Phosphofructokinase Isoenzymes Research Articles

Opening a Novel Biosynthetic Pathway to Dihydroxyacetone and Glycerol in Escherichia coli Mutants through Expression of a Gene Variant (fsaAA129S) for Fructose 6-Phosphate Aldolase

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h−1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h−1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.

The influence of PFK-II overexpression on neuroblastoma patients\u2019 survival may be dependent on the particular isoenzyme expressed, PFKFB3 or PFKFB4

Background/AimDuring cancer progression metabolic reprogramming is observed in parallel to the alternation in transcriptional profiles of malignant cells. Recent studies suggest that metabolic isoenzymes of phosphofructokinase II (PFK-II) – PFKFB3 and PFKFB4, often induced in hypoxic environment, significantly contribute to enhancement of glucose metabolism and in consequence cancer progression.Materials and methodsUsing the publicly available data deposited in the R2 data base we performed a Kaplan–Meyer analysis for cancer patients divided into groups with high and low expression levels of PFKFB3/4, determined based on the median.ResultsOur data showed that high PFKFB3/4 expression significantly correlates with shorter overall survival in several cancers. Moreover, we found that neuroblastoma patients with poor overall survival and evidence free survival are characterized by high PFKFB3 and at the same time low PFKFB4 expression, whereas patients with high PFKFB4 expressions are characterized by significantly better overall survival/evidence free survival rates.ConclusionOur analysis clearly indicates that expression of PFKFB3/4 isoenzymes may have a key prognostic value for several cancers. What’s more, it seems that in neuroblastoma the prognostic value of PFK-II may be dependent on the relation between PFKFB3 and PFKFB4 isoenzyme expression, indicating that further studies analyzing the role of both cancer specific PFK-II isoenzymes are highly desired.

Changes in activity and expression of phosphofructokinase in different rat brain regions after basal forebrain cholinergic lesion.

Selective lesion of rat basal forebrain by the cholinergic immunotoxin 192IgG-saporin was used as an animal model to address the question of whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease may be related to impaired cholinergic transmission. At different times after creating the immunolesion, the isoenzyme pattern and steady-state mRNA levels of the key glycolytic enzyme phosphofructokinase were determined in cortex, hippocampus, basal forebrain and nucleus caudatus. The loss of cholinergic input was accompanied by a persistent decrease in choline acetytransferase and acetylcholine esterase activities in the cortical target areas similar to the cholinergic malfunction seen in Alzheimer's dementia. The basal forebrain lesion induced by the immunotoxin resulted in a transient increase in phosphofructokinase activity peaking on day 7 after inducing the lesion in cortical areas. In parallel, an increased steady-state level of phosphofructokinase mRNA was determined by RT/real-time PCR and in situ hybridization. In contrast, analysis by western blotting and quantitative PCR revealed no changes in the phosphofructokinase isoenzyme pattern after immunolesion. It is concluded that common metabolic mechanisms may underlie the degenerative and repair processes in denervated rat brain and in the diseased Alzheimer's brain.

Different physiological roles of ATP- and PP(i)-dependent phosphofructokinase isoenzymes in the methylotrophic actinomycete Amycolatopsis methanolica.

Cells of the actinomycete Amycolatopsis methanolica grown on glucose possess only a single, exclusively PP(i)-dependent phosphofructokinase (PP(i)-PFK) (A. M. C. R. Alves, G. J. W. Euverink, H. J. Hektor, J. van der Vlag, W. Vrijbloed, D.H.A. Hondmann, J. Visser, and L. Dijkhuizen, J. Bacteriol. 176:6827-6835, 1994). When this methylotrophic bacterium is grown on one-carbon (C(1)) compounds (e.g., methanol), an ATP-dependent phosphofructokinase (ATP-PFK) activity is specifically induced, completely replacing the PP(i)-PFK. The two A. methanolica PFK isoenzymes have very distinct functions, namely, in the metabolism of C(6) and C(1) carbon substrates. This is the first report providing biochemical evidence for the presence and physiological roles of PP(i)-PFK and ATP-PFK isoenzymes in a bacterium. The novel ATP-PFK enzyme was purified to homogeneity and characterized in detail at the biochemical and molecular levels. The A. methanolica ATP-PFK and PP(i)-PFK proteins possess a low level of amino acid sequence similarity (24%), clearly showing that the two proteins are not the result of a gene duplication event. PP(i)-PFK is closely related to other (putative) actinomycete PFK enzymes. Surprisingly, the A. methanolica ATP-PFK is most similar to ATP-PFK from the protozoon Trypanosoma brucei and PP(i)-PFK proteins from the bacteria Borrelia burgdorferi and Treponema pallidum, both spirochetes, very distinct from actinomycetes. The data thus suggest that A. methanolica obtained the ATP-PFK-encoding gene via a lateral gene transfer event.

Deafferentation of the septo-hippocampal pathway in rats as a model of the metabolic events in Alzheimer's disease

Changes in the metabolic activity within the brain of patients suffering from Alzheimer's disease (AD) were investigated and compared with biochemical alterations in the hippocampus induced by fimbria/fornix transection in the rat. The deafferentation of the hippocampus results in a degeneration of cholinergic septo-hippocampal terminals accompanied by a persistent decrease of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) activities similar to the cholinergic malfunction in AD. In the animal model the [3H]-cytochalasin B binding to the glucose transporters was elevated up to the day 7 after surgery as was the activity of the phosphofructokinase (PFK) on day 3. A reactive astrogliosis could be evidenced by the upregulation of glial fibrillary acidic protein (GFAP). An increase of the PFK activity was also found in AD being accompanied by enhanced level of GFAP as well. A higher concentration of mRNA for all three isoenzymes of PFK was shown by reverse transcription (RT)-real time polymerase chain reaction (PCR) amplification. However, the pattern of PFK isoenzyme proteins and mRNAs did neither change in diseased human nor in the lesioned rat brain. The activities of the mitochondrial enzymes pyruvate dehydrogenase complex (PDHC) and cytochrome c oxidase (CO) were diminished in the lesioned rat hippocampus on day 7 as well as in AD brain. Subcellular fractionation showed that the activity of these enzymes was affected in the synaptosomal as well as in the extrasynaptosomal mitochondria indicating a loss of neuronal input and also a vulnerability of intrinsic hippocampal neurons and/or non-neuronal cells. The recovery of the mitochondrial enzyme activity in the animal model at later post lesion intervals may be the result of compensatory responses of surviving cells or of sprouting of other non-affected inputs. It is concluded that common metabolic mechanisms may underlie the concurrent degenerative and repair processes in the denervated hippocampus and the diseased Alzheimer brain.

Altered phosphofructokinase mRNA levels but unchanged isoenzyme pattern in brains from patients with Alzheimer's disease

In order to find out whether the increased phosphofructokinase (PFK) activities observed in brains from Alzheimer's disease (AD) patients are associated with alterations in PFK mRNA levels, we determined total PFK mRNA and the three different PFK isoenzyme mRNAs in AD and control patients by ribonuclease protection assay (RPA) and quantitative RT-PCR. PFK mRNA levels were found increased in some brain areas in AD patients. While all three PFK isoenzyme mRNAs were detectable in every studied brain sample, no changes of the PFK isoenzyme pattern were observed in patients with AD.

A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli.

Strains of Escherichia coli bearing different forms of phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source. A model was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model were experimentally confirmed in a strain with a mutant phosphofructokinase-2 (phosphofructokinase-2*) which is not inhibited by MgATP. The intracellular fructose 1, 6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either phosphofructokinase-1 or -2. Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1, 6-bisphosphate, from a trace of [U-14C]glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions. Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with [U-14C]glycerol as the only carbon source. Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules. Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-14C]glucose or [6-14C]glucose. It was found that, whereas in the strains with either of the wild-type phosphofructokinase isoenzymes, radioactive CO2 output from [1-14C]glucose was higher than with [6-14C]glucose, the reverse is found for the strain with phosphofructokinase-2*. This result also agrees with the corresponding prediction of the model. Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced. Finally, on the basis of these results it is proposed that strains bearing phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of phosphofructokinase. The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.

Kinetic Properties of the ATP-Dependent Phosphofructokinase Isoenzymes from Cucumber Seeds

Kinetic Properties of the ATP-Dependent Phosphofructokinase Isoenzymes from Cucumber Seeds

Properties of the Phosphofructokinase Isoenzymes from Germinating Cucumber Seeds

Summary Both the cytosolic and plastid PFK isoenzymes of cucumber are inhibited by sulphate at pH 7.2 but not at pH 8.0. Both the purine and pyrimidine triphosphates can be used as the phosphoryl donor to phosphorylate fructose 6-phosphate. Both isoenzymes exhibit lower Km-values for the pyrimidine triphosphates than purine triphosphates, while the maximum catalytic activity is higher with the purine triphosphates. The cytosolic PFK isoenzyme is not sensitive to GTP inhibition in contrast to the plastid isoenzyme where GTP is as inhibitory as ATP. Phosphoenol pyruvate and ADP are the strongest inhibitors of the plastid and cytosolic isoenzymes, respectively. Depending on the pH citrate, 2-phosphoglycerate, 3-phosphoglycerate and A TP also inhibit both isoenzymes. In most cases the inhibition caused by these compounds can be alleviated by the addition of phosphate. Fructose-2,6-bisphosphate has no effect on the activity of either isoenzymes in the presence and absence of the mentioned effectors.

The distribution of phosphofructokinase isoenzymes in the liver of camel, rat and rabbit

1. 1. The distribution of phosphofructokinase isoenzymes have been compared among camel, rat and rabbit livers. 2. 2. Only a single phosphofructokinase isoenzyme is present in the camel liver which has shown different physical and regulatory properties from the isoenzymes of rat and rabbit liver. 3. 3. The ammonium sulphate precipitation curves of the camel and rabbit enzymes were monophasic, whereas the rat enzyme was biphasic. 4. 4. Rabbit liver phosphofructokinase was slightly more anodic than the rat enzyme, whereas the camel enzyme was the least anodic as shown by the techniques of DEAE-cellulose chromatography and cellulose acetate electrophoresis. 5. 5. Partially purified camel liver phosphofructokinase showed different regulatory properties from the rabbit and rat isoenzymes as the apparent K m values were 0.58, 0.45 and 0.82 mM respectively.

Isoenzymes of phosphofructokinase in the rat. Demonstration of the three non-identical subunits by biochemical, immunochemical and kinetic studies.

In man and the rabbit, 6-phosphofructokinase (PFK, EC 2.7.1.11) exists in tetrameric isoenzymic forms composed of muscle (M or A), liver (L or B) and platelet or brain (P or C) subunits, which are under separate genetic control. In contrast, the genetic control of the rat PFK has not yet been conclusively established; it is unclear whether the P-type or C-type subunit exists in this species. To resolve this question, we investigated the enzyme from the skeletal muscle, liver and brain of rats of Wag/Rij strain. Our studies demonstrate that the rat PFK is also under the control of three structural loci and that the homotetramers M4, P4 and L4 exhibit unique chromatographic, immunological and kinetic-regulatory properties. Skeletal-muscle and brain PFKs consist of isolated M4 and P4 homotetramers respectively. Although liver PFK consists predominantly of L4 homotetramer, it also contains small amounts of PL3 and P2L2 species. All three PFKs exhibit allosteric properties: co-operativity with fructose 6-phosphate and inhibition by ATP decrease in the order P4 greater than L4 greater than M4. P4 and M4 tetramers are the most sensitive to citrate inhibition, whereas L4 tetramer is the least sensitive. More importantly, P4 and L4 isoenzymes are the most sensitive to activation by fructose 2,6-bisphosphate, whereas M4 isoenzyme is the least sensitive. These results indicate that the brain PFK in this strain of rat is a unique tetramer, P4, which also exhibits allosteric kinetics, as do the well-studied M4 and L4 isoenzymes. The reported differences in the number and nature of isoenzymes present in the rat brain and liver most probably reflect the differences in the strains studied by previous investigators. Since the nature of the rat PFK isoenzymes and nomenclatures reported by previous investigators have been now reconciled, it is proposed that, for the sake of uniformity, only well-established nomenclatures used for the rabbit or human PFK isoenzymes be used for the rat isoenzymes.

Analysis of isoenzymes in normal and carcinogen-treated human endometrial stromal cells in culture.

The isoenzyme patterns of lactate dehydrogenase (LDH), hexokinase, phosphofructokinase, and aldolase were investigated in cultured normal and carcinogen-treated human endometrial stromal cells. Both normal and carcinogen-treated cells had similar phosphofructokinase and aldolase isoenzymes. Distinctive changes in hexokinase and LDH isoenzyme patterns were found in the carcinogen-treated stromal cells. The LDH isoenzyme patterns of the carcinogen-treated stromal cells were shifted toward the muscle LDH forms. This is comparable to the alteration of LDH isoenzyme profiles observed in cell lines established from human uterine sarcomas. The two tissue culture media used affected the LDH isoenzyme patterns of endometrial stromal cells but differences between the LDH isoenzyme patterns of control and carcinogen-treated cells were detected regardless of the growth medium used. Total LDH activity was not significantly different in control and carcinogen-treated stromal cells. The hexokinase isoenzyme patterns expressed by the carcinogen-treated stromal cells were distinctly different from the normal hexokinase patterns. The treated stromal cells contained both hexokinase I and II, whereas the normal cells contained only hexokinase I. Hexokinase and LDH isoenzyme patterns may serve as markers with which to evaluate carcinogen-induced neoplastic changes in cultured endometrial stromal cells.

The effect of glucose on the activity of phosphofructokinase in the mucosa of rat small intestine.

In common with other phosphofructokinase isoenzymes, phosphofructokinase in the epithelial cells of rat small-intestinal mucosa is activated by fructose 2,6-bisphosphate. However, fructose 2,6-bisphosphate was found not to be present in mucosa as judged by three criteria: (1) chromatography on Sephadex G-25 of crude mucosal extracts from fed rats did not result in a decrease, or indeed any change, in the activity of phosphofructokinase under suboptimal conditions at pH7; (2) ultrafiltrates of mucosal extracts did not possess any acid-labile activating activity when tested against chromatographed liver phosphofructokinase; (3) phosphofructokinase-2 activity was not detectable in mucosal extracts. Furthermore, the perfusion in vitro of isolated loops of jejunum or the incubation of mucosal scrapings from either fed rats or rats starved for 48 h showed that the activity of mucosal phosphofructokinase is not subject to short-term regulation by glucose. These observations are consistent with the view that phosphofructokinase is the rate-limiting enzyme of glycolysis in intestinal mucosa and account for the fact that the rate of glucose utilization by rat small intestine is not very responsive to changes in the concentration of glucose in the lumen.

Alterations in phosphofructokinase isoenzymes during early human development. Establishment of adult organ-specific patterns.

Human 6-phosphofructokinase (EC 2.7.1.11) exists in tetrameric isoenzymic forms composed of muscle (M), liver (L) and platelet (P) subunits, which are under separate genetic control. In the adult, the proportion of these subunits in different organs reflects the relative activity of glycolysis versus gluconeogenesis. To elucidate the developmental basis for the observed distribution, we investigated the isoenzymic transitions of phosphofructokinase in human foetuses (12-40 weeks' gestation) by using high-resolution chromatography and monoclonal antibodies. We studied skeletal muscle, heart, liver and brain because these organs show very different glycolytic fluxes and isoenzymic patterns in adult individuals. Our results demonstrate that there is no unique 'foetal' form of phosphofructokinase in humans, but all three loci are variably expressed in all foetal organs during early gestation. As development proceeds, muscle and liver isoenzyme patterns show dramatic changes, with disappearance of P and L subunits in muscle and transient reappearance of M and P subunits in liver; in contrast, phosphofructokinase isoenzymes change little in brain and heart. Most changes occur at mid-gestation and near term, and adult isoenzyme patterns are expressed at birth, indicating that organ differentiation is complete. These studies show that phosphofructokinase undergoes changes of isoenzyme patterns similar to, but not identical with, those of other multilocus isoenzyme systems of glycolysis. The observed changes probably reflect changing patterns of gene expression, with repression of some loci and activation of others.

The isolation and characterization of phosphofructokinase from the epithelial cells of rat small intestine.

1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.

A review of animal phosphofructokinase isozymes with an emphasis on their physiological role.

Phosphofructokinase (PFK) isozymes and their physiological significance have been the focus of extensive research. The majority of this work has been centered around the PFK isozymes of rat, human and rabbit tissues. Consequently, this review emphasizes these studies. Additionally, a review of PFK isozymes in chickens, mice, guinea pig, and pig is presented. The relationship of the properties of each PFK isozyme in different tissues to the rates of glycolysis and/or gluconeogenesis in those tissues is discussed where possible. Moreover, the contribution of the different PFK isoenzymes to alterations of the glycolytic rate in various tissues is discussed in relationship to variations in nutritional, hormonal, developmental or pathological status of the animal.

An alteration in phosphofructokinase 2 of Escherichia coli which impairs gluconeogenic growth and improves growth on sugars.

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.

Plastid and cytosolic phosphofructokinases from the developing endosperm of ricinus communis: II. Comparison of the kinetic and regulatory properties of the isoenzymes

A steady-state kinetic analysis of plastid phosphofructokinase at pH 8.2 is consistent with the enzyme having a sequential reaction mechanism. Cytosolic phosphofructokinase probably has a similar mechanism. At pH 7.0 plastid phosphofructokinase shows cooperative binding of fructose 6-phosphate and is inhibited by higher concentrations of ATP. In contrast cytosolic phosphofructokinase shows normal kinetics at both pH 8.2 and 7.0 with respect to fructose 6-phosphate and is not inhibited by ATP. In the case of plastid phosphofructokinase the affinity for fructose 6-phosphate increases as the pH is raised from 7 to 8.2 whereas cytosolic phosphofructokinase is affected in an opposite manner. Phosphate is the principal activator of plastid phosphofructokinase since the cooperative kinetics toward fructose 6-phosphate are shifted toward Michaelis-Menten kinetics by 1 m m sodium phosphate and this concentration of phosphate relieves the inhibition by ATP. Both isoenzymes are inhibited by phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate at pH 7.2. Plastid phosphofructokinase is most strongly inhibited by phosphoenol pyruvate with the I 0.5 value varying from 0.08 to 0.5 μ m depending on substrate concentrations; phosphate reverses this inhibition. In contrast cytosolic phosphofructokinase is much less inhibited by phosphoenolpyruvate with an I 0.5 approximately 1000-fold higher. Cytosolic phosphofructokinase is powerfully inhibited by 3-phosphoglycerate with an I 0.5 value of 60 μ m and this appears to be the principal regulator of this isoenzyme. The two isoenzymes of phosphofructokinase in the endosperm appear, therefore, to be regulated differently. Plastid phosphofructokinase is inhibited by phosphoenolpyruvate and ATP and is activated by phosphate; whereas the cytosolic enzyme is inhibited principally by 3-phosphoglycerate and this inhibition is only partially relieved by phosphate. Some of the differences reported previously for phosphofructokinases from different plant tissues may, therefore, be due to varying ratios of the cytosolic and plastid isoenzymes.

Plastid and cytosolic phosphofructokinases from the developing endosperm of Ricinus communis: I. Separation, purification, and initial characterization of the isozymes

Isoenzymes of phosphofructokinase are present in the endosperm of developing seeds of Ricinus communis L. DEAE-Sephacel chromatography will separate the isoenzymes and can be used to identify them as cytosolic and plastid activities. Plastid phosphofructokinase represents approximately 60% of the total phosphofructokinase activity in this tissue. The isoenzymes have different kinetic properties. The plastid phosphofructokinase is activated by sodium phosphate at pH 6.8 and inhibited by sodium sulfate at pH 7.2, whereas the activity of cytosolic phosphofructokinase is much less sensitive to these anions under the same assay conditions. A procedure has been developed to purify the endosperm plastid phosphofructokinase using ion-exchange chromatography and 5′-AMP affinity chromatography. The molecular weight of plastid phosphofructokinase is 175,000 as estimated by gel filtration.

Increased ATP inhibition of liver phosphofructokinase from genetically diabetic mice.

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was partially purified from the livers of genetically diabetic mice (C57BL/KsJ-db) and their lean littermates (C57BL/KsJ). These genetically diabetic mice have been shown to be hyperglucagonemic and to exhibit symptoms resembling those of maturity-onset diabetes in humans. Two isoenzymes of phosphofructokinase were obtained after DEAE-Sephadex chromatography of extracts of livers from either normal or diabetic animals. One of these isozymes, peak II, from the genetically diabetic mice was shown to be more sensitive to ATP inhibition at physiological pH than the peak II isozyme from the normal animals. In addition, the peak II isozyme from the diabetic mice exhibited decreased affinity for fructose 6-phosphate. The altered kinetic properties of phosphofructokinase from diabetic animals are markedly similar to those recently reported for liver phosphofructokinase isolated from normal animals after glucagon treatment. Our results suggest that increased glucagon levels in diabetes may lead to altered regulation of phosphofructokinase in this disease.