Fructose 6-phosphate 1-phosphotransferase Research Articles

SNP Markers Associated with Some Root, Stolon, and Tuber Traits in Tetraploid Potatoes (Solanum tuberosum L.) Grown Under Diverse Growing Systems

AbstractNumerous loci, environmental factors, and their interactions have an impact on the phenotypic diversity of several significant traits in plants. One approach put forth in recent years for genetic research and finding quantitative trait loci (QTLs) responsible for the specific trait is association mapping. The purpose of the current study was to pinpoint the genetic underpins of significant underground traits in potato. A panel of 192 diverse tetraploid potato genotypes from different countries were grown under different growing conditions (i.e., aeroponics and pot) to study root, stolon and tuber traits. Significant differences (P ≤ 0.01) were found between the genotypes for all examined traits, and the heritability (H2) of the traits ranged from 0.74 to 0.94. Genotyping was carried out using the SolCAP 25K array. 21,226 polymorphic SNPs were used for association mapping of underground traits. A GWASpoly R package was implemented for the marker-trait associations, and 78 genomic regions were found associated with the traits under investigation.. The history of potato breeding was reflected in LD patterns. The identified SNPs have their putative gene functions related to the root and stolon architecture and tuber growth (i.e., WRKY transcription factor, MAPK, the GTP cyclohydrolase 1 (i.e., GTPCHI), Glutathionyl-hydroquinone reductase, and pyrophosphate—fructose 6-phosphate 1-phosphotransferase subunit alpha (PFPase). The results of the present study provides a framework that could be helpful for future potato breeding programs to increase tuber production and reduce the challenges of feeding the world's population in the years to come.

Upregulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in strawberry

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) is a cytosolic enzyme catalyzing the first committed step in glycolysis by reversibly phosphorylating fructose-6-phosphate to fructose-1,6-bisphosphate. The position of PFP in glycolytic and gluconeogenic metabolism, as well as activity patterns in ripening strawberry, suggest that the enzyme may influence carbohydrate allocation to sugars and organic acids. Fructose-2,6-bisphosphate activates and tightly regulates PFP activity in plants and has hampered attempts to increase PFP activity through overexpression. Heterologous expression of a homodimeric isoform from Giardia lamblia, not regulated by fructose-2,6-bisphosphate, was therefore employed to ensure in vivo increases in PFP activity. The coding sequence was placed into a constitutive expression cassette under control of the cauliflower mosaic virus 35S promoter and introduced into strawberry by Agrobacterium tumefaciens-mediated transformation. Heterologous expression of PFP resulted in an up to eightfold increase in total activity in ripe berries collected over two consecutive growing seasons. Total sugar and organic acid content of transgenic berries harvested during the first season were not affected when compared to the wild type, however, fructose content increased at the expense of sucrose. In the second season, total sugar content and composition remained unchanged while the citrate content increased slightly. Considering that PFP catalyses a reversible reaction, PFP activity appears to shift between gluconeogenic and glycolytic metabolism, depending on the metabolic status of the cell.

Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity was successfully down-regulated in sugarcane using constitutively expressed antisense and untranslatable forms of the sugarcane PFP-beta gene. In young internodal tissue activity was reduced by up to 70% while no residual activity could be detected in mature tissues. The transgenic plants showed no visible phenotype or significant differences in growth and development under greenhouse and field conditions. Sucrose concentrations were significantly increased in the immature internodes of the transgenic plants but not in the mature internodes. This contributed to an increase in the purity of the immature tissues, resembling an early ripening phenotype. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. These findings suggest that PFP influences the ability of young, biosynthetically active sugarcane culm tissue to accumulate sucrose but that the equilibrium of the glycolytic intermediates, including the stored sucrose, is restored when ATP-dependent phosphofructokinase and the residual PFP activity is sufficient to sustain the required glycolytic flux as the tissue matures. Moreover, it suggests a role for PFP in glycolytic carbon flow, which could be rate limiting under conditions of high metabolic activity.

Pyrophosphate:fructose 6-phosphate 1-phosphotransferase is involved in the mobilization of sugar reserves in the taproots of cold- and drought-stressed carrot plants

The purpose of this work was to further investigate the regulatory interplay between pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) and its positive effector, fructose 2,6-bisphosphate (Fru-2,6-P2), in the storage organs of cold- and drought-stressed plants. Since there is no detectable cytoplasmic fructose-1,6-bisphosphatase (cytFBPase) activity in the taproots of carrot plants, PFP is the only enzyme that can replace its function when stored starch is converted to transportable sucrose. The working hypothesis was that PFP is likely to be involved in the mobilisation of energy reserves and might have a special role in storage organs such as carrot taproots upon stress. Both cold and drought stress resulted in a marked increase in the endogenous Fru-2,6-P2levels. It is suggested that the significant changes in photosynthate allocation are the direct results of the stimulation of PFP activity by elevated Fru-2,6-P2levels. PFP stimulated by Fru-2,6-P2operated in the gluconeogenic direction in the taproots of stressed carrot plants, whereas the glycolytic direction was dominant in the non-stressed controls. This suggests that the metabolic status determining the net activity of PFP depends on the physiological stress situation, making PFP an important sensor of environmental changes. The experimental data indicated that PFP is involved in the mobilisation of energy reserves during unfavourable environmental changes by promoting the re-synthesis of transportable sucrose in taproots.

Protein synthesis by rice coleoptiles during prolonged anoxia: implications for glycolysis, growth and energy utilization.

Anoxia-tolerant plant tissues synthesize a number of proteins during anoxia, in addition to the 'classical anaerobic proteins' involved in glycolysis and fermentation. The present study used a model system of rice coleoptile tips to elucidate patterns of protein synthesis in this anoxia-tolerant plant tissue. Coleoptile tips 7-11 mm long were excised from intact seedlings exposed to anoxia, or excised from hypoxically pre-treated seedlings and then exposed to anoxia for 72 h. Total proteins or 35S-labelled proteins were extracted, separated using two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis and analysed using mass spectrometry. The coleoptile tips excised after intact seedlings had been exposed to anoxia for 72 h had a similar proteome to tips that were first excised and then exposed to anoxia. After 72 h anoxia, Bowman-Birk trypsin inhibitors and a glycine-rich RNA-binding protein decreased in abundance, whereas a nucleoside diphosphate kinase and several proteins with unknown functions were strongly enhanced. Using [35S]methionine as label, proteins synthesized at high levels in anoxia, and also in aeration, included a nucleoside diphosphate kinase, a glycine-rich RNA-binding protein, a putative elicitor-inducible protein and a putative actin-depolymerizing factor. Proteins synthesized predominately in anoxia included a pyruvate orthophosphate dikinase (PPDK), alcohol dehydrogenase 1 and 2, fructose 1,6-bisphosphate aldolase and a protein of unknown function. The induction of PPDK in anoxic rice coleoptiles might, in combination with pyruvate kinase (PK), enable operation of a 'substrate cycle' producing PPi from ATP. Production of PPi would (a) direct energy to crucial transport processes across the tonoplast (i.e. the H+-PPiase); (b) be required for sucrose hydrolysis via sucrose synthase; and (c) enable acceleration of glycolysis, via pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) acting in parallel with phosphofructokinase (PFK), thus enhancing ATP production in anoxic rice coleoptiles; ATP production would need to be increased if there was a substantial requirement for PPi.

Physiological relevance of fructose 2,6-bisphosphate in the regulation of spinach leaf pyrophosphate:fructose 6-phosphate 1-phosphotransferase.

A major problem in defining the physiological role of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) is the 1,000-fold discrepancy between the apparent affinity of PFP for its activator, fructose 2,6-bisphosphate (Fru-2,6-P2), determined under optimum conditions in vitro and the estimated concentration of this signal metabolite in vivo. The aim of this study was to investigate the combined influence of metabolic intermediates and inorganic phosphate (Pi) on the activation of PFP by Fru-2,6-P2. The enzyme was purified to near-homogeneity from leaves of spinach (Spinacia oleracea L.). Under optimal in vitro assay conditions, the activation constant (Ka) of spinach leaf PFP for Fru-2,6-P2 in the glycolytic direction was 15.8 nM. However, in the presence of physiological concentrations of fructose 6-phosphate, inorganic pyrophosphate (PPi), 3-phosphoglycerate (3PGA), phosphoenolpyruvate (PEP), ATP and Pi the Ka of spinach leaf PFP for Fru-2,6-P2 was up to 2000-fold greater than that measured in the optimised assay and Vmax decreased by up to 62%. Similar effects were observed with PFP purified from potato (Solanum tuberosum L.) tubers. Cytosolic metabolites and Pi also influenced the response of PFP to activation by its substrate fructose 1,6-bisphosphate (Fru-1,6-P2). When assayed under optimum conditions in the gluconeogenic direction, the Ka of spinach leaf PFP for Fru-1,6-P2 was approximately 50 microM. Physiological concentrations of PPi, 3PGA, PEP, ATP and Pi increased Ka up to 25-fold, and decreased Vmax by over 65%. From these results it was concluded that physiological concentrations of metabolites and Pi increase the Ka of PFP for Fru-2,6-P2 to values approaching the concentration of the activator in vivo. Hence, measured changes in cytosolic Fru-2,6-P2 levels could appreciably alter the activation state of PFP in vivo. Moreover, the same levels of metabolites increase the Ka of PFP for Fru-1,6-P2 to an extent that activation of PFP by this compound is unlikely to be physiologically relevant.

31P and 13C nuclear magnetic resonance studies of metabolic pathways in Pasteurella multocida characterization of a new mannitol-producing metabolic pathway.

Glucose metabolism of Pasteurella multocida was examined in resting cells in vivo using 13C NMR spectroscopy, in cell-free extracts in vitro using 31P NMR spectroscopy and using enzyme assays. The NMR data indicate that glucose is converted by the Embden-Meyerhof and pentose phosphate pathways. The P. multocida fructose 6-phosphate phosphotransferase activity (the key enzyme of the Embden-Meyerhof pathway) was similar to that of Escherichia coli. Nevertheless, and in contrast to that of E. coli, its activity was inhibited by alpha glycerophosphate. This inhibition is consistent with the very low fructose 6-phosphate phosphotransferase activity found in cell-free extracts of P. multocida using a spectrophotometric method. The dominant end products of glucose metabolism were mannitol, acetate and succinate. Under anaerobic conditions, P. multocida was able to constitutively produce mannitol from glucose, mannose, fructose, sucrose, glucose 6-phosphate and fructose 6-phosphate. We propose a new metabolic pathway in P. multocida where fructose 6-phosphate is reduced to mannitol 1-phosphate by fructose 6-phosphate reductase. Mannitol 1-phosphate produced is then converted to mannitol by mannitol 1-phosphatase.

The dominant developmental mutants of tomato, Mouse-ear and Curl, are associated with distinct modes of abnormal transcriptional regulation of a Knotted gene.

The Curl (Cu) and Mouse-ear (Me) mutations of tomato cause two seemingly unrelated developmental syndromes with a wide range of pleiotropic phenotypes. Yet, the distinct morphogenic alterations in shoots, leaves, and inflorescences conferred by the two mutations appear to be caused by unchecked meristematic activity that characterizes dominant mutations in Knotted1 (Kn1)-like genes of monocot plants. We have been unable to separate the two closely linked Cu and Me mutations, and they may lie in the same gene. A homeobox-containing class I Kn1-like gene, TKn2, also maps to the same location. Significantly, the dominant mutations are associated with two aberrant modes of TKn2 transcription. Overexpression of the two in-frame wild-type transcripts of TKn2 is associated with the Cu mutation, whereas misexpression of an abundant and oversized fusion mRNA is associated with the Me mutation. Available molecular evidence strongly suggests that the defective Me-TKn2 transcript is generated via a novel splicing event that merges transcripts of two closely linked genes. The translated fusion product is comprised of most of the 5' end of the adjacent PPi-dependent fructose 6-phosphate phosphotransferase (PFP) transcript spliced in-frame to coding position 64 of the TKn2 transcript, leaving the TKn2 homeobox intact. We suggest that class I Kn1-like genes were selected early during evolution to regulate basic programs of aerial meristems and that subtle alterations in their function may be the basis for the wide diversity in growth parameters of shoot systems, leaves, and inflorescences among plant species.

Respiratory enzyme activities during germination inBrassicaseed lots of differing vigour

AbstractThe rate of oxygen consumption by cabbage seeds increased on imbibition and there was a further sharp increase on germination. This was delayed in artificially aged seeds of low vigour. The increases in oxygen consumption reflect the increased oxidation of carbohydrates via respiratory pathways. The activities of key regulatory enzymes of glycolysis and the oxidative pentose phosphate pathway were measured in aged and unaged seed lots of cabbage. Differences in the activities of glucose 6-phosphate dehydrogenase and pyrophosphate:fructose 6-phosphate 1-phosphotransferase were correlated with the rate of germination (T50) in seed lots with large differences in seed vigour induced experimentally by artificial aging. However, the activities of these enzymes could not be used to distinguish between untreated seed lots which had smaller vigour differences apparent only under stress. The enzymes are presumably not controlling and determining seed vigour, but merely reflecting actual seed performance under the current conditions.

Possible involvement of fructose 1,6-bisphosphatase in cold-induced sweetening of potato tubers

In measuring fructose 1,6-bisphosphatase (EC 3.1.3.11; FBPase) in potato tubers, we used anti PFP antibodies to ensure that the assay was specific for FBPase and that PPi:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90: PFP) did not contribute to the production of fructose 6-phosphate (Fru-6-P). The involvement of cytosolic FBPase in cold sweetening was determined by monitoring enzyme activity during storage ofSolanum tuberosum cv. Erntestolz tubers at 2 or 8°C. In contrast to the rapid increase in sucrose and reducing sugars, the specific activity of FBPase showed no change and PFP rose slightly during storage at 2°C. Sugar concentrations and enzyme activities remained virtually unchanged during storage at 8°C. These data allow the interconversion of fructose 1.6-bisphosphate (Fru-1,6-P2) to Fru-6-P and vice versa by FBPase and PFP, respectively, in potato tubers, but do not support a causal relationship between coarse metabolic control of FBPase and cold-induced sweetening.

Anti-calmodulin potency of indol alkaloids in in vitro systems

We have demonstrated that bis-indol Vinca alkaloids of anti-mitotic activities (vinblastine, vincristine and navelbine) bind to calmodulin in a Ca 2+ -dependent manner. We designed direct binding tests (flourescence energy transfer and circular dichroism measurements) to quantify the interactions of bis-indol derivatives with calmodulin. The dissociation constants of calmodulin-navelbine and calmodulin-vinblastine complexes with 1:1 stoichiometry are 0.5 μM and 3 μM, respectively. These values indicate that the binding affinities of these Vinca alkaloids to calmodulin and tubulin are comparable. Immunological, enzyme kinetic and fluorescence anisotropy measurements showed that bis-indol alkaloids inhibit the interactions of calmodulin with target proteins. The results of indirect enzyme-linked immunosorbent assay showed that bis-indol alkaloids effectively antagonize with anti-calmodulin antibody for calmodulin binding (IC 50 = 90 μ M and 430 μ M for navelbine, vincristine and vinblastine, respectively). According to the fluorescence anisotropy and enzyme kinetic measurements, vinblastine, vincristine and navelbine, similarly to trifluoperazine, the classica calmodulin antagonist, compete with target enzyme [phosphofructokinase (ATP: D -fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11)] for calmodulin binding and inhibit the calmodulin-mediated inactivation of the enzyme. Monomers, catharantine and vindoline, do not have an inhibitory effect either on immunocomplex formation or on calmodulin-enzyme interaction. Navelbine appeared in our tests as the most potent drug in inhibiting the association of calmodulin to target proteins in comparison to other bis-indol derivatives. Since navelbine and vinblastine posses identical vindoline moiety, although they differ in the catharantine part, the difference in anti-calmodulin potencies is suggested to reside predominantly on this portion of the molecules. These findings might establish the pharmacological importance of these activities in the specificity and toxicity of the drugs.

Role of Pyrophosphate:D-Fructose 6-Phosphate 1-Phosphotransferase during Germination of Rice Seeds

The pyrophosphate: D-fructose 6-phosphate 1-phosphotransferase (PPi-PFK) and 6-phospho-fructokinase (ATP-PFK) activities were measured during germination of rice seeds. In plumule and radicle, the PPi-PFK activity was the highest just at the appearance of these organs. On the other hand, the activity of ATP-PFK was almost constant, and was always lower than that of PPi-PFK. Furthermore, the PPi-PFK activity of the apex including a growing point of plumule and of radicle was much higher than that of each base. On the contrary, the ATP-PFK activity did not show any significant difference between the apex and base, and was about equal to the PPi-PFK activity detected in the base of these organs. When these enzyme activities were measured in embryo, the highest activity of PPi-PFK was detected just before the onset of decrease of the seed weight. However, the activity of ATP-PFK did not show any significant change, and was always lower than that of PPi-PFK.

Effects of anoxia on growth and carbohydrate metabolism in suspension cultures of soybean and rice

The aim of this work was to compare the carbohydrate metabolism of suspension cultures of soybean ( Glycine max), intolerant of anoxia, with that of cultures of rice ( Oryza sativa), tolerant of anoxia. Soybean cells in anoxia showed no increase in fresh weight, dry weight or extractable protein, and labelled few proteins when supplied with [ 35S]methionine. There were modest (50%) increases in the maximum catalytic activities of sucrose synthase, phosphofructokinase, pyruvate kinase and phosphoenolpyruvate carboxylase. There was a three-fold increase in alcohol dehydrogenase and no detectable change in lactate dehydrogenase and pyrophosphate:fructose 6-phosphate 1-phosphotransferase [PFK(PP i)]. The rates of respiration ( O 2 uptake and CO 2 production in air) and fermentation (CO 2 production in nitrogen), all declined with time in anoxia and the cells died after six days in anoxia. Rice cells in anoxia showed small increases in weight and protein content, and labelled many proteins with [ 35S]methionine. Increased maximum catalytic activities were found for sucrose synthase (× 2), PFK(PP i) (× 6), pyruvate kinase (× 2), alcohol dehydrogenase (× 5) and lactate dehydrogenase (× 2). When rice cells were grown in anoxia, respiration declined steadily. Fermentation increased after four days in anoxia and then declined steadily. However, both respiration and fermentation were still appreciable even after 52 days in anoxia.

Essential Arginyl Residue at the Active Site of Pyrophosphate:Fructose 6-Phosphate 1-Phosphotransferase from Potato (Solanum tuberosum) Tuber.

The aim of this work was to test the proposal that the active site of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) contains an essential arginyl residue. Enzyme activity was inhibited equally in the glycolytic and gluconeogenic directions by arginine-modifying reagents. The second-order rate constants for 2,3-butanedione and phenylglyoxal were 13.1 [plus or minus] 0.45 and 55.3 [plus or minus] 1.3 M-1 min-1, respectively. The corresponding values for the kinetic order of inactivation by these modifying reagents were 0.84 [plus or minus] 0.049 for 2,3-butanedione and 0.89 [plus or minus] 0.052 for phenylglyoxal. The substrates, fructose 6-phosphate and pyrophosphate, and a range of substrate analogs protected the enzyme from inactivation by 2,3-butanedione. These data suggest that modification of no more than one arginyl residue at, or close to, the active site is required to inhibit the enzyme. This result supports the proposal that the active site of PFP in plants is equivalent to that of the bacterial ATP-phosphofructokinase (S.M. Carlisle, S.D. Blakeley, S.M. Hemmingsen, S.J. Trevanion, T. Hiyoshi, N.J. Kruger, and D.T. Dennis [1990] J Biol Chem 265: 18366-18371).

Substrate Specificity of Pyrophosphate:Fructose 6-Phosphate 1-Phosphotransferase from Potato Tuber

The aim of this work was to establish the precise ionic form of the reactants used by pyrophosphate:fructose-6-phosphate phosphotransferase. The enzyme was purified to near-homogeneity from potato (Solanum tuberosum L.) tubers. Changes in enzyme activity when the pH of the assay and the concentration of fructose 6-phosphate, pyrophosphate, and magnesium are varied independently indicate that fructose 6-phosphate(2-) and MgP(2)O(7) (2-) are the reacting species in the glycolytic direction. Analogous experiments with fructose 1,6-bisphosphate, inorganic phosphate, and magnesium demonstrate that the enzyme uses fructose 1,6-bisphosphate(4-), HPO(4) (2-), and Mg(2+) in the gluconeogenic direction. The ionic species used in the glycolytic direction are comparable with those required by bacterial ATP-dependent phosphofructokinase. This is consistent with the proposal that the active site of pyrophosphate:fructose-6-phosphate phosphotransferase in plants is equivalent to that of the bacterial phosphofructokinase (SM Carlisle et al. [1990] J Biol Chem 265: 18366-18371).

Purification and Characterization of Pyrophosphate: D-Fructose 6-Phosphate 1-Phosphotransferase from Rice Seedlings

Pyrophosphate: D-fructose 6-phosphate 1-phosphotransferase was purified to homogeneity from rice seedlings. The enzyme had an apparent molecular weight of 100,000 and consisted of a single protein. The pH optimum for the forward and reverse reactions was 8.1 and 7.9, respectively. Fructose-2, 6-bisphosphate (Fru-2, 6-P2) shifted the pH optimum for both the forward and reverse reactions to slightly acidic. Half-maximum activation of the enzyme for Fru-2, 6-P2 in the forward and reverse reactions was observed at 16 and 520 nM, respectively. The enzyme had normal Michaelis-Menten kinetics in both the presence and absence of Fru-2, 6-P2. Fru-2, 6-P2 stimulated the enzyme activity by increasing Vmax for Fru-6-P and PPi, and by raising the affinity for Fru-6-P, Fru-1, 6-P2, and PPi. At a saturated concentration of Fru-2, 6-P2 Pi strongly inhibited the enzyme in the forward reaction. The Dixon plots with Pi indicated that the inhibition behavior was of the mixed type with respect to both Fru-6-P and PPi with Ki ...

Inhibition of pyrophosphate:Fructose-6-phosphate 1-phosphotransferase by imidodiphosphate

The aim of this work was to examine the effect of imidodiphosphate on the kinetic properties of pyrophosphate:fructose 6-phosphate 1-phosphotransferase. The enzyme was purified about 700-fold from mature tubers of Solanum tuberosum cv. Maris Piper. Imidodiphosphate was not metabolised by this enzyme. The apparent ability of imidodiphosphate to act as a phosphoryl donor was due to contamination of this compound by inorganic pyrophosphate. When enzyme activity was measured in the glycolytic direction, imidodiphosphate per se was a mixed inhibitor with respect to both fructose 6-phosphate and inorganic pyrophosphate. Comparison of the kinetic constants with the estimated concentration of reactants in vivo indicates that enzyme activity could be substantially inhibited in spinach leaves fed with imidodiphosphate (H.E. Neuhaus and M. Stitt, Plant Sci.m 76 (1991) 49–55).

Potential Role of Pyrophosphate:Fructose 6-Phosphate Phosphotransferase in Carbohydrate Metabolism of Cold Stored Tubers of Solanum tuberosum cv Bintje

To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2 degrees C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4 degrees C. During storage at 8 degrees C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6-phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8 degrees C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8 degrees C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4 degrees C but remained the same throughout storage at 8 degrees C. After exposure to 2 degrees C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4 degrees C but decreased slightly at 8 degrees C. All observed changes became annulled after transfer of cold stored tubers to 18 degrees C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation.

Effect of Temperature on the Kinetic Properties of Pyrophosphate: Fructose 6-phosphate Phosphotransferase from Potato Tuber

Summary The aim of this work was to investigate the potential role of pyrophosphate: fructose 6-phosphate phosphotransferase during cold-induced sweetening in potatoes. The enzyme purified to near homogeneity from mature tubers of Solanum tuberosum cv. Record was reversibly inhibited by low temperature. Under optimal assay conditions the temperature coefficient of the enzyme increased from 1.3 at 25 °C to 2.6 at 5 °C. Low temperature had no significant effect on the affinity of the fully-activated enzyme for its substrates fructose 6-phosphate, pyrophosphate, fructose 1,6-bisphosphate and phosphate. However, the concentration of fructose 2,6-bisphosphate required for half-maximal activation increased about 8-fold as the temperature was lowered from 25 °C to 2 °C. In addition, the extent of activation by this metabolite increased from about 5-fold to 50-fold over the same temperature range. These effects may restrict the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate at low temperature, and thus contribute to the accumulation of hexose phosphates and subsequent sucrose synthesis in the cold.

Pyrophosphate-dependent phosphofructokinase. Conservation of protein sequence between the alpha- and beta-subunits and with the ATP-dependent phosphofructokinase.

Full-length cDNA clones for the alpha- and beta-subunits of pyrophosphate-fructose 6-phosphate 1-phosphotransferase have been isolated from a cDNA expression library derived from potato tuber poly(A)+ RNA. The nucleotide sequences indicate that the alpha- and beta-subunits are related with about 40% of amino acid residues being identical. A comparison of the deduced amino acid sequences of both subunits of this enzyme with that of the major ATP-dependent fructose 6-phosphate 1-phosphotransferase from Escherichia coli (Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994) showed little homology between the proteins except for regions involved in the binding of fructose 6-phosphate/fructose, 1,6-bisphosphate and possibly between regions binding pyrophosphate and the beta- and gamma-phosphates of ADP/ATP. A comparison of the derived secondary structures of the two subunits of the PPi-dependent enzyme with the known secondary structure of the E. coli ATP-dependent enzyme indicated that the overall structure of these enzymes is similar. These data suggest that catalytic activity resides on the beta-subunit of the pyrophosphate-dependent enzyme.