Fructokinase Activity Research Articles

A 31P NMR study of the GI tract: effect of fructose loading and measurement of transverse relaxation times.

The effect of fructose loading on high-energy phosphates in the jejunum, ileum, and large intestine of rats was studied using 31P NMR. Following fructose loading, an increase in the intensity of the PME resonance was observed in the jejunum, indicating an accumulation of fructose-1-phosphate. There were no significant changes in ATP or Pi. This demonstrates that the activity of fructokinase in the jejunum can be monitored by 31P NMR. Fructose loading had no detectable effect on metabolite levels in the ileum and large intestine. Resolution of intestinal spectra was poor due to unusually large linewidths and the presence of broad underlying signals. To study the mechanism of line broadening, the T2's of the phosphorus resonances were measured using a solenoidal coil. The T2's of the ATP, Pi, PME, and PCr resonances were much longer than the T2's, suggesting that the linewidths of these resonances are primarily due to susceptibility gradients and/or compartmentation of metabolites. Other signals, particularly in the PDE region, were homogeneously broadened and had very short T2's. Spin echoes obtained with evolution times of 1 to 4 ms suppressed these broad components, with little loss of intensity in the inhomogeneously broadened resonances; as a result, resolution was improved.

Enzymes of Sucrose Metabolism in Bryophytes

Enzymes associated with sucrose metabolism in higher plants were found in the eight species ofbryophytes examined here. Seven species of moss and a liverwort, Conocephalum conicum, were all found to have sucrose synthase, glucokinase, fructokinase, UDP-glucopyrophosphorylase, and phosphoglucomutase activities. A TP-dependent phosphofructokinase activity was found in all but Atrichum angustatum while PPi-dependent phosphofructokinase activity was found in over half the species examined. Fructose 2,6-bisphosphate, at a concentration of one micromolar, had no activating effect on the PPi-dependent phosphofructokinase activity seen in this study. In extracts from Aulacomnium palustre and Mnium affine, we found neutral invertase activity but not acid invertase. Both gametophytic and sporophytic tissues ofBryum capillare and Funaria hygrometrica were found to contain the same glycolytic enzymes; however, different levels of activity were detected in the different tissues. Collectively, these results demonstrate that the enzymes ofthe sucrose synthase pathway are present in bryophytes for feeding sucrose into their cellular metabolism.

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.

The relationship between the activities of the pentose phosphate pathway and glycolysis during early stages of floral induction in spinach.

A quantitative cytochemical study was made of fructokinase, glucokinase, and fructokinase (both PFK-ATP and PFK-PP + F-2:6-P) activities in shoot apices of 4-week old Spinacia oleracea. The rates of activity of these enzymes in the central zone of the shoot apex of plants kept on a short day regime were compared with those from plants transferred from a range of timing up to 24 h to a continuous light regime when floral induction occurred. A mechanism is suggested explaining how no measurable change in activities of the enzymes assayed could still account for the availability of adequate levels G-6-P as substrate for pentose pathway activity which is almost doubled early on in cells of the central zone of shoot apices induced to flower.

A Novel Sucrose Synthase Pathway for Sucrose Degradation in Cultured Sycamore Cells

Enzymes of sucrose degradation and glycolysis in cultured sycamore (Acer pseudoplatanus L.) cells were assayed and characterized in crude extracts and after partial purification, in an attempt to identify pathways for sucrose catabolism. Desalted cell extracts contained similar activities (20-40 nanomoles per milligram protein per minute) of sucrose synthase, neutral invertase, glucokinase, fructokinase, phosphofructokinase, and UDPglucose pyrophosphorylase (assayed with 2 micromolar pyrophosphate (PPi). PPi-linked phosphofructokinase activity was virtually dependent upon fructose 2,6-bisphosphate, and the maximum activity exceeded that of ATP-linked phosphofructokinase. Hexokinase activity, with glucose as substrate, was highly specific for ATP, whereas fructokinase activity was relatively nonspecific. At 1 millimolar nucleoside triphosphate, fructokinase activity decreased in the order: UTP > ATP > CTP > GTP. We propose two pathways for sucrose degradation. One involves invertase action, followed by classical glycolysis of hexose sugars, and the other is a novel pathway initiated by sucrose synthase. The K(m) for sucrose of sucrose synthase was severalfold lower than that of neutral invertase (15 versus 65 millimolar), which may determine carbon partitioning between the two pathways. The sucrose synthase pathway proposed involves cycling of uridylates and PPi. UDPglucose pyrophosphorylase, which is shown to be an effective ;PPi-scavenger,' would consume PPi and form UTP. The UTP could be then utilized in the UTP-linked fructokinase reaction, thereby forming UDP for sucrose synthase. The source of PPi is postulated to arise from the back reaction of PPi-linked phosphofructokinase. Sycamore cells contained a substantial endogenous pool of PPi (about 3 nanomoles per gram fresh weight, roughly 1/10 the amount of ATP in these cells), and sufficient fructose 2,6-bisphosphate (0.09 nanomole per gram fresh weight) to activate the PPi-linked phosphofructokinase. Possible regulation and energetic differences between the sucrose synthase and invertase pathways are discussed.

The Agaricus bisporus mannitol pathway during sporophore growth

Fructose 6-phosphatase and fructokinase activities were identified in Agaricus bisporus fruit body extracts. These enzymes and mannitol dehydrogenase (MDH; E.C. 1.1.1.138) were assayed during the growth of fruit bodies on compost. MDH activity was high at early stages of development, and declined during growth to very low levels after sporulation. Phosphatase activity rose to a maximum during late fruit body development, and then fell to a low level after sporulation. Fructokinase activity was present throughout development and maximum activity was observed at late development. The results are discussed in relation to previously observed changes in fruit body mannitol content during development.

Zymomonas mobilis mutants blocked in fructose utilization

A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.

The mechanism of guanosine triphosphate depletion in the liver after a fructose load. The role of fructokinase.

A Sephadex G-25 filtrate of a 100 000g supernatant of rat liver homogenate was shown to be able to phosphorylate fructose, with GTP as the phosphate donor. Attempts to separate ATP- and GTP-dependent fructokinase activities failed, indicating that there is a single enzyme able to use both nucleotides. With a partially purified enzyme, Km values for fructose of 0.83 and 0.56 mM were found with ATP and GTP as substrates respectively. Km values of 1.53 and 1.43 mM were found for GTP and ATP respectively. Both ADP and GDP inhibited the GTP- and ATP-dependent fructokinase activity. We conclude that the depletion of hepatic GTP caused by intravenous administration of fructose to mice and rats can be explained simply by the utilization of the nucleotide by fructokinase.

Effect of aging on changes in substrate and effector levels of rat-liver glycolytic and lipogenic enzymes during induction

When 1-, 2- and 9-month-old rats adapted to a stock diet were fed a fatfree/highsucrose diet, the changes in the substrate and effector levels of glycolytic and lipogenic enzymes in liver, and the effects of aging on the changes were investigated. By the sucrose diet feeding, the activities of phosphofructokinase, pymvate kinase and fructokinase were increased, whereas hexokinase activity was markedly decreased. The fructose 1-phosphate levels were significantly increased in the liver, while conversely, the glucose 6-phosphate and fructose 6-phosphate levels were decreased. As a result of sucrose diet feeding, the changed enzyme activities would appear to be consistent with the metabolic pathway for fructose being predominant over that for glucose. Consequently, the concentrations of acetyl-CoA, citrate and malate were increased and would in turn appear to favour an increase in lipogenesis. The changes in the substrate and effector levels of lipogenic enzymes were reduced with aging, as were those in the enzyme activities. However, the changes in enzyme activities and metabolite levels of glycolysis were not much affected with aging.

Response of hepatic fructokinase to long-term sucrose diets and diabetes in spiny mice, albino mice and rats

1. 1. The activity of hepatic fructokinase increased about 2-fold in desert-derived spiny mice ( Acomys cahirinus) and laboratory bred albino mice and rats, maintained on a 50% sucrose diet for 3 months. 2. 2. The role of fructose as the specific inducer was apparent, as 25% fructose diet produced activity increases similar to those of sucrose in contrast to 25% glucose diet. 3. 3. The activity of hexokinase was not affected by the sucrose diet, that of glucokinase rose marginally but those of pyruvate kinase and NADP-malate dehydrogenase rose pronouncedly, especially in the spiny mice. 4. 4. Fructokinase activity increased significantly only after 2 weeks on the diet and continued to rise gradually. The activities of other glycolytic enzymes rose markedly already after 3 days and peaked at about 14 days. 5. 5. Fasting for 48 hr did not influence fructokinase activity while markedly reducing that of glucokinase, pyruvate kinase and NADP-malate dehydrogenase. 6. 6. Streptozotocin diabetes in rats resulted in a 40% reduction in fructokinase activity after 14 days which was restored after 6 days of insulin treatment. The activity increases of other glycolytic enzymes were more marked. However, the fructokinase induction on the sucrose diet was evident also in diabetic rats, suggesting that the insulin and substrate effects are independent. The preference of fructose over glucose phosphorylation capacity was clearly demonstrable in the non-diabetic and diabetic rats and became enhanced on sucrose feeding. 7. 7. The activity of triokinase also increased on the sucrose diet in the 3 rodent species, suggesting a coordinative substrate effect on the induction of these two rate-limiting fructolysis enzymes. 8. 8. It is concluded that the hepatic fructokinase, similarly to the intestinal fructokinase, should be regarded as an adaptive enzyme, responding to substrate availability and to endocrine changes.

Chromosomal localization of gut, fruC, and pfk mutations affecting genes involved in Bacillus subtilis D-glucitol catabolism.

Mutations affecting the genes involved in B. subtilis D-glucitol catabolism were mapped either by PBS1-mediated transduction or DNA-mediated transformation. It was shown that the genes gutA and gutB coding for the D-glucitol permease and the D-glucitol dehydrogenase, respectively, and regulatory locus gutR are clustered in a gut operon localized between purB and dal close to the pha marker. A mutation affecting fructokinase activity (fruC) was mapped near the gut markers. The fruC gene does not belong to the operon. A mutation affecting phosphofructokinase activity (pfk) was mapped between the leuA and aroG markers.

Biochemical characterization of a fructokinase mutant of Rhizobium meliloti.

A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium. UR3 lacked both phosphoglucose isomerase and fructokinase activity. A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol. A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity. A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium. No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4. Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level. A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products. Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium. The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.

Partial purification and properties of a mannofructokinase from Streptococcus mutans SL-1.

Fructokinase activity was demonstrated in seven strains of oral streptococci. The enzyme purified from Streptococcus mutans SL-1 was capable of phosphorylating both D-fructose and D-mmannose to their respective 6-phosphates. Phosphorylation of both fructose and mannose was dependent on adenosine 5'-triphosphate and a divalent metal ion. The molecular weight of the purified enzyme was estimated to be 49,000. The apparent Km of the enzyme for fructose was 0.63 mM. This enzyme also utilized mannose as a substrate, with an apparent Km for mannose of 0.37 mM. Since the activities of the enzyme toward mannose and fructose were not separated upon purification of the enzyme and since mannose was a competitive inhibitor of fructose phosphorylation, the purified kinase is a single enzyme, mannofructokinase, with dual specificity for both mannose and fructose. A role for this enzyme in carbohydrate metabolism in S. mutans is postulated.

Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.

An alternative pathway for the degradation of endogenous fructose during the catabolism of sucrose in Rhodopseudomonas capsulata.

Sucrose catabolism was studied in Rhodopseudomonas capsulata. Sucrose was hydrolysed by the action of a constitutive cytoplasmic sucrase. The use of a glucose-6-phosphate dehydrogenase-deficient mutant and radiorespirometric experiments demonstrated that both the glucose and fructose moieties of sucrose were catabolized via the Entner-Doudoroff pathway. This result was confirmed by enzyme analysis and studies on sugar assimilation. All the enzymes of the Entner-Doudoroff pathway were present in bacteria grown on secrose but fructokinase (EC 1.7.1.4) activity was relatively low. In contrast, phosphoenolpyruvate:fructose phosphotransferase and 1-phosphofructokinase, the key enzymes for the catabolism of exogenous fructose, were only partially induced. Bacteria grown on sucrose and treated with chloramphenicol were, therefore, not able to assimilate exogenous fructose. We conclude that under these conditions endogenous fructose is catabolized via the Entner-Douboroff pathway, while exogenous fructose is degraded via fructose 1-phosphate and the Embden-Meyerhof pathway.

Development and control of intestinal and hepatic fructokinase.

Extract: The patterns of development of intestinal and hepatic fructokinase have been studied in the rat, in the rabbit, and in man, and information regarding the mechanism of control of this enzyme in mature organs has been obtained. Fructokinase activity was detectable 4 days before term in fetal rat jejunum (1.0 nmol/min/mg protein) and rose progressively to a plateau 20-fold greater by the 20th day of life (20 nmol/min/mg protein). The enzyme in rat ileum and liver and in rabbit liver behaved similarly (rising from 0.5 to 4.0 nmol/min/mg protein (ileum) and from 0.5 to 8.5 nmol/min/mg protein (liver)). In human fetal liver, there was a threefold rise in fructokinase activity from approximately the 10th to 24th week of gestation (increasing from 2.1 to 7.8 nmol/min/mg protein); jejunal activity rose slightly, but not significantly, during this period. As expected, feeding of mature rats with sucrose after prolonged carbohydrate starvation led to a twofold increase in jejunal (from 6.05 to 13.0 nmol/min/mg protein) and hepatic (from 14.1 to 24.2 nmol/min/mg protein) fructokinase activity. The administration of actinomycin before the introduction of sucrose inhibited the carbohydrate-stimulated rise in fructokinase activity in both organs. Similarly, injection of actinomycin during sucrose feeding produced a significant fall in enzyme activity which greatly exceeded that produced by fasting alone. The data suggest that the substrate-dependent activity of this enzyme may be regulated at the level of transcription. Speculation: These studies support the hypothesis that the genetic control of certain enzymes involved in carbohydrate metabolism can be influenced by exogenous factors, among them dietary carbohydrate.

Additional Studies on the Regulation of Carbohydrate-Dependent Enzymes in the Jejunum: Changes in Amino Acid Pools, Protein Synthesis, and the Effect of Actinomycin-D.

Adaptive changes in rat jejunal disaccharidase levels during carbohydrate starvation and carbohydrate feeding were studied in relation to protein synthesis, amino acid pools, and cell migration. When animals were fed a sucrose-rich, normal protein diet for 24 hr after being on a casein-rich, carbohydrate-free diet for 7 days, there was 50% reduction in the size of the total amino acid pool without a significant change in the proportions of individual amino acids in the pool. The specific radioactivity of the leucine pool increased whenever carbohydrate was added to the diet and protein intake reduced. The incorporation of radioactive precursors into homogenate and brush border proteins reflected the characteristics of the pools, and was thus higher during carbohydrate feeding when the pool size was smaller. The rate of cell migration was unaffected by diet. Disaccharidase activities fell during prolonged carbohydrate starvation. When animals were treated with actinomycin-D, ribonucleic acid synthesis measured 18 hr later was reduced 50%, and cell migration was arrested. The drug did not inhibit the sucrose-stimulated rise in homogenate sucrase activity, although it did abolish the expected increase in fructokinase activity. With actinomycin-D treatment, brush border sucrase activity did not parallel the increases seen in the homogenate, but rather fell markedly. The data demonstrate that dietary carbohydrate increases jejunal disaccharidase activity without elevations in the rate of protein synthesis and that the effect is also independent of ribonucleic acid synthesis and cell migration. By contrast, jejunal fructokinase activity is probably regulated at the level of transcription.

Metabolism of fructose in the small intestine II. The effect of fructose feeding on fructose transport and metabolism in guinea pig small intestine

1. The metabolism and transport of fructose in guinea pig small intestine following fructose feeding for up to 20 days has been measured. A preparation of villus epithelial cells was used for the measurements. 2. In confirmation of the results of other workers, most of the fructose metabolized by guinea pig small intestine appears as glucose, the rest appearing as lactate. 3. Fructose feeding had no effect on the protein content of the epithelial cell preparation, however, the DNA content decreased markedly after 10 days on the fructose diet. 4. Fructose metabolism decreased initially on fructose feeding. Subsequently a gradual increase occurred which became very pronounced after 10 days on the fructose diet. 5. Fructose feeding for 3 days resulted in no change in the activities of fructo-kinase and fructose-1-phosphate aldolase in the guinea pig small intestine. 6. Measurements of the rate of fructose uptake in segments of intestine taken from guinea pigs fed on normal laboratory chow or the fructose diet were made. The rate of fructose uptake in segments of intestine taken from animals fed for 3 days was the same as that of animals fed on normal laboratory chow. There was a slight increase in the rate of fructose uptake in intestinal segments taken from animals fed the fructose diet for 20 days. 7. Histological section of intestine taken from animals fed the fructose diet for 20 days showed an accumulation of a yellowish material in the sub-epithelial region at the tips of the villi. This material appeared to be present within macrophages. Preliminary evidence indicates that the material is a protein containing a carbohydrate component.

Metabolism of fructose in the small intestine I. The effect of fructose feeding on fructose transport and metabolism in rat small intestine

1. The metabolism and transport of fructose in rat small intestine following fructose feeding for up to 20 days has been measured. A preparation of villus epithelial cells was used for the measurements. 2. In confirmation of the results of other workers fructose was poorly metabolised in rat small intestine. Small conversion to lactate and glucose was observed. 3. Fructose feeding had virtually no effect on the conversion of fructose to lactate or glucose. 4. Fructose feeding for 3 days resulted in a significant increase (approx. 2-fold) in the activities of fructokinase (EC 2.7.1.1) and fructose-1-phosphate aldolase (EC 4.1.2.7) in rat small intestine whereas after fructose feeding for 15 days these enzyme activities were not significantly different from enzyme activities in the small intestine of animals fed on normal laboratory chow. 5. Measurements of the rate of fructose transport in segments of intestine taken from rats fed on normal laboratory chow or the fructose diet were made. The rate of fructose uptake in segments of intestine taken from animals fed for 3 days on the fructose diet was about twice that of animals fed on normal laboratory chow. The rate of fructose uptake was not further altered if segments were obtained from intestine of animals which had received the fructose diet for 15 days.

Factors affecting hexose phosphorylation in Acetobacter xylinum.

Fructose was oxidized and converted to cellulose by cells of Acetobacter xylinum grown on fructose or succinate, but not by cells grown on glucose. In resting fructose-grown cells, glucose strongly suppressed fructose utilization. Extracts obtained from fructose- or succinate-grown cells catalyzed the adenosine triphosphate (ATP)-dependent formation of the 6-phosphate esters of glucose and fructose, whereas glucose-grown cell extracts phosphorylated glucose but not fructose. Fructokinase and glucokinase activities were separated and partially purified from cells grown on glucose, fructose, or succinate. Whereas fructokinase phosphorylated fructose only, glucokinase was active towards glucose and less active towards mannose and glucosamine. The optimal pH for the fructokinase was 7.4 and for the glucokinase was 8.5. The K(m) values for the fructokinase were: fructose, 6.2 mm; and ATP, 0.83 mm. The K(m) values for the glucokinase were: glucose, 0.22 mm; and ATP, 4.2 mm. Fructokinase was inhibited by glucose, glucosamine, mannose, and deoxyglucose in a manner competitive with respect to fructose, with K(i) values of 0.1, 0.14, 0.5, and 7.5 mm, respectively. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP) inhibited both kinases noncompetitively with respect to ATP. The K(i) values were: 1.8 mm (ADP) and 2.1 mm (AMP) for fructokinase, and 2.2 mm (ADP) and 9.6 mm (AMP) for glucokinase. Fructose metabolism in A. xylinum appears to be regulated by the synthesis and activity of fructokinase.