Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fructokinases”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed1980

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.

Biological Transport, Active↗
PubMed1982

Enzymes related to fructose utilization in Pseudomonas cepacia.

Growth of Pseudomonas cepacia on fructose, mannitol, or sorbitol depended on formation of an inducible fructokinase (forming fructose-6-phosphate) and the presence of enzymes of the Entner-Doudoroff pathway. Mutants deficient in any of these enzymes failed to utilize the aforementioned carbohydrates. Fructokinase deficiency did not affect growth of the bacteria on glucose. Fructose was accumulated intracellularly by active transport. Mutants blocked in transport of fructose grew normally on mannitol or sorbitol despite their inability to utilize fructose. Growth on either of these hexitols or on galactitol was accompanied by induction of two hexitol dehydrogenases, one active primarily with mannitol and the other active with sorbitol and galactitol. As expected, a mutant deficient in mannitol dehydrogenase failed to utilize mannitol as a carbon and energy source but grew normally on sorbitol and galactitol. Extracts of bacteria grown on fructose, mannitol, or sorbitol and higher levels of phosphoglucose isomerase than extracts of bacteria grown on alternate carbon sources such as citrate or phthalate. The higher levels were due to appearance of a second phosphoglucose isomerase species not present in cells with the lower activity. The results indicate that the initial steps in fructose utilization by P. cepacia differ from those of most other pseudomonads, which transport fructose by phosphoenolpyruvate-dependent translocation, forming fructose-1-phosphate, and suggest that degradation of fructose, mannitol, and sorbitol occurs primarily via the Entner-Doudoroff pathway.

Aldehyde-Lyases↗
PubMed1993

Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.

Amino Acid Sequence↗
PubMed1977

Pathways of D-fructose catabolism in species of Pseudomonas.

Cell-free extracts of D-fructose grown cells of Pseudomonas putida, P. fluorescens, P. aeruginosa, P. stutzeri, P. mendocina, P. acidovorans and P. maltophila catalyzed a P-enolpyruvate-dependent phosphorylation of D-fructose and contained 1-P-fructokinase activity suggesting that in these species fructose-1-P and fructose-1,6-P2 were intermediates of D-fructose catabolism. Neither the 1-P-fructokinase nor the activity catalyzing a P-enolpyruvate-dependent phosphorylation of D-fructose was present in significant amounts in succinate-grown cells indicating that both activities were inducible. Cell-free extracts also contained activities of fructose-1,6-P2 aldolase, fructose-1,6-P2 phosphatase, and P-hexose isomerase which could convert fructose-1,6-P2 to intermediates of either the Embden-Meyerhof pathway or Entner-Doudoroff pathway. Radiolabeling experiments with 1-14C-D-fructose suggested that in P. putida, P. aeruginosa, P. stutzeri, and P. acidovorans most of the alanine was made via the Entner-Doudoroff pathway with a minor portion being made via the Embden-Meyerhof pathway. An edd- mutant of O. putida which lacked a functional Entner-Doudoroff pathway but was able to grow on D-fructose appeared to make alanine solely via the Embden-Meyerhof pathway.

Alanine↗
PubMed1975

Catabolism of D-fructose and D-ribose by Pseudomonas doudoroffii. I. Physiological studies and mutant analysis.

Pseudomonas doudoroffii, a strict aerobe of marine origin, was able to utilize fructose and ribose but not glucose, gluconate, or other hexoses, pentoses, or sugar alcohols as sole sources of carbon and energy. Evidence was presented indicating that in this organism fructose was utilized via an inducible P-enolpyruvate: fructose phosphotransferase system (FPTS) which catalyzed the phosphorylation of fructose in the 1 position. The resulting fructose-1-P (F-1-P) was converted to fructose-1,6-P2 (FDP) by means of an inducible 1-P-fructokinase (1-PFK). The subsequent conversion of FDP to pyruvate involved enzymes of the Embden-Meyerhof pathway (EMP) which, with the exception of glyceraldehyde-3-P dehydrogenase (G3PDH), were constitutive. Two G3PDH activities were detected, one of which was inducible and NAD-dependent while the other was constitutive and NADP-dependent. Cell-free extracts of P. doudoroffii also contained enzymes of the methylglyoxal pathway (MGP) which converted dihydroxyacetone-P to pyruvate. The low specific activities of enzymes of this pathway as compared to the EMP suggested that the major route of FDP catabolism was via the latter pathway. 2. Ribose catabolism appeared to involve an inducible uptake system and an inducible ribokinase, the resulting ribose-5-P being converted to glyceraldehyde-3-P and fructose-6-P (F-6-P) by means of constitutive activities of the pentose-P pathway. The F-6-P formed as a result of these reactions was converted to FDP by means of a constitutive 6-P-fructokinase (6-PFK). Since no activity converting fructose or F-1-P to F-6-P could be detected in cell-free extracts of P. doudoroffii, the results suggested that fructose and ribose were catabolized via 1-PFK and 6-PFK, respectively, the two pathways converging at the level of FDP. Further evidence for this suggestion was obtained from a mutant which lacked an NAD-dependent G3PDH, accumulated FDP from both fructose and ribose, and was not able to grow on either of these compounds. 3. Ribose grown cells had increased amounts of the fructose uptake system and 1-PFK suggesting that a compound (or compounds) common to the catabolism of both fructose and ribose acted as the inducer(s) of these activities. Evidence was presented suggesting that the probable inducer(s) of 1-PFK and FPTS could be FDP, glyceraldehyde-3-P, or dihydroxyacetone-P. 4. A mutant unable to grow on fructose was characterized and found to lack FPTS while retaining 1-PFK and other enzyme activities of the EMP and MGP, indicating that a functional FPTS was essential for growth on fructose and suggesting that all or most of this sugar was catabolized via F-1-P.

Dihydroxyacetone Phosphate↗
PubMed1975

Catabolism of D-fructose and D-ribose by Pseudomonas doudoroffii. II. Properties of 1-phosphofructokinase and 6-phosphofructokinase.

1. The 1-P-fructokinase (1-PFK) and 6-P-fructokinase (6-PFK) from Pseudmonas doudoroffii were partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. The pH optima of these enzymes were 9.0 and 8.5, respectively. 2. When the concentrations of the substrates of the 1-PFK reaction were varied, Michaelis-Menten kinetics were observed. The Kms for D-fructose-1-P (F-1-P) and ATP were 3.03 X 10(-4) M and 3.39 X 10(-4) M, respectively. Variation of MgCl2 at fixed concentrations of F-1-P and ATP resulted in sigmoidal kinetics; about 10 mM MgCl2 was necessary for maximal activity. Activity of 1-PFK was inhibited when the ratio of ATP:Mg++ was higher than 0.5, suggesting that ATP:2Mg++ was the substrate and that free ATP was inhibitory. Although an absolute requirement for K+ or NH4+ could not be demonstrated, these cations stimulated the rate of the reaction. Activity of 1-PFK was not significantly affected by 3 mM AMP, cyclic-AMP, Pi, D-fructose-6-P (F-6-P), ADP, P-enolpyruvate (PEP), pyruvate, citrate, or L-gluamate. 3. Sigmoidal kinetics were observed for 6-PFK when the concentration of F-6-P was increased and the level of ATP was kept constant. Activity of 6-PFK was increased by ADP, inhibited by PEP, and unaffected by 3 mM AMP, cyclic-AMP, Pi, F-1-P, pyruvate, or citrate.

Adenosine Triphosphate↗
PubMed2004

Fructose metabolizing enzymes from mouse liver: influence of age and caloric restriction.

The influence of caloric restriction (CR) on the activities of liver fructose metabolizing enzymes and metabolite levels were studied in young (3 months) and old (30 months) mice. Fructokinase activity was increased (P<0.05) in both young and old CR mice when compared to controls while triokinase activity was increased (P<0.05) only in old CR versus control mice. Aldolase was not altered by CR in either old or young mice. No age-related differences in activities were observed in controls although a trend towards an increase was observed for triokinase, while significant age-related increases were observed for fructokinase and triokinase, but not aldolase, in CR mice. Both young and old mice on CR showed significant decreases in fructose and fructose-1-phosphate, however, no age-related changes in metabolite levels were observed for either control or CR mice. A fructose-1-phosphate kinase activity was also measured and found to be unchanged in both young and old mice on CR, but the activity was significantly lower in the old mice compared with young. We show here that the enzymes involved in fructose metabolism are influenced by CR and that this could contribute to alterations in gluconeogenesis and glycolysis observed with CR.

Adaptor Proteins, Signal Transducing↗
PubMed2002

In situ analysis of enzymes involved in sucrose to hexose-phosphate conversion during stolon-to-tuber transition of potato.

An in situ study of enzymes involved in sucrose to hexose-phosphate conversion during in vitro stolon-to-tuber transition of potato (Solanum tuberosum L. cv. Bintje) was employed to follow developmental changes in spatial patterns. In situ activity of the respective enzymes was visualized by specific activity-staining techniques and they revealed distinct spatially and developmentally regulated patterns. Two of the enzymes studied were also subject to in situ investigations at the transcriptional level. During the stages of stolon formation high hexokinase (EC 2.7.1.1) and acid (cell wall-bound) invertase (EC 3.2.1.26) activities were restricted to the mitotically active (sub)apical region, suggesting a possible importance of these enzymes for cell division. At the onset of tuberization sucrose synthase (EC 2.4.1.13) and fructokinase (EC 2.7.1.4) were strongly induced (visualized at transcriptional and translational level) and the acid invertase activities disappeared from the swelling subapical region as expected. The high degree of similarity in the spatial pattern and the temporal induction of sucrose synthase and fructokinase suggests a tightly co-ordinated coarse (up)regulation, which may be subject to a sugar-modulated mechanism(s) by which genes involved in the metabolic sucrose-starch converting potential are co-ordinately regulated during tuber growth. The overall activity of uridine-5-diphosphoglucose pyrophosphorylase (EC 2.7.7.9) was present in all tissues during stolon and tuber development, implying that its coarse control is not subject to (in)direct developmental regulation.

Journal Article↗
PubMed1984

Enzymes of sucrose breakdown in soybean nodules: alkaline invertase.

The specific activities of acid and alkaline invertases (beta-d-fructofuranoside fructohydrolase, EC 3.2.1.26), sucrose synthase (UDPglucose: d-fructose 2-alpha-d-glucosyltransferase, EC 2.4.1.13), hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), and fructokinase (ATP: d-fructose 6-phosphotransferase, EC 2.7.1.4) were determined in soybean (Glycine max L. Merr cv Williams) nodules at different stages of development and, for comparison, in roots of nonnodulated soybeans. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodules, but there was only a small amount of acid invertase present. The nodules contained more phosphorylating activity with fructose than glucose. Essentially all of the alkaline invertase, sucrose synthase, and fructokinase were in the soluble fraction of nodule extracts whereas hexokinase was in the bacteroid, plant particulate, and soluble fractions.Soybean nodule alkaline invertase was partially purified and shown to be a beta-d-fructofuranosidase which was specific for sucrose. The pH optimum was 7.6 and the K(m) for sucrose was 10 millimolar. Fructose was a competitive inhibitor. Tris was a noncompetitive inhibitor and the enzyme was very sensitive to inhibition by heavy metals.

Journal Article↗
PubMed1988

Enzymes of sucrose and hexose metabolism in developing kernels of two inbreds of maize.

Tissue distribution and activity of enzymes involved in sucrose and hexose metabolism were examined in kernels of two inbreds of maize (Zea mays L.) at progressive stages of development. Levels of sugars and starch were also quantitated throughout development. Enzyme activities studied were: ATP-linked fructokinase, UTP-linked fructokinase, ATP-linked glucokinase, sucrose synthase, UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, PPi-linked phosphofructokinase, ATP-linked phosphofructokinase, NAD-dependent sorbitol dehydrogenase, NADP-dependent 6-P-gluconate dehydrogenase, NADP-dependent Glc-6-P dehydrogenase, aldolase, phosphoglucoisomerase, and phosphoglucomutase. Distribution of invertase activity was examined histochemically. Hexokinase and ATP-linked phosphofructokinase activities were the lowest among these enzymes and it is likely that these enzymes may regulate the utilization of sucrose in developing maize kernels. Most of the hexokinase activity was found in the endosperm, but the embryo had high activity on a dry weight basis. The endosperm, which stores primarily starch, contained high PPi-linked phosphofructokinase and low ATP-linked phosphofructokinase activities, whereas the embryo, which stores primarily lipids, had much higher ATP-linked phosphofructokinase activity than did the endosperm. It is suggested that PPi required by UDP-Glc pyrophosphorylase and PPi-linked phosphofructokinase in the endosperm may be supplied by starch synthesis. Sorbitol dehydrogenase activity was largely restricted to the endosperm, whereas 6-P-gluconate and Glc-6-P dehydrogenase activities were highest in the base and pericarp. A possible metabolic pathway by which sucrose is converted into starch is proposed.

Journal Article↗
PubMed1989

Separation and characterization of four hexose kinases from developing maize kernels.

Four forms of hexose kinase activity from developing maize (Zea mays L.) kernels have been separated by ammonium sulfate precipitation, gel filtration chromatography, blue-agarose chromatography, and ion exchange chromatography. Two of these hexose kinases utilized d-glucose most effectively and are classified as glucokinases (EC 2.7.1.2). The other two hexose kinases utilized only d-fructose and are classified as fructokinases (EC 2.7.1.4). All hexose kinases analyzed had broad pH optima between 7.5 and 9.5 with optimal activity at pH 8.5. The two glucokinases differed in substrate affinities. One form had low K(m) values [K(m)(glucose) = 117 micromolar, K(m)(ATP) = 66 micromolar] whereas the other form had much higher K(m) values [K(m)(glucose) = 750 micromolar, K(m)(ATP) = 182 micromolar]. Both fructokinases had similar substrate saturation responses. The K(m)(fructose) was about 130 micromolar and the K(m)(ATP) was about 700 micromolar. Both exhibited uncompetitive substrate inhibition by fructose [K(i)(fructose) = 1.40 to 2.00 millimolar]. ADP inhibited all four hexose kinase activities, whereas sugar phosphates had little effect on their activities. The data suggest that substrate concentrations are an important factor controlling hexose kinase activity in situ.

Journal Article↗
PubMed1997

Assessment of energy status and fructose metabolism of liver in obstructive jaundice by 31P magnetic resonance spectroscopy.

The effect of cholestasis on hepatic energy status and fructose metabolism in jaundiced rats and patients was investigated using 31P magnetic resonance spectroscopy. Rats with obstructive jaundice (OJ group) were studied 7 days after bile duct ligation. Drainage rats were studied at 3 days (DR3 group) and 7 days (DR7 group) after the relief of 1 week obstruction of the common bile duct. In the bile duct ligated rat, the beta-adenosine triphosphate (ATP)/Pi (inorganic phosphate) ratio was significantly lower than in sham-operated controls. This ratio recovered rapidly in the DR3 and DR7 groups. The maximum increase in the phosphomonoester peak (PMEmax) after an intravenous bolus of fructose was significantly reduced in both the OJ and DR3 groups, and was accompanied by a decrease in hepatic fructokinase activity. The PMEmax and the fructokinase activity recovered in the DR7 group. In a clinical study, the beta-ATP/Pi ratio in six healthy volunteers was comparable to that of 15 patients with obstructive jaundice, regardless of their biliary drainage status. The PMEmax in all patients (serum bilirubin > or = 5 mg/dL), irrespective of biliary drainage, was significantly lower than in volunteers. Furthermore, the PMEmax in four of the eight patients with biliary drainage (serum bilirubin < 5 mg/dL) was lower than in volunteers. It is concluded that while energy status in jaundiced patients is well maintained, fructose phosphorylation is inhibited and recovery is delayed after the relief of obstruction compared with serum bilirubin. For the non-invasive evaluation of damaged liver function in jaundice, 31P magnetic resonance spectroscopy is a useful technique.

Adenosine Triphosphate↗
PubMed1997

Kinetics of Sugar Transport and Phosphorylation Influence Glucose and Fructose Cometabolism by Zymomonas mobilis.

The competitive inhibition of fructokinase by glucose has been proposed as the mechanism by which Zymomonas mobilis preferentially consumes glucose from mixtures of glucose and fructose and accumulates fructose when growing on sucrose. In this study, incorporation of radioactive fructose into biomass was used as a measure of fructose catabolism. It was determined that the rate of fructose incorporation by Z. mobilis CP4 was somewhat lower in the presence of an equimolar concentration of glucose but that the inhibition of fructokinase by glucose was not nearly as severe in vivo as was predicted from in vitro studies. Interestingly, addition of glucose to a culture of Z. mobilis CP4-M2, a glucokinaseless mutant, resulted in an immediate and nearly complete inhibition of fructose incorporation. Furthermore, addition of nonmetabolizeable glucose analogs had a similar effect on fructose catabolism by the wild-type Z. mobilis CP4, and fructose uptake by Z. mobilis CP4-M2 was shown to be severely inhibited by equimolar amounts of glucose. These results suggest that competition for fructose transport plays an important role in preferential catabolism of glucose from sugar mixtures. Indeed, the apparent K(infm) values for sugar uptake by Z. mobilis CP4 were approximately 200 mM for fructose and 13 mM for glucose. Other experiments supported the conclusion that a single facilitated diffusion transport system, encoded by the glf gene, is solely responsible for the uptake of both glucose and fructose. The results are discussed with regard to the hypothesis that the kinetics of sugar transport and phosphorylation allow the preferential consumption of glucose and accumulation of fructose, making the fructose available for the enzyme glucose-fructose oxidoreductase, which forms sorbitol, an important osmoprotectant for Z. mobilis when growing in the presence of high sugar concentrations.

Journal Article↗
PubMed1992

Sucrose fermentation by Fusobacterium mortiferum ATCC 25557: transport, catabolism, and products.

Studies of sucrose utilization by Fusobacterium mortiferum ATCC 25557 have provided the first definitive evidence for phosphoenolpyruvate-dependent sugar:phosphotransferase activity in the family Bacteroidaceae. The phosphoenolpyruvate-dependent sucrose:phosphotransferase system and the two enzymes required for the dissimilation of sucrose 6-phosphate are induced specifically by growth of F. mortiferum on the disaccharide. Monomeric sucrose 6-phosphate hydrolase (M(r), 52,000) and a dimeric ATP-dependent fructokinase (subunit M(r), 32,000) have been purified to electrophoretic homogeneity. The physicochemical and catalytic properties of these enzymes have been examined, and the N-terminal amino acid sequences for both proteins are reported. The characteristics of sucrose 6-phosphate hydrolase and fructokinase from F. mortiferum are compared with the same enzymes from both gram-positive and gram-negative species. Butyric, acetic, and D-lactic acids are the end products of sucrose fermentation by F. mortiferum. A pathway is proposed for the translocation, phosphorylation, and metabolism of sucrose by this anaerobic pathogen.

Acetates↗
PubMed1994

Changes of liver metabolite concentrations in adults with disorders of fructose metabolism after intravenous fructose by 31P magnetic resonance spectroscopy.

A novel 31P magnetic resonance spectroscopy procedure allows the estimation of absolute concentrations of certain phosphorus-containing compounds in liver. We have validated this approach by measuring ATP, phosphomonesters, and inorganic phosphate (Pi) during fasting and after an i.v. fructose bolus in healthy adults and in three adults with disorders of fructose metabolism and by comparing results with known metabolic concentrations measured chemically. During fasting, the ATP concentration averaged 2.7 +/- 0.3 (SD, n = 9) mmol/L, which, after due correction for other nucleoside triphosphates, was 2.1 mmol/L and corresponded well with known concentrations. Fructose-1-phosphate (F-1-P) could not be measured during fasting; its concentration after fructose was calculated from the difference of the phosphomonester signals before (2.9 +/- 0.2 mmol/L) and after fructose. Pi was 1.4 +/- 0.3 mmol/L and represented the one fourth of Pi visible in magnetic resonance spectra. In the three healthy controls after fructose (200 mg/kg, 20% solution, 2.5 min), the fructokinase-mediated increase of F-1-P was rapid, reaching 4.9 mmol/L within 3 min, whereas the uncorrected ATP decreased from 2.7 to 1.8 mmol/L and the Pi from 1.4 to 0.3 mmol/L. The subsequent decrease of F-1-P, mediated by fructaldolase, was accompanied by an overshooting rise of Pi to 2.7 mmol/L. In the patient with essential fructosuria, the concentrations of F-1-P, ATP, and Pi remained unchanged, confirming that fructokinase was indeed inactive. In the patient with hereditary fructose intolerance, initial metabolic changes were the same as in the controls, but baseline concentrations were not yet reestablished after 7 h, indicating weak fructaldolase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1981

Regulation of glucose metabolism in pancreatic islets.

We evaluated the possible role of islet glucokinase in controlling the rate of islet glucose metabolism, and thereby the rate of glucose-induced insulin release. The activities of glucokinase, hexokinase, P-fructokinase, and glyceraldehyde-P dehydrogenase were quantitated in sonicated or isotonically homogenized islet preparations using pyridine nucleotide-dependent fluorometric assays. In sonicates, about 1/4 of the islet glucose phosphorylating activity was due to an enzyme with kinetic properties similar to glucokinase; 3/4 of the activity was due to hexokinase. The procedure for determining islet glucokinase activity was improved by centrifuging isotonic islet homogenates at 12,000 x g. The supernatant fraction was enriched for glucokinase. About 1/2 of the glucose phosphorylating activity in this fraction was due to glucokinase and 1/2 was due to hexokinase. The glucokinase activity in islet homogenates was !23 of the activity of hexokinase, 1/40 of the activity of P-fructokinase, and 1/400 of the activity of glyceraldehyde-P dehydrogenase. Detailed concentration dependency curves of glucose and mannose utilization were also obtained with intact isolated pancreatic rat islets. Glucose and mannose usage in islets was governed by two superimposed hyperbolic systems differing in Km and Vmax. A high Km system (Km for glucose 11 mM and for mannose 21 mM) predominated. A low Km system (Km for glucose 215 and for mannose 530 microM) contributed about 15% to the total activity. The available data with intact islets could be rationalized by the existence of two distinct hexose phosphorylating enzymes with differing capacities and kinetic properties. These enzymes, tentatively identified as glucokinase and hexokinase, could coexist in the same cell or could be distributed among different cell types. The possible physiologic significance of these results is discussed, emphasizing the idea of dual control of glycolysis and insulin release by glucokinase and hexokinase. An earlier proposal that glucokinase serves as glucoreceptor of beta-cells [J. Biol. Chem. 243:2730 (1968)] is greatly strengthened by the present studies.

Animals↗
PubMed1978

Measurement of metabolites in single preimplantation embryos; a new means to study metabolic control in early embryos.

Methods are described for preparing and analyzing single preimplantation mouse embryos for a variety of metabolites and cofactors (glucose-6-P, fructose-6-P, fructose-1,6-bisphosphate, ATP, AMP, Pi, citrate, isocitrate, alpha-ketoglutarate, and malate). Oil-well and enzymatic cycling techniques are combined to provide the sensitivity needed to measure the amounts present (10(-12) to 1o(-15) moles). After experimental treatment, embryos are collected on glass slides and freeze-dried. They can then be stored indefinitely under vacuum at -25 degrees C without deterioration. With these procedures, the embryos were collected at successive stages of development and subjected to starvation and refeeding with glucose, pyruvate or both. The results confirm the existence of a block at early stages at the P-fructokinase step. This may be due to inhibition by the very high citrate levels present. The data suggest that glycolysis is turned on late in preimplantation development by the rise in fructose-6-P, a deinhibitor of P-fructokinase. In the citrate cycle, no step between citrate and alpha-ketoglutarate is rate-limiting, but a step between alpha-ketaglutarate and malate appears to impede the flux at early embryonic stages.

Adenosine Monophosphate↗
PubMed2006

The expression of ketohexokinase is diminished in human clear cell type of renal cell carcinoma.

For identification and targeting of tumor-associated marker proteins, the proteome of clear cell type of renal cell carcinoma (RCC) and normal kidney tissues was analyzed by 2-DE. Ketohexokinase (also called fructokinase), which catalyzes the phosphorylation of fructose to fructose 1-phosphate, was identified by MALDI-TOF MS and found to be expressed at low rates in the renal tumor tissues. We found a decreased amount of ketohexokinase mRNA in RCC compared to that observed in the normal kidney tissues by Northern blot. The activity of ketohexokinase in 20 clear cell RCC specimens and the 20 corresponding normal kidneys was investigated, and its activity was shown to be approximately 1.4-fold lower in the RCC specimens than in the normal kidney. Ketohexokinase activity in tumor stage pT3 RCC was 1.5-fold lower than in pT1 RCC. The level of ketohexokinase activity in histological grade 3 RCC was 1.8-fold lower than that in grade 1 cancer. In addition, using in situ hybridization, it was revealed that ketohexokinase in the normal kidney tissue was confined to the proximal tubular epithelial cells, while the expression of ketohexokinase in RCC tissues was extremely low. Our research results show that the expression of human ketohexokinase was diminished in clear cell RCC.

Adenocarcinoma, Clear Cell↗