Phosphofructokinase activities within the order Spirochaetales and the characterisation of the pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila

The subtype of phosphofructokinase activity, either ATP-, ADP- or pyrophosphate-dependent, present in members of three genera from the Spirochaetales was investigated. The individual species/strains examined included Spirochaeta alkalica, S. asiatica, S. halophila, S. isovalerica, S. litoralis, S. zuelzerae, S. thermophila, two thermophilic spirochetes, Treponema bryantii, T. denticola, paragraph signT. pectinovorum, Leptospira biflexa and L. interrogans. All of the Spirochaeta strains, regardless of their phenotype, possessed primarily a pyrophosphate-dependent phosphofructokinase. In contrast, T. bryantii, T. denticola and L. biflexa had predominantly an ATP-dependent activity, whereas no activity was detected in T. pectinovorum or paragraph signL. interrogans. The results suggest that pyrophosphate-dependent phosphofructokinase activity may be a reliable phenotypic marker for the genus Spirochaeta and that there are potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales. The pyrophosphate-dependent phosphofructokinase from S. thermophila strain RI 19.B1 was purified (303-fold) to homogeneity and biochemically characterised. The S. thermophila enzyme displayed hyperbolic kinetics with respect to both the forward and reverse cosubstrates and was not significantly affected by traditional activators or inhibitors of phosphofructokinase. The biochemical characterisation represents the first spirochete phosphofructokinase to be described.     

Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila.

The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity. The enzyme purified from E. coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M. thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E. coli or M. thermophila. Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures. No esterase activity was detected with p-nitrophenylacetate as the substrate. Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit. As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer. Western blot (immunoblot) analysis of cell extract proteins from M. thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.     

A thermostable acid alpha-glucosidase from Tetrahymena thermophila: purification and characterization

An acid alpha-glucosidase (EC 3.2.1.20) was purified to homogeneity from the culture medium of Tetrahymena thermophila CU 399. Its general molecular, catalytic and immunological properties were compared to those of the T. pyriformis W enzyme. The enzyme from T. thermophila was a 105-kD monomer and the N-terminus (25 amino acid residues) displayed some homology with that of T. pyriformis enzyme. The purified enzyme was most active at 56 degrees C and showed resistance to thermal inactivation. The acid alpha-glucosidase appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. The Km values determined with p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose and glycogen were 0.7 mM, 2.5 mM, 28.5 mM and 18.5 mg/ml, respectively. The enzyme was antigenically distinct from T. pyriformis acid alpha-glucosidase.     

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

1. The distribution of phosphofructokinase isoenzymes have been compared among camel, rat and rabbit livers. 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. The ammonium sulphate precipitation curves of the camel and rabbit enzymes were monophasic, whereas the rat enzyme was biphasic. 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. Partially purified camel liver phosphofructokinase showed different regulatory properties from the rabbit and rat isoenzymes as the apparent Km values were 0.58, 0.45 and 0.82 mM respectively.     

A Thermostable Acid α-Glucosidase from Tetrahymena thermophila: Purification and Characterization

An acid α-glucosidase (EC 3.2.1.20) was purified to homogeneity from the culture medium of Tetrahymena thermophila CU 399. Its general molecular, catalytic and immunological properties were compared to those of the T. pyriformis W enzyme. The enzyme from T. thermophila was a 105-kD monomer and the N-terminus (25 amino acid residues) displayed some homology with that of T. pyriformis enzyme. The purified enzyme was most active at 56° C and showed resistance to thermal inactivation. The acid α-glucosidase appears to have α-1,6-glucosidase as well as α-1,4-glucosidase activity. The Km values determined with p-nitrophenyl-α-glucopyranoside, maltose, isomaltose and glycogen were 0.7 mM, 2.5 mM, 28.5 mM and 18.5 mg/ml, respectively. The enzyme was antigenically distinct from T. pyriformis acid α-glucosidase.     

Purification and partial characterization of glyceraldehyde-3-phosphate dehydrogenase from the ciliate Tetrahymena thermophila

In the present study, we purified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is involved in cellular energy production and has important housekeeping functions, from the ciliate Tetrahymena thermophila using a three-step procedure. The enzyme was purified ~68 folds by ammonium sulfate precipitation, followed by two steps of column chromatography (DEAE-cellulose and Mono-S). The purified enzyme is a homotetramer with a molecular weight of ~120 kDa. Isoelectric focusing analysis showed the presence of only one basic GAPDH isoform with an isoelectric point of 8.8. Western blot analysis showed a single 32-kDa band corresponding to the enzyme subunit using a monospecific polyclonal antibody against the T. thermophila GAPDH. The maximum of enzyme activity occurred at pH 8.0 and at 30-35°C. The apparent K(m) values for both NAD(+) and D-glyceraldehyde-3-phosphate were 0.102 ± 0.012 and 0.360 ± 0.018 mM, respectively. The maximal velocity (V(max)) was 39.40 ± 2.95 U/mg. The T. thermophila GAPDH is inhibited by oxidative and nitrosative stress reagents.     

Purification, microsequencing and cloning of spinach ATP-dependent phosphofructokinase link sequence and function for the plant enzyme

Despite its importance in plant metabolism, no sequences of higher plant ATP-dependent phosphofructokinase (EC 2.7.1.11) are annotated in the databases. We have purified the enzyme from spinach leaves 309-fold to electrophoretic homogeneity. The purified enzyme was a homotetramer of approximately 52 kDa subunits with a specific activity of 600 mU x mg(-1) and a Km value for ATP of 81 microm. The purified enzyme was not activated by phosphate, but slightly inhibited instead, suggesting that it was the chloroplast isoform. The inclusion of adenosine 5'-(beta,gamma-imido)triphosphate was conducive to enzyme activity during the purification protocol. The sequences of eight tryptic peptides from the final protein preparation, which did not utilize pyrophosphate as a phosphoryl donor, were determined and an exactly corresponding cDNA was cloned. The sequence of enzymatically active spinach ATP-dependent phosphofructokinase suggests that a large family of genomics-derived higher plant sequences currently annotated in the databases as putative pyrophosphate-dependent phosphofructokinases according to sequence similarity is misannotated with respect to the cosubstrate.     

Purification and identification of intermediate catabolic products in the in vivo degradation of pig liver phosphofructokinase

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to homogeneity from pig livers. Polyclonal antibody against the enzyme was induced in a rabbit, and the IgG fraction was obtained by chromatography on a Protein A-Sepharose CL-4B column. The specific antibody was purified further by immunoaffinity chromatography on a phosphofructokinase-conjugated affinity column. Intermediate catabolic products of phosphofructokinase were extracted from fresh pig livers under conditions of inhibition of proteinases, concentrated by chromatography on an anti-phosphofructokinase IgG-conjugated affinity column, and purified by two-dimensional polyacrylamide gel electrophoresis. Their cross-reactivities to the purified phosphofructokinase were assessed by an immunoelectrotransfer blot method. The intact form of phosphofructokinase in pig liver was demonstrated as the major spot of 84 kDa on the blot. Polypeptides of 68, 64, 56, and 51 kDa showed apparent cross-reactivities to phosphofructokinase. The structural homology among them was confirmed by proteinase V8 digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility of artifacts in preparation was ruled out by an internal tracer method. Thus, it is concluded that the predominant isozyme of phosphofructokinase in pig liver (84 kDa) is in vivo degraded via intermediate catabolic products of 68, 64, 56, and 51 kDa.     

Activation of acetate by Methanosarcina thermophila. Purification and characterization of phosphotransacetylase

Phosphotransacetylase (EC 2.3.1.8) was purified 83-fold to a specific activity of 2.5 mmol of acetyl-CoA synthesized per min/mg of protein from Methanosarcina thermophila cultivated on acetate. This rate was 10-fold greater than the rate of acetyl phosphate synthesis. The native enzyme (Mr 42,000-52,000) was a monomer and was not integral to the membrane. Activity was optimum at pH 7.0, and 35-45 degrees C. The enzyme was stable to air and to temperatures up to 70 degrees C, but was inactivated at higher temperatures. Phosphate and sulfate partially protected against heat inactivation. Potassium or ammonium ion concentrations above 10 mM were required for maximum activity of the purified enzyme; the intracellular potassium concentration of M. thermophila approximated 175 mM. Sodium, phosphate, sulfate, and arsenate ions were inhibitory to enzyme activity. Western blots of cell extracts showed that phosphotransacetylase was synthesized in higher quantity in acetate-grown cells than in methanol-grown cells.     

Purification and partial characterization of different forms of phosphofructokinase in man.

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from human muscle, brain, heart and granulocytes has been purified using a two or three step purification procedure. The main step is Blue Dextran-Sepharose 4B chromatography with selective elution of phosphofructokinase by formation of the ternary complex ADP or ATP-fructose-6-P-enzyme. Muscle and heart contain only enzyme subunits with a molecular weight of 85,000. This type of subunit is predominnant in brain, where it co-exists with subunits of about 80,000 daltons. A single type of subunits is found in the granulocytes, with a molecular weight of 80,000. Anti-muscle phosphofructokinase antiserum reacts only with M-type enzyme. Anti-granulocyte enzyme antiserum, absorbed by pure brain phosphofructokinase, exhibits a narrow specificity against the so-called L-type enzyme. Anti-brain antiserum, absorbed by pure muscle phosphofructokinase and partly purified red cell enzyme, exhibits a narrow specificity against a phosphofructokinase form predominant in fibroblasts and present in brain (F-type).     

Phosphofructokinase in the liver fluke Fasciola hepatica. Purification and kinetic changes by phosphorylation

Phosphofructokinase from the liver fluke Fasciola hepatica was purified from extracts of the whole organisms. The molecular weight of the protomer as determined by sodium dodecyl sulfate-gel electrophoresis is 83,000. Phosphorylation of the liver fluke phosphofructokinase by the catalytic subunit of cAMP-dependent protein kinase occurred at a rate that was at least an order of magnitude greater than that observed with mammalian heart phosphofructokinase. The maximum level of phosphate incorporated was 0.22 mol P/mol of protomer. The kinetic properties of the enzyme were greatly altered as a result of phosphorylation. Compared to native enzyme, phosphorylated enzyme had a greater affinity for its substrate Fru-6-P and a decreased sensitivity to inhibition by ATP. These kinetic changes were similar to those of native enzyme in the presence of positive modifiers such as AMP. AMP also activated the phosphorylated enzyme. Activation of the phosphorylated enzyme by AMP was characterized by a further increase in affinity for Fru-6-P and a further decrease in sensitivity to ATP inhibition. Thus, the liver fluke phosphofructokinase can be modulated by covalent phosphorylation as well as noncovalent binding of different modifier ligands.     

Partial proteolysis of human L-type phosphofructokinase

L (liver) type phosphofructokinase subunits purified from human leukocytes are slightly lighter than L subunits from liver and red blood cells. A mild treatment of red blood cell L4 enzyme with subtilisin converts its subunits into forms of similar molecular weight to leukocyte enzyme. From a kinetical point of view, subtilisin-treated L4 phosphofructokinase and leukocyte enzymes are characterized by a decrease of the allosteric properties as compared to non-treated red cell L4 phosphofructokinase.     

ADP-dependent glucokinase/phosphofructokinase, a novel bifunctional enzyme from the hyperthermophilic archaeon Methanococcus jannaschii

A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.     

The purification, characterization and regulatory properties of liver phosphofructokinase in the Arabian one-humped camel (Camelus dromedarius)

1. Phosphofructokinase from camel liver was purified to homogeneity more than 3600-fold, and the yield of the preparation was 46%. 2.The sodium dodecyl sulphate-treated purified enzyme migrated as a single band in 10% polyacrylamide gel. 3. The enzyme is a tetramer, with a monomer Mr 90,000. 4. The regulatory properties of the purified enzyme from camel liver were studied at pH 7.0. 5. The enzyme displayed cooperativity with respect to fructose 6-phosphate and was inhibited by high concentrations of ATP. 6. The enzyme was also inhibited by citrate, phosphocreatine and 2,3-bisphosphoglycerate. 7. On the other hand, ADP, AMP, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate were all found to be strong activators for camel liver phosphofructokinase.     

Phosphofructokinase from Dirofilaria immitis. Stimulation of activity by phosphorylation with cyclic AMP-dependent protein kinase

Phosphofructokinase has been partially purified from the filariid helminth, Dirofilaria immitis, using ion exchange and affinity chromatography. The D. immitis phosphofructokinase cross-reacted with antibodies prepared against the phosphofructokinase from Ascaris suum. These antibodies had been bound to agarose beads. The enzyme was eluted from the immobilized antigen-antibody complex by denaturing agents, and the subunit molecular weight determined by sodium dodecyl sulfate gel electrophoresis was identical to that of the ascarid enzyme, 90,000. At pH 6.8, substrate saturation curves of the filarial phosphofructokinase with ATP revealed that the enzyme was inhibited by ATP. The fructose-6-P saturation curve was sigmoid at all ATP levels tested. Phosphorylation of the D. immitis phosphofructokinase by the catalytic subunit of beef heart cyclic AMP-dependent protein kinase resulted in incorporation of 0.8 mol of phosphate/mol of subunit and in a 3-4-fold increase in catalytic activity when measured at pH 6.8 at inhibitory levels of ATP. Additional kinetic studies revealed that the phosphorylated enzyme was less susceptible to ATP inhibition than was the nonphosphorylated form. It is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of carbohydrate metabolism in the filarial as well as the intestinal-dwelling nematodes.     

Properties of phosphofructokinase from rat liver and their relation to the control of glycolysis and gluconeogenesis

1. Phosphofructokinase from rat liver has been partially purified by ammonium sulphate precipitation so as to remove enzymes that interfere in one assay for phosphofructokinase. The properties of this enzyme were found to be similar to those of the same enzyme from other tissues (e.g. cardiac muscle, skeletal muscle and brain) that were previously investigated by other workers. 2. Low concentrations of ATP inhibited phosphofructokinase activity by decreasing the affinity of the enzyme for the other substrate, fructose 6-phosphate. Citrate, and other intermediates of the tricarboxylic acid cycle, also inhibited the activity of phosphofructokinase. 3. This inhibition was relieved by either AMP or fructose 1,6-diphosphate; however, higher concentrations of ATP decreased and finally removed the effect of these activators. 4. Ammonium sulphate protected the enzyme from inactivation, and increased the activity by relieving the inhibition due to ATP. The latter effect was similar to that of AMP. 5. Phosphofructokinase was found in the same cellular compartment as fructose 1,6-diphosphatase, namely the soluble cytoplasm. 6. The properties of phosphofructokinase and fructose 1,6-diphosphatase are compared and a theory is proposed that affords dual control of both enzymes in the liver. The relation of this to the control of glycolysis and gluconeogenesis is discussed.     

Enzyme concentration affects the allosteric behavior of yeast phosphofructokinase

The influence of enzyme concentration on the kinetic behavior of yeast phosphofructokinase has been examined. A marked decrease in the ATP inhibition was observed when the enzyme activity was studied in permeabilized cells (in situ) as well as when the kinetic study was carried out with the purified yeast enzyme at a concentration of 120 micrograms/ml as compared to a 100-fold diluted enzyme. A similar result was obtained by adding polyethylene glycol either to a cell free extract or to the diluted pure enzyme to increase the local protein concentration. However, enzyme concentration had no significant effect on the fructose-6-P saturation curve. These results provide evidence that the allosteric behavior of yeast phosphofructokinase is affected by enzyme concentration.     

Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.     

Glucose catabolism by Spirochaeta thermophila RI 19.B1.

Spirochaeta thermophila RI 19.B1 (DSM 6192) fermented glucose to lactate, acetate, CO2, and H2 with concomitant formation of cell material. The cell dry mass yield was 20.0 g/mol of glucose. From the fermentation balance data and knowledge of the fermentation pathway, a YATP of 9.22 g of dry mass per mol of ATP was calculated for pH-uncontrolled batch-culture growth on glucose in a mineral medium. Measurement of enzyme activities in glucose-grown cells revealed that glucose was taken up by a permease and then subjected to ATP-dependent phosphorylation by a hexokinase. Glucose-6-phosphate was further metabolized to pyruvate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase activity was PPi rather than ATP. This was also found for the type strain of S. thermophila, Z-1203 (DSM 6578). PPi was probably formed by pyrophosphoroclastic cleavage of ATP, with recovery of the resultant AMP by the activity of adenylate kinase. All other measured kinase activities utilized ATP as the phosphoryl donor. Pyruvate was further metabolized to acetyl coenzyme A with concomitant production of H2 and CO2 by pyruvate synthase. Lactate was also produced from pyruvate by a fructose-1,6-diphosphate-insensitive lactate dehydrogenase. Evidence was obtained for the transfer of reducing equivalents from the glycolytic pathway to hydrogenase to produce H2. No formate dehydrogenase or significant ethanol-producing enzyme activities were detected.     

Ribose 1,5-bisphosphate regulates rat kidney cortex phosphofructokinase

Phosphofructokinase (EC 2.7.1.11) is a major enzyme of the glycolytic pathway, catalyzing the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate. In this study, we demonstrated the effect of ribose 1,5-bisphosphate on phosphofructokinase purified from rat kidney cortex. Ribose 1,5-bisphosphate relieved the phosphofructokinase from ATP inhibition and increased the affinity for fructose 6-phosphate at nanomolar concentrations. These activating effects of ribose 1,5-bisphosphate were enhanced in the presence of AMP. Ribose 1,5-bisphosphate reduced the inhibition of the phosphofructokinase induced by citrate. These results suggest that ribose 1,5-bisphosphate is an activator of rat kidney cortex phosphofructokinase and synergistically regulates the enzyme activity with AMP.