Aldehyde-induced adenosine triphosphatase activities of fructose 6-phosphate and fructose kinases

Chitose-6-P (2,5-anhydromannose-6-P) induces ATPase activity of fructose-6-P kinase with a Vmax 2-3% that of the normal kinase reaction with fructose-6-P or 2,5-anhydromannitol. Chitose (and presumably also chitose-6-P) is 52% hydrated in water while chitose deuterated at C-1 is 60% hydrated because of the equilibrium isotope effect of 0.73 on aldehyde hydration. Deuterated chitose-6-P gave a normal isotope effect on V/K of 1.23, but no effect on Vmax, showing that the free aldehyde is the activator and the hydrated form does not bind appreciably. With fructokinase, chitose can act either as a substrate, being phosphorylated at C-6 when adsorbed with C-6 next to MgATP, or as an inducer of ATPase activity when adsorbed with C-1 next to MgATP. The ATPase has a rate about 25% that of the kinase.     

The cause of hepatic accumulation of fructose 1-phosphate on fructose loading

1. The changes in the metabolite content in freeze-clamped livers of fed rats occurring on perfusion with 10mm-d-fructose have been examined. 2. The most striking effects of fructose were an accumulation of fructose 1-phosphate, as already known, up to 8.7mumol/g of liver within 10min, a loss of total adenine nucleotides (up to 35% after 40min) with a decrease in the ATP content to 23% within 10min, a sevenfold rise in the concentration of IMP to 1.1mumol/g and an eightfold rise of alpha-glycerophosphate to 1.1mumol/g. 3. There was a transient decrease in P(i) from 4.2 to 1.7mumol/g. Within 40min the P(i) content recovered to the normal value, probably because of an uptake of P(i) from the perfusion medium. 4. The degradation of the adenine nucleotides beyond the stage of AMP can be accounted for by the decrease of ATP and P(i). As ATP inhibits 5-nucleotidase, and as P(i) inhibits AMP deaminase any AMP arising in the tissue is liable to undergo dephosphorylation or deamination under the conditions occurring after fructose loading. 5. The content of lactate increased to 4.3mumol/g at 80min; pyruvate also increased and the [lactate]/[pyruvate] ratio remained within physiological limits. 6. The concentration of free fructose within the liver remained much below that in the perfusion medium, indicating that the rate of penetration of fructose into the tissue was lower than the rate of utilization. 7. The fission of fructose 1-phosphate by liver aldolase is inhibited by several phosphorylated intermediates, especially by IMP. This inhibition is competitive with a K(i) of 0.1mm. 8. The maximal rates of the enzymes synthesizing and splitting fructose 1-phosphate are about equal. The accumulation of fructose 1-phosphate on fructose loading is due to the inhibition of the fission of fructose 1-phosphate by the IMP arising from the degradation of the adenine nucleotides.     

Regulation of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle by enzyme phosphorylation and sn-glycerol 3-phosphate

The regulation of the Fru-6-P/Fru-2,6-P2 cycle by the cooperation of allosteric and covalent mechanisms was investigated in a reconstituted enzyme system under in vitro conditions. Phosphorylation of the bifunctional enzyme exerts a much stronger effect than sn-glycerol 3-phosphate in lowering the quasi-stationary concentration of fructose 2,6-bisphosphate and in increasing the critical concentration of the fructose phosphates, respectively. However, sn-glycerol 3-phosphate is able to strongly amplify the decrease of the quasi-stationary concentration of fructose 2,6-bisphosphate due to phosphorylation. The experiments can be described by a mathematical model involving rate equations for the dephosphorylated and the phosphorylated PFD-2 and FBPase-2. The results are compared with data from the literature obtained under in vivo conditions.     

Reversible unfolding of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino) ethyl)naphthalene-1-sulfonic acid) probes. The unfolding reaction is described minimally as a 4-state transition from folded dimer-->partially unfolded dimer-->monomer-->unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.     

Tissue-specific alternative splicing of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase

We have reported the occurrence of eight splice variants of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase (RB2K). In the present study, we quantified these splice variants in various tissues using a RNAse protection assay and found a tissue-specific pattern of alternative splicing of the RB2K gene. Splice variants containing exon F were specifically expressed in brain. Moreover, exons D and E were spliced in brain, skeletal muscle and heart. Consequently, eight, six, four and two splice variants were expressed in brain, skeletal muscle, heart and liver plus testis, respectively. These results suggest that distinct RB2K isoforms could be involved in regulation of glycolysis in a tissue-specific manner.     

A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate

At a concentration of 1 mM, fructose 1-phosphate stimulated about twofold, and glucose 6-phosphate inhibited by about 30%, the phosphorylation of 5 mM glucose in high-speed supernatants prepared from rat liver or from isolated hepatocytes, but did not affect, or barely so, the activity of a partially purified preparation of glucokinase. Anion-exchange chromatography of liver extracts separated glucokinase from a fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive inhibitor of that enzyme. This inhibitor could be further purified by chromatography on phospho-Ultrogel. It was destroyed by trypsin and was heat-labile. It inhibited glucokinase competitively with respect to glucose and its inhibitory effect was greatly reinforced by fructose 6-phosphate although not by glucose 6-phosphate. Fructose 1-phosphate relieved the enzyme of the inhibitory effect of the regulator and antagonised the effect of fructose 6-phosphate in a competitive manner. It is concluded that the regulator plays a role in the physiological control of the activity of glucokinase, particularly with respect to the stimulatory effect of fructose in isolated hepatocytes (see preceding paper in this journal).     

Fructose 2,6-bisphosphate as a contaminant of commercially obtained fructose 6-phosphate: Effect on PPi:fructose 6-phosphate phosphotransferase

Fructose 6-phosphate from several commercial sources was shown to be contaminated with fructose 2,6-bisphosphate. This contaminant was identified by its activation of PP_i:fructose 6-phosphate phosphotransferase, extreme acid lability and behaviour on ion-exchange chromatography. The apparent kinetic properties of PP_i:fructose 6-phosphate phosphotransferase from castor bean endosperm were considerably altered when contaminated fructose 6-phosphate was used as a substrate. Varying levels of fructose 2,6-bisphosphate in the substrate may account for differences that have been observed in the properties of the above enzyme from several plant sources.     

Identification of sorbitol 3-phosphate and fructose 3-phosphate in normal and diabetic human erythrocytes

Using 31P NMR spectroscopy, we have identified sorbitol 3-phosphate and fructose 3-phosphate in normal human erythrocytes wherein their concentrations are estimated to be 13 mumol/liter cells. Incubation of hemolysates with sorbitol, fructose and ATP suggest that both sorbitol and fructose are phosphorylated separately and directly at the 3-hydroxyl position suggesting the presence in these cells of a novel and specific kinase(s). In addition to sorbitol 3-phosphate and fructose 3-phosphate which were previously identified in the mammalian lens and sciatic nerve, erythrocytes have two extra metabolites resonating at 6.7 and 6.8 ppm in the 31P NMR spectrum. Although not identified in this study, the unusual chemical shifts of these compounds, their low pKa values and the fact that they appear as doublet in proton-coupled 31P NMR spectra, suggest that these phosphomonoesters belong to the same class of metabolites as sorbitol 3-phosphate and fructose 3-phosphate. Preliminary studies of erythrocytes from an unselected group of diabetic subjects showed an overall increase in the concentration of all four metabolites, although an overlap with normal values was noted.     

The rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle.

Substrate cycling of fructose 6-phosphate through reactions catalysed by 6-phosphofructokinase and fructose-1,6-bisphosphatase was measured in skeletal muscles of the rat in vitro. The rate of this cycle was calculated from the steady-state values of the 3H/14C ratio in hexose monophosphates and fructose 1,6-bisphosphate after the metabolism of either [5-3H,6-14C]glucose or [3-3H,2-14C] glucose. Two techniques for the separation of hexose phosphates were studied; t.l.c. chromatography on poly(ethyleneimine)-cellulose sheets or ion-exchange chromatography coupled with enzymic conversion. These two methods gave almost identical results, suggesting that either technique could be used for determination of rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling. It was found that more than 50% of the 3H was retained in the fructose 1,6-bisphosphate; it is therefore probable that previous measurement of cycling rates, which have assumed complete loss of 3H, have underestimated the rate of this cycle. The effects of insulin, adrenaline and adrenergic agonists and antagonists on rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling were investigated. In the presence of insulin, adrenaline (1 microM) increased the cycling rate by about 10-fold in epitrochlearis muscle in vitro; the maximum rate under these conditions was about 2.5 mumol/h per g of tissue. The concentration of adrenaline that increased the cycling rate by 50% was about 50 nM. This effect of adrenaline appears to be mediated by the beta-adrenergic receptor, since the rate was increased by beta-adrenergic agonists and blocked by beta-adrenergic antagonists. From the knowledge of the precise rate of this cycle, the possible physiological importance of cycling is discussed.     

Occurrence and characterization of fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase in Euglena gracilis

1. Fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase occurred in Euglena gracilis SM-ZK, and is located in cytosol. 2. Fructose 6-phosphate, 2-kinase and fructose 2,6-bisphosphatase were partially purified, and both enzyme activities were not separated during the partial purification. 3. The pH optimum for fructose 6-phosphate, 2-kinase activity was 7.0. The saturation curve of the enzyme activity for ATP concentration was hyperbolic, and the Km value for the substrate was 0.88 mM. On the other hand, the saturation curve of the enzyme activity for fructose 6-phosphate concentration was sigmoidal, and the K0.5 value for the substrate was 70 microM. 4. The pH optimum for fructose 2,6-bisphosphatase activity was 6.5. The saturation curve for fructose 2,6-bisphosphate concentration was sigmoidal, and the K0.5 value for the substrate was 1.29 microM. Fructose 2,6-bisphosphate showed a substrate inhibition at high concentration over 5 microM, and the enzyme activity was completely inhibited by 20 microM of fructose 2,6-bisphosphate.     

Phosphofructokinase 2: the enzyme that forms fructose 2,6-bisphosphate from fructose 6-phosphate and ATP

The polypeptide composition of plasmalemma, mitochondria and endoplasmic reticulum, isolated from maize coleoptile, was determined using polyacrylamide gel electrophoresis in the presence of lithium dodecyl sulfate. A polypeptide with an apparent molecular weight of 90,000 was found to be the major polypeptide associated with plasmalemma. This polypeptide is firmly attached to the membrane since it was not extracted by KCl. However, after a treatment with Triton X100, the polypeptide was detached from the membrane. It was also found that a heat treatment of the membrane partly destroyed this polypeptide. This effect of heat treatment was also observed after isolation of the polypeptide by electrophoresis.     

Study of the fructose 6-phosphate/fructose 1,6-bi-phosphate cycle in the liver in vivo.

1. The method proposed by Rognstad & Katz [(1976) Arch, Biochem, Biophys, 177, 337-345] for the determination of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle by the randomization of carbon between C-1 and C-6 of glucose glucose formed from [1-14C] galactose was applied to anaesthetized rats and conscious mice. 2. It was checked that the hydrolysis of fructose 6-phosphate by glucose 6-phosphatase is too weak to invalidate the method. The participation of the Cori cycle in the randomization was negligible within the short experimental period used (2-4 min). 3. No detectable randomization of carbon was observed in starved animals, indicating that phosphofructokinase is inactive in this experimental condition. 4. Randomization of carbon was detected as soon as 1 min after administration of [1-14C] galactose to fed animals and was maximal at about 3-4 min. It was calculated that on average 15% of the glucose formed by the liver to fed rats was recycled through the triose phosphates. The extent of cycling was quite variable. Recycling was also observed in starved rats in which glucose had been administered intravenously 10 min previously. In these animals, recycling was completely inhibited by glucagon. 5. The main factors that appear to be responsible for the very large changes in recycling observed in various experimental conditions are the concentrations of fructose 1,6-bisphosphate and of fructose 6-phosphate and also the affinity of phosphofructokinase for fructose 6-phosphate. The concentration of nucleotides does not seem to play a role.     

Crystallization and preliminary X-ray analysis of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.     

Characterization of two isozymic forms of heart fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Occurrence of two isozymic forms of fructose 6-P, 2-kinase: fructose 2,6-bisphosphatase in bovine heart was investigated by transcribing mRNAs and amplifying the cDNAs with polymerase chain reactions. Analysis of the PCR products revealed 1.7 Kb and 1.5 Kb DNAs, and the determination of their nucleotide sequences showed that these DNAs are identical except for the lack of 180 base pairs near the 3' of the bovine heart enzyme DNA previously reported (6). This missing nucleotide sequence encodes Asn451-Gln510 and contains the phosphorylation sites for cAMP dependent protein kinase and protein kinase C.     

Effects of fructose 6-phosphate and adenylates on the activities of rabbit liver fructose bisphosphatase and phosphofructokinase.

The kinetics of induction of cytosolic DT-diaphorase (NAD(P)H dehydrogenase-quinone, EC 1.6.99.2) by benzo(a)pyrene (BP) in the liver of the 8-day-old rat has been studied. After a lag phase of 8 h, DT-diaphorase reaches its maximum activity in three waves, with plateau levels of activity between 15–18, 26–36, and 40 h after administration of BP, at 4, 15, and 26 times the basal activity, respectively. A lower degree of induction of DT-diaphorase could be observed in the kidney cortex of the young rat and in the liver of the adult rat. No induction was observed in the fetal liver and in the adult kidney cortex. Lead acetate treatment of the adult rat resulted in induction of DT-diaphorase by BP in the liver and in the kidney cortex. Induction could not be observed in the regenerating liver of the adult rat. Experiments with picolinic acid (PA)—as a G₁ inhibitor—administered simultaneously or at different time intervals after BP administration resulted in an inhibition of induction, depending on the time of administration of picolinic acid. It is concluded that a mitotic cell cycle is necessary for DT-diaphorase induction by BP. Evidence is presented that BP acts in late G₁. The kinetics of induction of aryl hydrocarbon hydroxylase (AHH) by BP in liver microsomes of the 8-day-old rat has been compared with the induction of cytosolic DT-diaphorase. The effect of PA on the induction of AHH has also been studied. In view of the differences in kinetics of induction and in the effects of PA, it is concluded that the induction of AHH and that of DT-diaphorase are dissociated. AHH induction may take place in all hepatocytes, in contrast to DT-diaphorase induction.     

Distribution of pyrophosphate:fructose 6-phosphate phosphotransferase in maize leaves

In leaves of maize (Zea mays) the activity of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) is much less than that of ATP:fructose 6-phosphate 1-phosphotransferase. A sequential extraction technique was used to study the location of PFP in this tissue. When compared with enzymes known to be restricted to specific locations in maize, the distribution of PFP activity in the sequential extracts indicated that PFP is located predominantly, if not exclusively, in the mesophyll cytoplasm. Although confined to the same site as sucrose synthesis, the level of PFP activity is inadequate to contribute significantly to the gluconeogenic flux from fructose 1,6-bisphosphate to fructose 6-phosphate. The absence of PFP activity from the bundle-sheath demonstrates that this activity is not essential for glycolysis in higher plants.