Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In liver, the 470-residue bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyses the synthesis and degradation of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. In rat hepatoma (HTC) cells, this enzyme has kinetic, antigenic, and regulatory properties, such as insensitivity to cyclic AMP-dependent protein kinase and lack of associated FBPase-2 activity, that differ from those in liver. To compare the sequence of the HTC enzyme with that of the liver enzyme, we have cloned the corresponding fully-coding cDNA from HTC cells. This cDNA predicts a protein of 448 residues in which the first 32 residues of liver PFK-2/FBPase-2 including the cyclic AMP target sequence have been replaced by a unique N-terminal decapeptide. The rest of the protein is identical with the liver enzyme. An N-terminally truncated recombinant peptide of 380 residues containing the PFK-2 and FBPase-2 domains was expressed in Escherichia coli as a beta-galactosidase fusion protein. It was recognized by anti-PFK-2 antibodies but its enzymic activities were barely detectable. In contrast, a cDNA fully-coding for the HTC enzyme could be expressed in E. coli as a beta-galactosidase-free peptide that exhibited both PFK-2 and FBPase-2 activities. This peptide had those PFK-2 kinetic properties of the HTC enzyme that differ from the liver enzyme. These data, together with immunoblot experiments, suggest that the lack of associated FBPase-2 activity in HTC cells results from a post-translational modification of the enzyme rather than from the difference in amino acid sequence. As well as this peculiar type of PFK-2/FBPase-2 mRNA, HTC cells also contained low concentrations of the liver-type mRNA. Unlike in liver, neither mRNA was induced by dexamethasone in these cells.     

Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library. Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner

The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSAXXVAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5'-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion.     

Contractile activity modifies Fru-2,6-P(2) metabolism in rabbit fast twitch skeletal muscle.

Modification of muscular contractile patterns by denervation and chronic low frequency stimulation induces structural, physiological, and biochemical alterations in fast twitch skeletal muscles. Fructose 2,6-bisphosphate is a potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. The concentration of Fru-2,6-P(2) depends on the activity of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which catalyzes the synthesis and degradation of this metabolite. This enzyme has several isoforms, the relative abundance of which depends on the tissue metabolic properties. Skeletal muscle expresses two of these isoforms; it mainly contains the muscle isozyme (M-type) and a small amount of the liver isozyme (L-type), whose expression is under hormonal control. Moreover, contractile activity regulates expression of muscular proteins related with glucose metabolism. Fast twitch rabbit skeletal muscle denervation or chronic low frequency stimulation can provide information about the regulation of this enzyme. Our results show an increase in Fru-2,6-P(2) concentration after 2 days of denervation or stimulation. In denervated muscle, this increase is mediated by a rise in liver PFK-2/FBPase-2 isozyme, while in stimulated muscle it is mediated by a rise in muscle PFK-2/FBPase-2 isozyme. In conclusion, our results show that contractile activity could alter the expression of PFK-2/FBPase-2.     

6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase in Trypanosomatidae. Molecular characterization, database searches, modelling studies and evolutionary analysis

Fructose 2,6-bisphosphate is a potent allosteric activator of trypanosomatid pyruvate kinase and thus represents an important regulator of energy metabolism in these protozoan parasites. A 6-phosphofructo-2-kinase, responsible for the synthesis of this regulator, was highly purified from the bloodstream form of Trypanosoma brucei and kinetically characterized. By searching trypanosomatid genome databases, four genes encoding proteins homologous to the mammalian bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) were found for both T. brucei and the related parasite Leishmania major and four pairs in Trypanosoma cruzi. These genes were predicted to each encode a protein in which, at most, only a single domain would be active. Two of the T. brucei proteins showed most conservation in the PFK-2 domain, although one of them was predicted to be inactive due to substitution of residues responsible for ligating the catalytically essential divalent metal cation; the two other proteins were most conserved in the FBPase-2 domain. The two PFK-2-like proteins were expressed in Escherichia coli. Indeed, the first displayed PFK-2 activity with similar kinetic properties to that of the enzyme purified from T. brucei, whereas no activity was found for the second. Interestingly, several of the predicted trypanosomatid PFK-2/FBPase-2 proteins have long N-terminal extensions. The N-terminal domains of the two polypeptides with most similarity to mammalian PFK-2s contain a series of tandem repeat ankyrin motifs. In other proteins such motifs are known to mediate protein-protein interactions. Phylogenetic analysis suggests that the four different PFK-2/FBPase-2 isoenzymes found in Trypanosoma and Leishmania evolved from a single ancestral bifunctional enzyme within the trypanosomatid lineage. A possible explanation for the evolution of multiple monofunctional enzymes and for the presence of the ankyrin-motif repeats in the PFK-2 isoenzymes is presented.     

Expression and regulation of 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase isozymes in white adipose tissue.

The aim of this work was to identify the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) isozyme(s) present in white adipose tissue. Ion-exchange chromatography of PFK-2 from rat epididymal fat pads yielded an elution pattern compatible with the presence of both the L (liver) and M (muscle) isozymes. This was consistent with a study of the phosphorylation of the purified adipose tissue enzyme by cAMP-dependent protein kinase, by specific labelling of the preparation with [2-32P]fructose 2,6-bisphosphate and by reaction with antibodies. Characterization of the PFK-2/FBPase-2 mRNAs showed that mature adipocytes express the mRNA that codes for the L isozyme and the two mRNAs that code for the M isozyme. Preadipocytes expressed mRNA that codes for the M isozyme. Incubation of rat epididymal fat pads with adrenaline stimulated glycolysis but decreased fructose 2,6-bisphosphate concentrations without significant inactivation of PFK-2. These results support previous findings showing that fructose 2,6-bisphosphate is not involved in the adrenaline-induced stimulation of glycolysis in white adipose tissue.     

Cloning, expression and chromosomal localization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene

6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional enzyme responsible for the synthesis and breakdown of Fru-2,6-P2, a key metabolite in the regulation of glycolysis. Several genes encode distinct PFK-2/FBPase-2 isozymes that differ in their tissue distribution and enzyme regulation. In this paper, we present the isolation of a cDNA from a human testis cDNA library that encodes a PFK-2/FBPase-2 isozyme. Sequencing data show an open reading frame of 1407 nucleotides that codifies for a protein of 469 amino acids. This has a calculated molecular weight of 54kDa and 97% similarity with rat testis PFK-2/FBPase-2, with complete conservation of the amino acid residues involved in the catalytic mechanism. Fluorescence in-situ hybridization (FISH) localized testis PFK-2/FBPase-2 gene (PFKFB4) in human chromosome 3 at bands p21-p22. A Northern blot analysis of different rat tissues showed the presence of a 2.4-kb mRNA expressed specifically in testis. In mammalian COS-1 cells, the human testis cDNA drives expression of an isozyme with a molecular weight of 55kDa. This isozyme shows clear PFK-2 activity. Taken together, these results provide evidence for a new PFK-2/FBPase-2 gene coding for a human testis isozyme.     

Characterization of distinct mRNAs coding for putative isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Three distinct clones encoding full-length 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) were characterized from a rat liver cDNA library. Clone 22c was 1859 bp long and coded for the 470 amino acids of the bifunctional subunit of the liver homodimer. This polypeptide is phosphorylated on serine 32 by cyclic-AMP-dependent protein kinase. Clone 4c (2681 bp) had a coding region identical to that of clone 22c but it included a putative intron of 959 bp. In clone 5c (1750 bp), the sequence upstream from amino acid 33 differed from that in clone 22c and coded for a unique N-terminal portion of 10 amino acids. Poly(A)-rich RNA from rat tissues was hybridized with cDNA probes corresponding to the unique N-terminal portions of clones 22c and 5c. Dot and Northern blots showed signals indicative of three distinct PFK-2/FBPase-2 mRNAs. There were a 6.8-kb mRNA typical of cardiac tissue, a 2.1-kb mRNA typical of liver, corresponding to clone 22c, and a 1.9-kb mRNA typical of skeletal muscle, corresponding to clone 5c. Primer extension analysis showed that clones 22c and 5c were nearly complete since their respective 5'-untranslated sequences were at most 96/97 bp and 44 bp shorter than the corresponding mRNAs. These data provide a molecular basis for the existence of PFK-2/FBPase-2 isozymes.     

Molecular forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expressed in rat skeletal muscle.

The rat cDNA for the muscle-type (M) isozyme of 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) contains two putative translation initiation sites. To determine whether the M isozyme expressed in rat skeletal muscle corresponds to the short (PFK2M-sf) or the long (PFK2M-lf) isoform, we have expressed them in Escherichia coli. A third construction was also expressed in which the second ATG codon was deleted (PFK2M-lf delta ATG) to ensure that initiation started at the first ATG. The properties of these recombinant proteins were compared with those of the PFK-2/FBPase-2 present in rat skeletal muscle and liver. The recombinant proteins displayed PFK-2 and FBPase-2 activities and the M(r) values of the subunits measured by SDS-polyacrylamide gel electrophoresis were compatible with the calculated ones. The purified recombinant lf form contained not only the expected lf band (54,500 M(r)) but also the sf band (52,000 M(r)), indicating that the expression system could synthesize the long and the short isoforms from the same mRNA. The kinetic properties of the recombinant sf form were not different from those of the rat muscle enzyme. By contrast, lf delta ATG PFK-2 displayed a higher Km for its substrates and a lower Vmax. Immunoblotting with an antibody directed against the long isoform revealed a 54,500 M(r) band both in the lf and the lf delta ATG recombinant, but no band in rat skeletal muscle extracts. In these extracts, one band of 52,000 and a minor one of 54,500 M(r) were detected by an anti PFK-2/FBPase-2 antibody. The 54,500 M(r) band was recognized by an antibody directed against the L isozyme, suggesting that a small amount of the latter is expressed in skeletal muscle. Thus, the M isozyme differs from the L isozyme by replacement of the first 32 amino acids of the L isozyme by an unrelated nonapeptide.     

Evolutionary analysis of fructose 2,6-bisphosphate metabolism

Fructose 2,6-bisphosphate is a potent metabolic regulator in eukaryotic organisms; it affects the activity of key enzymes of the glycolytic and gluconeogenic pathways. The enzymes responsible for its synthesis and hydrolysis, 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) are present in representatives of all major eukaryotic taxa. Results from a bioinformatics analysis of genome databases suggest that very early in evolution, in a common ancestor of all extant eukaryotes, distinct genes encoding PFK-2 and FBPase-2, or related enzymes with broader substrate specificity, fused resulting in a bifunctional enzyme both domains of which had, or later acquired, specificity for fructose 2,6-bisphosphate. Subsequently, in different phylogenetic lineages duplications of the gene of the bifunctional enzyme occurred, allowing the development of distinct isoenzymes for expression in different tissues, at specific developmental stages or under different nutritional conditions. Independently in different lineages of many unicellular eukaryotes one of the domains of the different PFK-2/FBPase-2 isoforms has undergone substitutions of critical catalytic residues, or deletions rendering some enzymes monofunctional. In a considerable number of other unicellular eukaryotes, mainly parasitic organisms, the enzyme seems to have been lost altogether. Besides the catalytic core, the PFK-2/FBPase-2 has often N- and C-terminal extensions which show little sequence conservation. The N-terminal extension in particular can vary considerably in length, and seems to have acquired motifs which, in a lineage-specific manner, may be responsible for regulation of catalytic activities, by phosphorylation or ligand binding, or for mediating protein-protein interactions.     

Overexpression of fructose 2,6-bisphosphatase decreases glycolysis and delays cell cycle progression

The ability to overexpress6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase(PFK-2)/(FBPase-2) or a truncated form of the enzyme with only thebisphosphatase domain allowed us to analyze the relative role of thekinase and the bisphosphatase activities in regulating fructose2,6-bisphosphate (Fru-2,6-P₂) concentration and toelucidate their differential metabolic impact in epithelial Mv1Lucells. The effect of overexpressing PFK-2/FBPase-2 resulted in a smallincrease in the kinase activity and in the activity ratio of thebifunctional enzyme, increasing Fru-2,6-P₂ levels, butthese changes had no major effects on cell metabolism. In contrast,expression of the bisphosphatase domain increased the bisphosphataseactivity, producing a significant decrease in Fru-2,6-P₂ concentration. The fall in the bisphosphorylated metabolite correlated with a decrease in lactate production and ATP concentration, as well asa delay in cell cycle. These results provide support for Fru-2,6-P₂ as a regulator of glycolytic flux and point outthe role of glycolysis in cell cycle progression.

     

光/暗对玉米叶片果糖-6-磷酸激酶-2和果糖-2,6-二磷酸酯酶活力的影响

比较了照光和黑暗条件下玉米叶片果糖—6—磷酸激酶—2(PFK-2)和果糖—2,6—二磷酸酯酶(FBPase-2)的活力变化。当玉米植株从暗中转入光下后,其叶片PFK—2的活力随光照时间的延长而逐渐降低,而FBPase-2活力变化不明显;从光下转入暗后叶片PFK-2活力明显上升,FBPase-2活力仍无明显变化;其PFK-2/FBPase-2比值在光处理时下降,暗处理时上升。同时叶片中果糖—2,6—二磷酸的含量与PFK-2/FBPase-2活力比值的变化趋势一致。连续光照 20 h,PFK-2活力持续下降,表明PFK-2的光钝化现象与玉米植株的昼夜节律变化无关。     

A Fundamental Trade-off in Covalent Switching and Its Circumvention by Enzyme Bifunctionality in Glucose Homeostasis

Covalent modification provides a mechanism for modulating molecular state and regulating physiology. A cycle of competing enzymes that add and remove a single modification can act as a molecular switch between “on” and “off” and has been widely studied as a core motif in systems biology. Here, we exploit the recently developed “linear framework” for time scale separation to determine the general principles of such switches. These methods are not limited to Michaelis-Menten assumptions, and our conclusions hold for enzymes whose mechanisms may be arbitrarily complicated. We show that switching efficiency improves with increasing irreversibility of the enzymes and that the on/off transition occurs when the ratio of enzyme levels reaches a value that depends only on the rate constants. Fluctuations in enzyme levels, which habitually occur due to cellular heterogeneity, can cause flipping back and forth between on and off, leading to incoherent mosaic behavior in tissues, that worsens as switching becomes sharper. This trade-off can be circumvented if enzyme levels are correlated. In particular, if the competing catalytic domains are on the same protein but do not influence each other, the resulting bifunctional enzyme can switch sharply while remaining coherent. In the mammalian liver, the switch between glycolysis and gluconeogenesis is regulated by the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). We suggest that bifunctionality of PFK-2/FBPase-2 complements the metabolic zonation of the liver by ensuring coherent switching in response to insulin and glucagon.     

Evidence for new phosphorylation sites for protein kinase C and cyclic AMP-dependent protein kinase in bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was phosphorylated by incubation with [gamma-32P]MgATP and cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). After digestion with chymotrypsin, the phosphorylation sites for the two protein kinases were identified by peptide mapping, and microsequencing. Evidence for new phosphorylation sites for PKA (Ser-483) and PKC (Ser-84 and Ser-466) was obtained.     

Regulation of fish glycolysis—gluconeogenesis: role of fructose 2,6 P2 and PFK-2

• 1.1. Fructose 2,6 P₂ and PFK-2 have a key role in the regulation of glycolysis-gluconeogenesis in fish
• 2.2. PFK-1 and FBPase-1, as in mammals, are the target enzymes for fructose 2,6 P₂, this in turn may be controlled by glucagon and insulin.
• 3.3. PFK-2 from fish liver seems to be a bifunctional enzyme regulated by phosphorylation/dephosphorylation.
• 4.4. Starvation, refeeding, diet composition and anoxia studies provide a general view of the fructose 2,6 P₂ fish system from which the differences between fish and mammal glycolysis-gluconeogenesis may be ascertained.

     

Cloning and expression of novel isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine heart

Distinct 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) cDNAs were cloned from bovine heart, showing that PFK-2/FBPase-2 gene B, which contains 16 exons, codes for at least five mRNAs. Three of them (B1, B2, B4) could encode the 58,000-M_r isozyme. In B2 mRNA, exon 15 encodes four more residues than in Bl. In B4 mRNA, exon 15 encodes six more residues than in B1, butexon 16 (20 residues) is missing. B3 mRNA corresponds to the 54,000-M_r isozyme. It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region. B5 mRNA encodes a truncated form. When expressed in E. coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK-2 activity.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog skeletal muscle: purification,kinetics and immunological properties

Fructose 2,6-bisphosphate is the most potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. This study was prompted by the finding that the content of fructose 2,6-bisphosphate in frog skeletal muscle was dramatically increased at the initiation of exercise and was closely correlated with the glycolytic flux during exercise. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, the enzyme system catalyzing the synthesis and degradation of fructose 2,6-bisphosphate, was purified from frog (Rana esculenta) skeletal muscle and its properties were compared with those of the rat muscle type enzyme expressed in Escherichia coli using recombinant DNA techniques. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was purified 5600-fold. 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities could not be separated, indicating that the frog muscle enzyme is bifunctional. The enzyme preparation from frog muscle showed two bands on sodium dodecylsulphate polyacrylamide gel electrophoresis. The minor band had a relative molecular mass of 55800 and was identified as a liver (L-type) isoenzyme. It was recognized by an antiserum raised against a specific amino-terminal amino acid sequence of the L-type isoenzyme and was phosphorylated by the cyclic AMP-dependent protein kinase. The major band in the preparations from frog muscle (relative molecular mass = 53900) was slightly larger than the recombinant rat muscle (M-type) isoenzyme (relative molecular mass = 53300). The pH profiles of the frog muscle enzyme were similar to those of the rat M-type isoenzyme, 6-phosphofructo-2-kinase activity was optimal at pH 9.3, whereas fructose-2,6-bisphosphatase activity was optimal at pH 5.5. However, the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle differed from other M-type isoenzymes in that, at physiological pH, the maximum activity of 6-phosphofructo-2-kinase exceeded that of fructose-2,6-bisphosphatase, the activity ratio being 1.7 (at pH 7.2) compared to 0.2 in the rat M-type isoenzyme. 6-Phosphofructo-2-kinase activity from the frog and rat muscle enzymes was strongly inhibited by citrate and by phosphoenolpyruvate whereas glycerol 3-phosphate had no effect. Fructose-2,6-bisphosphatase activity from frog muscle was very sensitive to the non-competitive inhibitor fructose 6-phosphate (inhibitor concentration causing 50% decrease in activity = 2 mol · l^-1). The inhibition was counteracted by inorganic phosphate and, particularly, by glycerol 3-phosphate. In the presence of inorganic phosphate and glycerol 3-phosphate the frog muscle fructose-2,6-bisphosphatase was much more sensitive to fructose 6-phosphate inhibition than was the rat M-type fructose-2,6-bisphosphatase. No change in kinetics and no phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from frog muscle was observed after incubation with protein kinase C and a Ca²⁺/calmodulin-dependent protein kinase. The kinetics of frog muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, although they would favour an initial increase in fructose 2,6-bisphosphate in exercising frog muscle, cannot fully account for the changes in fructose 2,6-bisphosphate observed in muscle of exercising frog. Regulatory mechanisms not yet studied must be involved in working frog muscle in vivo.Abbreviations BSA bovine serum albumin - Ca/CAMK Ca²⁺/calmodulin-dependent protein kinase (EC 2.7.1.37) - CL anti-l-type PFK-21 FBPase-2 antiserum - DTT dithiothreitol - EP phosphorylated enzyme intermediate - FBPase-2 fructose-2,6-bisphosphatase (EC 3.1.3.46) - F2,6P₂ fructose 2,6-bisphosphate - I_0,5 inhibitor concentration required to decrease enzyme activity by 50% - MCL-2 anti-PFK-2/FBPase-2 antiserum - M_r relative molecular mass - PEG polyethylene glycol - PFK-1 6-phosphofructo-1-kinase (EC 2.7.1.11) - PKF-2 6-phosphofructo-2-kinase (EC 2.7.1.105) - PKA protein kinase A = cyclic AMP-dependent protein kinase (EC 2.7.1.37) - PKC protein kinase C (EC 2.7.1.37) - SDS sodium dodecylsulphate - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - U unit of enzyme activity     

Does rat liver 6-phosphofructo-1-kinase exhibit 'half-of-the-sites-phosphorylation'?

6-Phosphofructo-1-kinase (PFK-1) from a variety of species and organs can undergo phosphorylation by cAMP-dependent protein kinase. In most studies the stoichiometry of the phosphorylation reaction was far below the expected minimum value of 4 mol phosphate/mol PFK-1 tetramer. The present study with rat liver PFK-1 and purified catalytic subunit of cAMP-dependent protein kinase was undertaken in order to find the maximum phosphorylation stoichiometry under well-defined conditions. Irrespective of whether PFK-1 had been first treated with purified protein phosphatase 2C or not, no more than 1.66 +/- 0.22 mol phosphate/mol PFK-1 tetramer was incorporated, the highest single value being 2 mol phosphate/PFK-1 tetramer. This stoichiometry was found to be independent from the method of protein evaluation (gel dye-binding assay or amino acid analysis) and from the concentration of PFK-1 in the phosphorylation system (15.6 nM-0.53 microM). The stoichiometry was not affected by the presence of allosteric ligands, fructose-1,6-bisphosphatase or the PFK-1-inactivating protein. The possibility could be excluded that partial proteolysis was responsible for the incomplete phosphorylation. Two-dimensional polyacrylamide gel electrophoresis gave no indication of the existence of two different subunits in rat liver PFK-1. Possible reasons why rat liver PFK-1 undergoes 'half-of-the-sites' phosphorylation are discussed.