to chronic strain on the clavicle. On follow-up the lesion's course was benign. Planar imaging with F-18 FDG was performed twice during follow-up, and on both occasions there was no accumulation of radioactivity over the suspicious area, indicating normal glucose consumption. This case demonstrates the differential diagnostic potential of F-18 FDG and shows that clinically useful information may be obtained without a position emission tomograph.

Adolescent↗
PubMed1974

Relationship between glucose and sodium excretion in the new-born dog.

1. The relationship between renal glucose and sodium excretion was studied in thirty-three new-born dogs aged 1-14 days and in ten adult dogs.2. Glucose was infused into the animals at rates sufficient to produce an amount of filtered glucose at least 1.5 times the tubular transport of glucose (saturating glucose load). In both puppies and adults tubular glucose reabsorption at saturating glucose loads varied directly with the glomerular filtration rate (r = 0.54 and 0.73 respectively, P < 0.01 for both).3. In the puppy, as the fraction of filtered sodium excreted (C(Na)/C(In)) increased from 0.05 to 0.45, the ratio, renal tubular glucose transport divided by glomerular filtration rate at saturating glucose loads, (T(G)/GFR)(m), decreased from 3.7 to 1.7 mg/ml. (r = -0.75, P < 0.01). In the adult C(Na)/C(In) was below 0.08 in all experiments and (T(G)/GFR)(m) was within the 95% confidence limits predicted by regression analysis of the data from puppies. Although mean (T(G)/GFR)(m) was greater in the adult than in the puppy (P < 0.01), when puppies had C(Na)/C(In) similar to that for adults, they had (T(G)/GFR)(m) values equivalent to that for the adult.4. There was excellent correlation between glucose excretion and water excretion for both adult and new-born dogs (r = 0.93 and 0.87, respectively). However, for any glucose loss, water loss was greater in the puppy than in the adult (P < 0.01).5. During the control period total sodium excretion (per gram kidney) and C(Na)/C(In) were similar in the new-born and adult dog. However, during glucose loading, the puppies excreted more sodium and had a higher C(Na)/C(In) than did the adult, although glucose excretion was greater in the adult than in the puppy (P < 0.01 for all comparisons).6. Glomerular blood flow, as measured by radioactive microspheres, was redistributed towards inner cortical nephrons during glucose loading in the puppy. There was no such redistribution of glomerular blood flow in the adult.7. Sodium reabsorption beyond the proximal tubule was blocked with ethacrynic acid and chlorothiazide. In the puppy, the increase of C(Na)/C(In) following a glucose load was the same whether the glucose load followed control or distal blockade collections, suggesting that reductions of sodium reabsorption following a glucose load probably came from the proximal tubule. C(Na)/C(In) during glucose loading plus distal blockade was significantly (P < 0.01) higher in the puppy (0.598) than in the adult (0.280), indicating that glucose diuresis produced a greater inhibition of proximal tubular sodium reabsorption in the new-born than in the adult dog. These results support the hypothesis that the high sodium excretion rate during glucose diuresis in the new-born dogs appears to be due to the greater sensitivity of the neonatal proximal tubule to the osmotic effect of glucose. When presented with a glucose osmotic load the new-born dog diminishes net proximal sodium reabsorption more than does the adult and thus depresses tubular glucose reabsorption to a greater extent. The lower values of maximal glucose transport rates found in new-born animals may be related, therefore, to the higher fractional sodium excretion rates during glucose diuresis rather than to a diminished intrinsic glucose transport capacity in the new-born kidney.

Age Factors↗
PubMed1977

Thyroid hormonelike actions of 3,3',5'-L-triiodothyronine nad 3,3'-diiodothyronine.

l-Thyroxine is converted to 3,5,3'-l-triiodothyronine (T(3)) as well as to 3,3',5'-l-triiodothyronine (reverse T(3)). One product of further deiodination is 3,3'-diiodothyronine (3,3'T(2)). The serum levels of reverse T(3) and 3,3'T(2) change considerably in various physiological and disease states. We previously found that reverse T(3) and 3,3'T(2) bind to the solubilized hepatic nuclear "receptors" for thyroid hormones. This led us to study binding and actions of these metabolites in cultured rat pituitary cells in which glucose consumption and growth hormone production are regulated by T(3) and l-thyroxine. Reverse T(3) and 3,3'T(2) stimulated growth hormone production and glucose consumption and inhibited nuclear binding of radioactive T(3). Either metabolite produced maximal effects that equaled those of T(3), and neither inhibited the T(3) response. Further, additive effects were observed when reverse T(3) was combined with submaximal concentrations of T(3). In serum-free and serum-containing media, concentrations of 3,3'T(2) 50- to 70- and 10- to 100-fold greater, respectively, than those of T(3) were required for equivalent stimulations and for inhibition of nuclear binding by T(3). The relative activity differences under the two conditions can be attributed to weaker serum protein binding of 3,3'T(2) than T(3). With cells in serum-free media, reverse T(3) was a less avid competitor than 3,3'T(2) for T(3) binding by the nuclear receptors, and was less potent than 3,3'T(2) (0.001 the potency of T(3)) in inducing growth hormone production or glucose oxidation. In incubations with serum-containing media, reverse T(3) was an ineffective competitor for T(3) binding, and had only 0.1 the inducing potency of 3,3'T(2) (0.001 the potency of T(3)). The weaker activity of reverse T(3) relative to 3,3'T(2) in serum-containing media could be explained by stronger serum binding of reverse T(3) than 3,3'T(2). In addition, after long-term incubation of cells with radioactive reverse T(3), much of the cell-associated radioactivity was recovered as 3,3'T(2). These studies suggest that reverse T(3) and 3,3'T(2) can stimulate thyroid hormone-regulated functions as weak agonists by acting via the same receptors that mediate T(3) actions. Moreover, some of the effects of reverse T(3) may be due to 3,3'T(2) produced by deiodination of reverse T(3).

Animals↗
PubMed1983

Effects of nutrient secretagogues upon phospholipid metabolism in rat pancreatic islets.

Rat pancreatic islets labelled with [32P]Pi were used to investigate the effects of nutrient secretagogues upon phospholipid metabolism. Stimulatory concentrations of glucose, 4-methyl-2-oxopentanoate, 3-phenylpyruvate and 2-endo-aminonorbornane-2-carboxylic acid caused a marked and specific stimulation of labelling with [32P]Pi of phosphatidylinositol, phosphatidic acid and the polyphosphoinositides. The effects of glucose were concentration-related and were inhibited by mannoheptulose and menadione. Enhanced phospholipid labelling persisted in the absence of added calcium ions but was abolished by excess EDTA. The time-course of labelling of these phospholipids in response to glucose contrasted with that previously observed using carbamylcholine in that the accumulation of radioactivity in phosphatidic acid and in phosphatidylinositol occurred in parallel. We conclude that glucose, in common with other nutrient secretagogues or those which promote the metabolism of endogenous nutrients, causes a marked stimulation of turnover in the phosphatidylinositol cycle. This effect requires the integrity of nutrient catabolism and is probably not dependent upon the stimulation of Ca2+ uptake from the extracellular medium.

Adenosine Triphosphate↗
PubMed1988

Effect of chloroquine on biosynthesis, release and degradation of insulin in isolated islets of rat pancreas.

Insulin has been reported to degrade inside the islets and islet lysosomal proteases have been thought to take part. As chloroquine is regarded as a potent lysosomotropic agent, an attempt has been made to see whether chloroquine has an influence on intrainsular degradation of insulin. After preculture of collagenase-isolated rat islets at 11 mM glucose together with [3H]leucine for 3 days for labelling newly synthesized insulin, islets were cultured for 1 day at 2.2 or 22 mM glucose with or without 0.02 mM chloroquine. Afterwards, radioactivity was measured in the proinsulin/insulin fraction. For control, the influence of chloroquine during 3-h incubation of both freshly isolated and precultured islets was also studied. During the 1-day culture at 2.2 mM glucose, prelabelled insulin was degraded significantly and addition of chloroquine did not alter the amount of insulin degraded. At 22 mM glucose, no significant amount of insulin had been degraded. During the 3-h incubation of freshly isolated as well as precultured islets, chloroquine was found to inhibit significantly glucose-induced biosynthesis of insulin. Glucose-induced release of insulin, however, was not influenced by chloroquine. It is concluded that chloroquine does not influence glucose-induced release or intra-insular degradation of insulin, but it interferes with the biosynthesis of insulin.

Animals↗
PubMed1968

The interaction of glycolysis, gluconeogenesis and the tricarboxylic acid cycle in rat liver in vivo.

1. The equations derived by Heath (1968) were applied to data from experiments on rats in four metabolic states: fed, post-absorptive, starved and 2hr. after an eventually lethal injury. The data used were: (a) The fractions of label injected as C1-, C2- and C3-pyruvate (where the prefix indicates the position of labelling) that are incorporated into carbon dioxide and glucose in post-absorptive and injured rats (yields). Yields could be corrected to yields on label taken up by the liver. (b) The (C5-label in glutamate)/(total label in glutamate) ratio in the liver after C2-pyruvate in rats in all four states. (c) The distribution of label within glutamate after C2-pyruvate or C2-alanine in the livers of fed, post-absorptive and starved rats. (d) The distribution of label within glucose after C2-lactate or C2-pyruvate in starved rats. (e) The relative specific radioactivities of pyruvate, aspartate, glutamate and (in two states only) of glucose 6-phosphate after injection of [U-(14)C]glucose into rats in all four states. These data were previously published, except those after (e) and some after (b) above, which are given in this paper. 2. In addition the concentrations of pyruvate, citrate, glutamate and aspartate in the livers of post-absorptive and injured rats were found. Injury decreased glutamate and citrate concentrations and to a smaller extent aspartate and pyruvate concentrations. 3. Non-steady-state theory showed that most of the data could be used without serious error in steady-state theory. Steady-state theory correlated all but one observation (the relative yields of (14)CO(2) from C2- and C3-pyruvate) listed after (a)-(e) above within the experimental errors, and gave rough estimates of the rates of pyruvate carboxylation, conversion of pyruvate and fat into acetyl-CoA and utilization of glutamate. The main conclusions were: (a) symmetrization of label in oxaloacetate both in the mitochondrion and in the cytoplasm was far from complete, because oxaloacetate did not equilibrate with fumarate in either. From this and other findings it was deduced: (b) that malate or fumarate or both left the mitochondrion, and not oxaloacetate; (c) that there was a loss from the mitochondrion of a fraction of the malate or fumarate or both formed from succinate, and (d) the resulting deficiency of oxaloacetate for the perpetuation of the tricarboxylic acid cycle was made up from pyruvate in fed and post-absorptive rats, but (e) in the starved rat could only be made up by utilization of glutamate. (f) In the fed rat the tricarboxylic acid cycle ran mostly on pyruvate, but in the post-absorptive and starved rat mostly on fat. (g) In the injured rat the tricarboxylic acid cycle was slowed, label in oxaloacetate was completely symmetrized (cf. conclusion a), and the tricarboxylic acid cycle utilized glutamate. (h) The conclusions were not invalidated by isotopic exchange, i.e. flux of label without net flux of compound, nor by interaction with lipogenic processes. (i) In the kidneys interaction between the tricarboxylic acid cycle and gluconeogenesis was different from in the liver, and was much less. The effects on the theory were roughly assessed, and were small. 4. The experiments and optimum experimental conditions required to check the theory are listed, and several predictions, open to experimental confirmation, are made.

Alanine↗
PubMed1987

Glucose and fatty acid metabolism in normal and diabetic rabbit cerebral microvessels.

Rabbit cerebral microvessels were used to study fatty acid metabolism and its utilization relative to glucose. Microvessels were incubated with either [6-14C]glucose or [1-14C]oleic acid and the incorporation of radioactivity into 14CO2, lactate, triglyceride, cholesterol ester, and phospholipid was determined. The inclusion of 5.5 mM glucose in the incubation mixture reduced oleate oxidation by 50% and increased esterification into both phospholipid and triglyceride. Glucose oxidation to CO2 was reduced by oleate addition, whereas lactate production was unaffected. 2'-Tetradecylglycidic acid, an inhibitor of carnitine acyltransferase I, blocked oleic acid oxidation in the presence and absence of glucose. It did not effect fatty acid esterification when glucose was absent and eliminated the inhibition of oleate on glucose oxidation. Glucose oxidation to 14CO2 was markedly suppressed in microvessels from alloxan-treated diabetic rabbits but lactate formation was unchanged. Fatty acid oxidation to CO2 and incorporation into triglyceride, phospholipid, and cholesterol ester remained unchanged in the diabetic state. The experiments show that both fatty acid and glucose can be used as a fuel source by the cerebral microvessels, and the interactions found between fatty acid and glucose metabolism are similar to the fatty acid-glucose cycle, described previously.

Alloxan↗
PubMed1990

Participation of glucose in the synthesis of glycoproteins in preimplantation mouse embryos.

Electrophoretic separation of solubilized embryos incubated for 24 h in the presence of [U-14C]glucose indicated incorporation of glucose carbon into a number of protein bands. Treatment of nitrocellulose blots of electrophoretograms with glucosidases had no effect on incorporated counts, confirming that the labelled bands were not due to protein bound glycogen. Furthermore, addition of 0.1 microgram mL-1 tunicamycin to the incubation medium virtually eliminated incorporation of glucose into the protein bands but had no effect on the pattern or rate of incorporation of labelled amino acids in parallel experiments. Also the pattern of labelling of protein by glucose was reflected in the pattern of binding of Con A to the nitrocellulose blots. There were quantitative and qualitative changes in labelling as development progressed. For embryos cultured from the 2-cell stage, a small amount of label was incorporated in two major bands at relative mobility (Mr) 69 and 97 K. With culture from the 8-cell stage, three additional major bands (33, 44 and 56 K) were labelled. Embryos cultured from the morula stage showed a different profile of incorporation; there was much more active labelling, and eight major and a number of minor radioactive bands were identified. Whilst tunicamycin suppressed glucose incorporation into glycoproteins and inhibited compaction of embryos, it had little effect on other parameters of metabolism during incubation in its presence for 24 h. No significant effects of the metabolite on protein synthesis, glycogen storage, lactate production or overall macromolecular synthesis were evident. By contrast, the anabolic metabolism of embryos decompacted by long periods of exposure to tunicamycin was severely reduced although glycolysis was still unaffected. Amphomycin at very high concentration (500 micrograms mL-1) was toxic to embryos but at concentrations up to 250 micrograms mL-1 had no effect on compaction and development of blastocysts. Addition of monensin to the incubation medium [16 micrograms mL-1] did not interfere with the development of either 2-cell or 8-cell embryos to blastocysts.

Amino Acids↗
PubMed2000

Fate of 2-deoxy-2-[18F]fluoro-D-glucose in control and diabetic rats.

Recent experiments conducted in vitro have documented a marked difference in the time course for D-[U-14C]glucose net uptake by pieces of pancreatic tissue versus isolated pancreatic islets. The present study aimed, therefore, at assessing whether the endocrine pancreas contributes to a detectable extent to the overall net uptake of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) by the pancreatic gland. For this purpose, the radioactive content of the pancreas was compared to that of plasma, erythrocytes, liver, brain, hypophysis and parotid gland 3 min, 15 min and 240 min after the intravenous injection of FDG to both control rats and animals injected with streptozotocin and later treated with insulin or not. In the control rats, the radioactive content (cpm/mg wet wt.) of erythrocytes was always lower than that of liver. In other organs, it displayed the following hierarchy pancreas < parotid < hypophysis < brain, the absolute values being either lower (3 min) or much higher (240 min) than in liver. In the diabetic rats, whether treated with insulin or not, the radioactive content of erythrocytes, pancreas, brain, hypophysis and parotid gland, relative to the paired value found in liver, was equal or lower than that of control rats when the animals were hyperglycemic and equal or higher than that of control rats when the animals became hypoglycemic as the result of intensive insulin treatment. Even only 3 min after the injection of FDG, and despite persistent hyperglycemia in the streptozotocin-injected and insulin-treated rats, the pancreas/ liver paired ratio in radioactive content failed to be significantly lower in the diabetic animals than in control rats. These findings indicate that 2-deoxy-2-[18F]fluoro-D-glucose is not a suitable tool to detect any preferential labelling of insulin-producing cells, relative to acinar cells, at least when considering only the total radioactive content of the pancreatic gland.

Animals↗
PubMed1990

Substantial glucose leakage from liposomes on filters and upon molecular-sieve chromatography in determinations of reconstituted glucose-transport activity and liposome volumes.

Transport-protein activities are often determined by procedures that involve isolation of liposomes containing the transported radioactive solute. We determined the activity of the human red cell glucose transporter in liposomes and, by similar procedures, internal volumes of liposomes. For these purposes, we isolated freeze-thawed liposomes loaded with [14C]glucose, either by filtration on cellulose-nitrate and cellulose-acetate filters, or by chromatography on Sephadex. The interaction of liposomes with filters caused substantial leakage of [14C]glucose. About half of the internal [14C]glucose was released on the filters from glucose-transporter liposomes with inhibited transport. Chromatography at high flow rate provided higher and more accurate values than did the filtration procedure. Leakage corrections could be made by use of flow-cell scintillation elution profiles. The ratios between the corrected chromatographic volume values and the filtration values were 1.4-3.0 for liposomes without protein, 2.4-4.0 for glucose-transporter liposomes and 3.6-7.9 for liposomes with several human red cell integral membrane proteins. The D-glucose equilibrium exchange with glucose-transporter liposomes at 50 mM D-glucose was 2.0 nmol D-glucose per microgram transporter per second as determined by use of chromatography at high flow rate. The filtration procedure gave only 0.6 nmol.microgram-1.s-1 due to the [14C]glucose leakage. In our experiments, the chromatographic procedure thus proved superior.

Biological Transport↗
PubMed2004

A novel enzymatic reaction for converting DNA to CO-DNA.

We have found previously that DNA from both the chick cerebrum and cardiac muscle has a modified structure. We named this novel DNA, CO-DNA. CO-DNA is a form of DNA in which a carbonyl group is attached to C-1 of the 2-deoxyribose and to the nitrogenous base. Therefore, 3-deoxyglucosone is the sugar constituent for CO-DNA. We found previously that the modification of the sugar moiety in DNA occurs around embryonic day 12 in the chick embryo. In this study, we isolated enzymes for the conversion of DNA to CO-DNA from chick cerebra. In our reaction system, uniformly labeled 14C-glucose was used as substrate. During incubation, the radioactivity was incorporated into DNA. From the analysis of 14C-labeled deoxynucleoside, the radioactive sugar was confirmed to be 3-deoxyglucosone. We propose a series of reactions involved in the conversion of DNA to CO-DNA: (1) DNA-enzyme complex is formed during preincubation, (2) 14C-glucose is transformed to 14C-3-deoxyhexonic acid, (3) 14C-3-deoxyhexonic acid is subsequently transformed into the sugar-phosphate, which is a mixture of phosphorylated 14C-3-deoxyhexonic acid and phosphorylated 14C-3-deoxyglucosone, (4) 2-deoxyribose in DNA is replaced with 14C-3-deoxyglucosone through its intermediate phosphorylated form, and (5) DNA is finally converted to CO-DNA.

Animals↗
PubMed1980

Biosynthesis of peripheral nervous system myelin proteins in vitro.

Myelin prepared from spinal roots and sciatic nerves of young rats (24 days of age) after incubation with [3H] amino acids in glucose-Krebs Ringer bicarbonate contained a considerable amount of radioactivity incorporated into the proteins. When these were separated on SDS-polyacrylamide gels, peaks of radioactivity were found to correspond to the P0, 23K, P1, and P2 proteins. Although very little Fast-Green-stained protein was visible in the high molecular weight region of the gels (> 27,000), much radioactivity was located in this area. Incorporation of [3H] amino acids into the myelin proteins continued up to 3 h and was dependent on glucose, but not on O2-CO2 replacement of air in the flask, whereas cycloheximide inhibited incorporation by 80%. Much less amino acid was incorporated into myelin protein of old rats. High molecular weight proteins appeared to be more metabolically active than other myelin proteins, as estimated by the percent of total counts incorporated/amount of Fast-Green staining, and their activity decreased less with age. The 23K and P1 proteins incorporated [3H] amino acid in approximate proportion to their content. P0 was less, and P2 somewhat more metabolically active than their contents would predict. [3H]fucose also was incorporated in vitro into P0 and P1 (probably 19K) proteins as well as into the high molecular weight proteins, while glucosamine served less well as a glycoprotein precursor. This preparation may be useful for examining further parameters of PNS myelin synthesis, assembly, and the effects of neurotoxic agents.

Aging↗
PubMed1979

Glucosyltransferase activity in kidney fractions of normal and streptozotocin-diabetic rats.

Glucosyltransferase (UDPglucose: galactosylhydroxylysine-basement membrane glucosyltransferase), an enzyme specifically involved in collagen synthesis, was measured in various kidney fractions of normal, diabetic and underfed rats, using as basis the incorporation of radioactivity into protein during incubation with UDP[U-14C]glucose and alkali-soluble fetal calf-skin collagen. Three criteria of enzyme activity were compared: A, total radioactivity of the washed protein precipitate; B, this figure minus activity incorporated in the absence of the collagen acceptor; and C, radioactivity incorporated into the mixed amino acid fraction, collected by elution with dilute NH4OH from a Dowex 50 resin column after alkaline hydrolysis of the protein. Method A was found satisfactory using whole medulla or isolated glomeruli, since the average proportions of total protein radioactivity recovered in the NH3 fraction were 0.81 and 0.87, respectively, and the deviations were small. There was a larger and variable proportion of nonspecific incorporation using whole cortex. Incubation of a control set of sample without added collagen was found to be unnecessary (Method B). Per mg protein, medulla and glomeruli had more enzyme than did whole cortex. In diabetes, activity was enhanced in the 10,000 X g supernatant fraction of cortex, as previously reported. However, the increase associated with diabetes was even more consistent in the medulla, averaging 3-fold in the 10,000 X g pellet fraction. No increase was found in isolated glomeruli in diabetes. Also, no increase was seen in the kidneys of non-diabetic rats with body weight similar to that of the diabetics.

Animals↗
PubMed1993

Glucose utilization and lactate production by Helicobacter pylori.

The transport and incorporation of D-glucose into the human pathogen Helicobacter pylori was investigated employing radioactive tracer analysis and 1H and 13C nuclear magnetic resonance spectroscopy. The bacterium was found to utilize D-glucose contrary to the accepted view that it cannot catabolize carbohydrates. Under the experimental conditions employed, the rate of transport of [14C]glucose was 3.24 mmol min-1 (g protein)-1, and the rate of incorporation into the cellular mass was 1.06 mumol h-1 (g protein)-1. The utilization of [13C]glucose showed biphasic characteristics with a slower initial period followed by a phase with a rate of utilization at least an order of magnitude faster. The apparent rates of decline of glucose levels during both phases varied between strains and depended on the growth conditions of the bacteria prior to harvesting. The main product of glucose catabolism was identified as lactate. These findings provide new perspectives into the physiology of H. pylori and have implications for the active search to develop appropriate therapies for the micro-organism.

Biological Transport, Active↗