Comparison of the Activities and Some Properties of Pyrophosphate and ATP Dependent Fructose-6-Phosphate 1-Phosphotransferases of Phaseolus vulgaris Seeds

The distribution of pyrophosphate: fructose 6-phosphate phosphotransferase (PFP) and ATP: fructose-6-phosphate 1-phosphotransferase (PFK) was studied in germinating bean (Phaseolus vulgaris cv Top Crop) seeds. In the cotyledons the PFP activity was comparable with that of PFK. However, in the plumule and radicle plus hypocotyl, PFP activity exceeds that of PFK. Approximately 70 to 90%, depending on the stage of germination, of the total PFP and PFK activities were present in the cotyledons. Highest specific activity of both enzymes, however, occurred in the radicle plus hypocotyl (64-90 nanomoles·min·milligram protein). Fractionation studies indicate that 40% of the total PFK activity was associated with the plastids while PFP is apparently confined to the cytoplasm. The cytosolic isozyme of PFK exhibits hyperbolic kinetics with respect to fructose 6-P and ATP with K_m values of 320 and 46 micromolar, respectively. PFP also exhibits hyperbolic kinetics both in the presence and absence of the activator fructose-2,6-P₂. The activation is caused by lowering the K_m for fructose 6-P from 18 to 1.1 millimolar and that for pyrophosphate (PPi) from 40 to 25 micromolar, respectively. Levels of fructose 2,6-P₂ and PPi in the seeds are sufficient to activate PFP and thereby enable a glycolytic role for PFP during germination. However, the fructose 6-P content appears to be well below the K_m of PFP for this compound and would therefore preferentially bind to the catalytic site of PFK, which has a lower K_m for fructose 6-P. The ATP content appears to be at saturating levels for PFK.     

Molecular Comparison of Pyrophosphate- and ATP-Dependent Fructose 6-Phosphate 1-Phosphotransferases from Potato Tuber

The aim of this work was to compare the molecular properties of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) and ATP:fructose 6-phosphate 1-phosphotransferase (PFK). Both enzymes were purified to apparent homogeneity from potato tubers (Solanum tuberosum cv Record). Neither PFP nor PFK preparations contained detectable activity of the other enzyme. PFP was composed of two polypeptides of apparent molecular weight 58,000 and 55,700 whereas PFK contained four polypeptides of apparent molecular weight between 46,300 and 53,300. Chemical cleavage of individual PFP and PFK polypeptides gave a different set of fragments for each polypeptide. On Western blots antisera against PFP failed to cross-react with any of the four PFK polypeptides, and antibodies against PFK failed to bind to either of the PFP polypeptides. Antibodies that immunoprecipitate PFP activity had no effect on PFK activity. Conversely, antibodies against the four PFK polypeptides precipitated the activity of PFK, but not that of PFP. This work shows that potato tuber PFP and PFK are composed of distinct, unrelated polypeptides and indicate that interconversion between PFP and PFK is unlikely.     

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.     

Cytosolic ATP-Dependent Phosphofructokinase from Spinach

ATP-dependent 6-phosphofructokinase (PFK) activity is present in both chloroplastic and in nonchloroplastic fractions isolated from spinach protoplasts. The activity in the extra-chloroplastic fraction was stimulated 2- to 3.5-fold by 25 mm inorganic phosphate (Pi), the chloroplast-associated activity was inhibited 2- to 5-fold. The Pi stimulated activity was ATP-dependent and was not an artifact due to the presence of fructose 6-P, Pi, pyrophosphatase, and pyrophosphate fructose 6-P 1-phosphotransferase (PFP). PFK activities, which expressed characteristics similar to those separated from protoplasts, could be separated following ammonium sulfate fractionation of crude extracts; the ammonium sulfate treatment also separated both PFK activities from PFP. It is concluded that spinach leaves contain a cytosolic PFK. This activity is relatively stable, is stimulated by Pi over a wide pH range, is not a result of the transformation of another enzyme activity, and has an activity that is similar to, or slightly less than, that of the cytosolic PFP.     

Identification of two forms of PFK and a fructose-2,6-bisphosphate independent form of PFP in a green alga

Cell-free preparations from the green alga, Chlorella pyrenoidosa, contained two forms of phosphofructokinase (PFK), designated PFK I and PFK II. This represents the first evidence for a second form of PFK in green algae. A pyrophosphate D-fructose-6-phosphate, 1-phosphotransferase (PFP) activity, that was unaffected by the regulatory metabolite, fructose-2,6-bisphosphate, co-purified with PFK II through several steps. The data suggest that Chlorella pyrenoidosa resembles higher plants in containing two forms of PFK, but differs in containing an atypical form of PFP.Abbreviations PFK phosphofructokinase - PFP pyrophosphate D-fructose-6-phosphate, 1-phosphotransferase, Fru-2,6-P₂-fructose-2,6-bisphosphate - DEAE diethylaminoethyl-     

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.     

The catalytic direction of pyrophosphate:fructose 6-phosphate 1-phosphotransferase in rice coleoptiles in anoxia

The catalytic direction of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP; EC 2.7.1.90) in coleoptiles of rice ( Oryza sativa L.) seedlings subjected to anoxia stress is discussed. The stress greatly induced ethanol synthesis and increased activities of alcohol dehydrogenase (ADH; EC 1.1.1.1) and pyruvate decarboxylase (PDC; EC 4.1.1.1) in the coleoptiles, whereas the elevated PDC activity was much lower than the elevated ADH activity, suggesting that PDC may be one of the limiting factors for ethanolic fermentation in rice coleoptiles. Anoxic stress decreased concentrations of fructose 6-phosphate (Fru-6-P) and glucose 6-phosphate, and increased concentration of fructose 1,6-bisphosphate (Fru-1,6-bisP) in the coleoptiles. PFP activity in rice coleoptiles was low in an aerobic condition and increased during the stress, whereas no significant increase was found in ATP:fructose-6-phosphate 1-phosphotransferase (PFK; EC 2.7.1.11) activity in stressed coleoptiles. Fructose 2,6-bisphosphate concentration in rice coleoptiles was increased by the stress and pyrophosphate concentration was above the K_m for the forward direction of PFP and was sufficient to inhibit the reverse direction of PFP. Under stress conditions the potential of carbon flux from Fru-6-P toward ethanol through PFK may be much lower than the potential of carbon flux from pyruvate toward ethanol through PDC. These results suggest that PFP may play an important role in maintaining active glycolysis and ethanolic fermentation in rice coleoptiles in anoxia.     

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.     

The Respiratory Metabolism of Strelitzia juncea Ait. Seeds. The Effect of Dormancy Release by Oxygen on certain Glycolytic Enzyme Activities and Metabolite Concentrations

The activity of the glycolytic enzymes PFK, PFP, PK and aldolaseas well as the content of glucose, fructose, glucose-6-phosphateand fructose-6-phosphate were compared in the embryos of airand oxygen-incubated seeds of Strelitzia juncea. Determinationswere made during the first 4 d of incubation, prior to radicleemergence, which commences on day five for oxygen-treated seeds. No difference in PFK activity was found for the two treatments,and for both treatments PFK tended to increase with the incubationperiod. The fr2, 6P₂-stimulated PFP activity was slightly higherfor oxygen-incubated seeds, and showed a significant increasein activity over the 4 d incubation period for both treatments.No significant change in the general trend of PK and aldolaseactivity resulted from incubating the seeds in oxygen. The almost equimolar glucose and fructose contents of the embryoswere lower after 1 d of oxygen incubation of the seeds, andthe content decreased sharply during the incubation period.It is concluded that a moderate increase in the glycolytic capacityof embryos resulted from oxygen treatment of S. juncea seeds. Key words: Dormancy, glycolysis, Strelitzia juncea     

Sugar metabolism in developing tubers of Solanum tuberosum

Regulation of the sugar content of the developing tubers of three varieties (King Edward, Maris Bard, Pentland Javelin) of Solanum tuberosum was investigated. Sucrose, glucose, fructose, UDP-glucose and fructose-2,6-bispbosphate were measured during tuber development as were the maximum catalytic activities of acid invertase, alkaline invertase, sucrose synthase, α-glucan phosphorylase, hexokinase, phospbofructokinase and pyrophosphate: fructose 6-phosphate 1-phosphotransferase [PFK(PPi)]. Sucrose was the dominant sugar and there was less fructose than glucose; the amounts of all three per gram fresh weight fell during tuber development. The activity of hexokinase per gram fresh weight declined during development but those of the other enzymes listed did not alter significantly. No change in the amounts of fructose-2,6-bisphosphate or UDP-glucose per gram fresh weight were found. The above measurements suggest that much of the sucrose translocated to the developing tuber is metabolized via sucrose syntbase to UDP-glucose that is converted to glucose 1-phosphate by UDP-glucose pyrophosphorylase using pyrophosphate generated by PFK (PPi).     

Metabolite-mediated catalyst conversion of PFK and PFP: can PFK really be converted to PFP?

It has been suggested that in spinach leaves an enzyme able to catalyze the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate can exist in two different interconvertible forms which use ATP and pyrophosphate respectively as phosphoryl donors [FEBS Letters 169 (1984) 287-292]. However, the data presented to support this suggestion could also be interpreted without assuming such an unusual type of interconversion. This reinterpretation considers that PFK and PFP are two distinct enzymes which are differentially activated by incubation with various effectors such as UDPG, pyrophosphate, ATP, fructose 6-phosphate and fructose 2,6-bisphosphate.     

Glycolytic enzymes in non-photosynthetic plastids of pea (Pisum sativum L.) roots

The presence of the glycolytic enzymes from hexokinase to pyruvate kinase in plastids of seedling pea (Pisum sativum L.) roots was investigated. The recoveries, latencies and specific activities of each enzyme in different fractions was compared with those of organelle marker enzymes. Tryptic-digestion experiments were performed on each enzyme to determine whether activities were bound within membranes. The results indicate that hexokinase (EC 2.7.1.2) and phosphoglyceromutase (EC 5.4.2.1) are absent from pea root plastids. The possible function of the remaining enzymes is considered.Abbreviations GADPH glyceraldehyde 3-phosphate dehydrogenase - PFK phosphofructokinase - PFP pyrophosphate: fructose 6-phosphate 1-phosphotransferase Bronwen A. Trimming gratefully acknowledges the award of a studentship from the Science and Engineering Research Council     

Pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase activity patterns in relation to sucrose storage across sugarcane varieties

Pyrophosphate: d -fructose-6-phosphate 1-phosphotransferase (PFP; EC 2.7.1.90) and ATP: d -fructose-6-phosphate 1-phosphotransferase (PFK; EC 2.7.1.11) activities were determined in sugarcane varieties differing in sucrose content. For this purpose, activities were measured in those internodes where the maximum rate of sucrose accumulation occurs. The specific activity of internodal PFP varied significantly between the sugarcane varieties and was inversely correlated with the sucrose content. There was also a highly significant inverse correlation between PFP and sucrose content in a segregating F1 population. PFK activity was comparable to, or lower than, PFP activity and no relationship was evident between PFK activity and sucrose content. In all tissues investigated, the fructose 2,6-bisphosphate levels were probably sufficient to ensure full activation of PFP. The levels of PFP activity appear to be controlled by the expression of the β -subunit of the protein. The molecular mass of the PFP _β subunit polypeptide(s) was approximately 63 kDa. There was an inverse correlation between sucrose content and the partitioning of radiolabel into respiration in internodal tissue slices labelled with [U-¹⁴C]glucose across 3 sugarcane lines. The estimated flux of carbon into respiration correlated directly with PFP activity.     

Carbohydrate Metabolism and Activity of Pyrophosphate: Fructose-6-Phosphate Phosphotransferase in Photosynthetic Soybean (Glycine max, Merr.) Suspension Cells

Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Merr.) suspension cells. In the presence of exogenous sugars, log phase growth occurred and the cells displayed mixotrophic metabolism. During this stage, photosynthetic oxygen evolution was depressed and sugars were assimilated from the medium. Upon depletion of medium sugar, oxygen evolution and chlorophyll content increased, and cells entered stationary phase. Activities of various enzymes of glycolysis and sucrose metabolism, including PFP, sucrose synthase, fructokinase, glucokinase, UDP-glucose pyrophosphorylase, and fructose-1,6-bisphosphatase, changed as the cells went from log to stationary phases of growth. The largest change occurred in the activity of PFP, which was three-fold higher in log phase cells. PFP activity increased in cells grown on media initially containing sucrose, glucose, or fructose and began to decline when sugar in the medium was depleted. Western blots probed with antibody specific to the -subunit of potato PFP revealed a single 56 kilodalton immunoreactive band that changed in intensity during the growth cycle in association with changes in total PFP activity. The level of fructose-2,6-bisphosphate, an activator of the soybean PFP, increased during the first 24 hours after cell transfer and returned to the stationary phase level prior to the increase in PFP activity. Throughout the growth cycle, the calculated in vivo cytosolic concentration of fructose-2,6-bisphosphate exceeded by more than two orders of magnitude the previously reported activation coefficient (K_a) for soybean PFP. These results indicate that metabolism of exogenously supplied sugars by these cells involves a PFP-dependent step that is not coupled directly to sucrose utilization. Activity of this pathway appears to be controlled by changes in the level of PFP, rather than changes in the total cytosolic level of fructose-2,6-bisphosphate.     

Glycolytic activity in embryos of Pisum sativum and of non-dormant or dormant seeds of Avena sativa L. expressed through activities of PFK and PPi-PFK

Summary A quantative cytochemical assay for PP_i-PFK activity in the presence of Fru-2,6-P₂ is described along with its application to determine levels of activity in embryos of Pisum sativum and Avena sativa. The activity of ATP-PFK has also been studied in parallel as have PFK activities during the switch from dormant to non-dormant embryos in Avena sativa. PP_i-PFK activity, has been demonstrated in all tissues of Pisum sativum embryos and of Avena sativa embryos including the scutellum and the aleurone layers. The PP_i-PFK activity was greater than that of ATP-PFK in both dormant and non-dormant seeds though with only marginally more activity in the dormant as opposed to the non-dormant state.Abbreviations AMP adenosine monophosphate - ATP adenosine triphosphate - Fru-1,6-P₂ fructose 1,6-bisphosphate - Fru-2,6-P₂ fructose 2,6-bisphosphate - Fru-6-P fructose 6-phosphate - FB Pase 2 fructose 2,6-bisphosphatase (EC 3.1.3.46) - Gl-3-PD glyceraldehyde-3-phosphate dehydrogenase - NAD nicotinamide adenine dinucleotide - NBT nitroblue tetrazolium - PEP phosphoenolpyruvate - PFK 6-phosphofructokinase (EC 2.7.1.11) - PFK₂ 6-phosphofructo-2-kinase (EC 2.7.1.105) - PP_i pyrophosphate - PP_i-PFK pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) - PVA polyvinyl alcohol (G04/140 Wacke Chemical Company)     

Induction of PPi-dependent phosphofructokinase by phosphate starvation in seedlings of Brassica nigra

Activity of PPi-dependent phosphofructokinase (PFP) was monitored in Brassica nigra seedlings grown under nutrient-sufficient or phosphate (Pi)-starved conditions. Roots, stems and leaves of 50 d Pi-deficient seedlings displayed 4.0-, 3.7- and 2.3-fold greater PFP activity, respectively, than did nutrient sufficient controls. This induction was based primarily upon an increased susceptibility of PFP from the Pi-starved tissues to activation by fructose-2,6-bisphosphate. The ratio of PFP to ATP-dependent phosphofructokinase (PFK) was approximately 2:1 and 1:1 in the various organs of 50 d Pi-starved and Pi-fed plants, respectively. Immunoblots probed with anti-(potato PFP) immune serum revealed that the induction of PFP in Pi-starved B. nigra was coincident with an elevation in the amount of PFP α-subunit in the leaves as well as an increase in the α:β subunit ratio in the stems and roots. Induction of PFP in the various tissues was also accompanied by an appreciable decline in intracellular Pi level, decreased soluble protein content, and elevated phosphoenolpyruvate phosphatase activity. Time course studies revealed that these responses to Pi stress were significantly delayed in the leaves as compared to the roots and stems suggesting that Pi may be preferentially sequestered to the leaves during Pi starvation. These data also provide further evidence that B. nigra PFP is an adaptive enzyme that may function during Pi deprivation as: (1) a glycolytic bypass to PFK; and (2) a ‘Pi-recycling system’ that converts esterified-P to Pi that would be rapidly reassimilated into the metabolism of the Pi-deficient cells.     

Pyrophosphate:fructose 6-phosphate 1-phosphotransferase and glycolysis in non-photosynthetic tissues of higher plants.

The activity of pyrophosphate:fructose-6-phosphate 1-phosphotransferase [PFK (PPi); EC 2.7.1.90] in extracts of the storage tissues of leek (Allium porrum), beetroot (Beta vulgaris) and roots of darnel (Lolium temulentum) exceeded 0.15 mumol/min per g fresh wt. As net flux from fructose 1,6-bisphosphate to fructose 6-phosphate in these tissues is unlikely, it is suggested that PFK (PPi) does not contribute to gluconeogenesis or starch synthesis. The maximum catalytic activities of PFK (PPi) in apex, stele and cortex of the root of pea (Pisum sativum) and in the developing and the thermogenic club of the spadix of cuckoo-pint (Arum maculatum) were measured and compared with those of phosphofructokinase, and to estimates of the rates of carbohydrate oxidation. PPi and fructose 2,6-bisphosphate in Arum clubs were measured. The above measurements are consistent with a glycolytic role for PFK (PPi) in tissues where there is marked biosynthesis, but not in the thermogenic club of Arum. The possibility that PFK (PPi) is a means of synthesizing pyrophosphate is discussed.     

High-yield purification of potato tuber pyrophosphate: fructose-6-phosphate 1-phosphotransferase.

The procedure of Yuan et al. (1988, Biochem. Biophys. Res. Commun. 154, 111-117) for the isolation of potato pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) has been modified so that a high yield of homogeneous enzyme could be obtained. Modifications included a lower temperature heat step, a lower percentage initial polyethylene glycol fractionation step (0 to 4%, w/v), stepwise elution following an increase from 30 to 50 mM pyrophosphate during affinity chromatography on Whatman P11 phosphocellulose, anion-exchange chromatography using Q-Sepharose "Fast Flow," and gel filtration chromatography with Superose 6 "Prep grade." Our procedure resulted in an overall 42% yield and a final specific activity of 87 mumol fructose 1,6-bisphosphate produced per minute per milligram protein. Rabbit anti-(potato PFP) polyclonal antibodies effectively immunoprecipitated the activity of both the pure enzyme and the enzyme from a crude extract. Western blot analysis demonstrated that the antibodies were monospecific for PFP. A survey of various potato cultivars demonstrated significant differences in PFP activity with respect to fresh weight. This observation should be taken into consideration before any purification of potato PFP is undertaken.     

The subcellular location and characteristics of pyrophosphate-fructose-6-phosphate 1-phosphotransferase from suspension-cultured cells of soybean

The cytoplasm was identified as the probable location of pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) in suspension-cultured cells of soybean (Glycine max L.). The characteristics of the partially purified enzyme were investigated. The activity was strongly dependent on the presence of fructose 2,6-bisphosphate and this activator exerted its effects through a dramatic increase in the affinity of the enzyme for its substrates, fructose 6-phosphate and inorganic pyrophosphate. Saturation curves for all substrates were hyperbolic. The apparent molecular weight of the partially purified enzyme was 183000 by gel filtration chromatography and 128000 by sucrose-density-gradient centrifugation. The activation by fructose 2,6-bisphosphate was not accompanied by any measurable change in molecular weight. The possible role of this enzyme in the metabolism of non-photosynthetic sink tissues is discussed.Abbreviations PFP pyrophosphate-fructose-6-phosphate 1-phosphotransferase - P_i phosphate - PP_i pyrophosphate     

6-Phosphofructo-1-kinase of rat placenta

6-Phosphofructo-1-kinase (PFK) of rat placenta was purified to homogeneity with a recovery of 56% of the enzyme activity in the original extract. The purified enzyme is a tetramer and the M_r value of the subunit is 85000 ± 1500 as shown by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Considering the properties of the native rat placental PFK isoenzyme, it is clear that this tissue is a complex mixture of homotetramer and heterotetramer. Purified placenta PFK displayed little cooperativity at pH 7.0 with respect to fructose 6-phosphate and was markedly inhibited with high concentrations of ATP. The affinity of the enzyme for fructose 6-phosphate was increased by fructose 2,6-biphosphate. The purified enzyme was highly inhibited by citrate, whereas it was only slightly inhibited by phospho enolpyruvate. ADP, AMP and fructose 2,6-biphosphate showed little stimulation towards placental PFK. The present study suggests that the placental PFK is a relatively active enzymic form and it is also probably characterized with a high rate of glycolysis possibly because this tissue requires a high energy production for the development and maintenance of the fetus as the placenta tends to be a semipermeable membrane through which substances are exchanged between mother and fetus.