Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several previously untested proteins promote the reversible inactivation of rabbit skeletal muscle phosphofructokinase. Grouped in decreasing order of effectiveness, they include the following: skeletal muscle troponin C greater than troponin, the two smooth muscle myosin light chains, alpha-actinin, and S-100 much greater than parvalbumin and soybean trypsin inhibitor. The efficiency of troponin C in this process may even exceed that previously reported for calmodulin. Sequences near calcium binding site III are apparently involved in the troponin C-phosphofructokinase interaction. Troponin C and calmodulin exert calcium-dependent effects on the physical and chemical properties of muscle phosphofructokinase. When calcium is present, comigration with either protein allows the enzyme to enter the stacking gel during urea-polyacrylamide gel electrophoresis. Both enhance the phosphorylation of phosphofructokinase catalyzed by the cAMP-dependent protein kinase, with phosphate incorporations approaching 2 mol of P/mol of protomer. Reaction occurs at Ser774 and at Ser376--a novel site whose phosphorylation is highly sensitive to troponin C and less so to calmodulin. Maximum phosphorylation has slight effect on the catalytic activity of the enzyme under standard assay conditions. The troponin C induced or calmodulin-induced phosphorylation of phosphofructokinase requires calcium and is strongly inhibited by either fructose 2,6-bisphosphate or fructose 1,6-bisphosphate. Inactivation occurs in the presence or absence of calcium, with generally higher concentrations of effectors required for protection in the latter case. Liver and yeast phosphofructokinases shows little activity loss in the presence of either calmodulin or troponin C. We have developed and tested a general mathematical model for the protein-induced inactivation of phosphofructokinase which may find application to other systems.
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PMID:Protein-induced inactivation and phosphorylation of rabbit muscle phosphofructokinase. 182 8

We have reported previously that phosphofructokinase from the liver fluke Fasciola hepatica is activated by phosphorylation with cAMP-dependent protein kinase, and that this event appears to be important in vivo for regulation of PFK (E. S. Kamemoto, L. Lan, and T. E. Mansour (1989) Arch. Biochem. Biophys. 271, 553-559). Here, we report the amino acid sequence of the single tryptic phosphopeptide generated after phosphorylation of the purified enzyme with cAMP-dependent protein kinase and [gamma 32P]ATP. Through a combination of Edman microsequence analysis, fast atom bombardment mass spectroscopy, and phosphoamino acid analysis, the sequence of the phosphorylated peptide was determined to be: R-S-T(P)-M-M-I-P-G-M-E-G-K. This sequence is not homologous to any previously determined phosphofructokinase phosphopeptides. Regulatory differences between the mammalian and parasite enzymes are discussed with particular emphasis on regulation by protein phosphorylation.
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PMID:Phosphofructokinase from Fasciola hepatica: sequence of the cAMP-dependent protein kinase phosphorylation site. 183 41

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.
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PMID:The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase. 200 78

The level of phosphorylation and activation of phosphofructokinase by serotonin (5-hydroxytryptamine) was studied in intact liver flukes Fasciola hepatica. The enzyme was immunoprecipitated with antiserum prepared against pure enzyme from the liver flukes. The resuspended immunoprecipitated enzyme retained most of its original activity and its kinetic properties. The level of phosphorylation was determined by a "back phosphorylation" technique. According to this technique, the immunoprecipitated phosphofructokinase was phosphorylated with the catalytic subunit of pure cAMP-dependent protein kinase. Incubation of intact liver flukes with serotonin caused an increase in the level of enzyme phosphorylation which was concomitant with an increase in enzyme activity. The level of phosphorylation was increased by 0.08 mol per protomer as a result of maximal activation by serotonin. It is proposed that phosphorylation plays, at least in part, a functional role in the regulation of phosphofructokinase from the liver fluke F. hepatica under in vivo conditions.
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PMID:In vivo regulation of phosphorylation level and activity of phosphofructokinase by serotonin in Fasciola hepatica. 254

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.
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PMID:Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle. 282 70

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.
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PMID:Phosphofructokinase in the liver fluke Fasciola hepatica. Purification and kinetic changes by phosphorylation. 293 41

The consistent application of phosphatase inhibitors and a novel final purification step using a connected series of DE-51, DE-52, and DE-53 anion-exchange chromatography columns facilitate the preparation of electrophoretically homogeneous subpopulations of rabbit muscle phosphofructokinase which differ in their catalytic properties and endogenous covalent phosphate content. A band of "high"-phosphate enzyme (fraction II) flanked by regions of "low"-phosphate enzyme (fractions I and III) is an unusual feature of the final purification profile. Fractions I (containing in this case 0.42 mol of P/82 000 g of enzyme) and II (containing 1.26 mol of P/82 000 g of enzyme) exhibit the most pronounced functional differences of the fractions. Following our original report [Liou, R.-S., & Anderson, S. R. (1980) Biochemistry 19, 2684], both are activated by the addition of rabbit skeletal muscle F-actin. Under the assay conditions, half-maximal stimulation of phosphofructokinase activity occurs at 15.4 nM actin (in terms of monomer) for fraction I and 9.7 nM for fraction II. The low-phosphate enzyme is synergistically activated in the presence of 0.12 microM actin plus 3.0 microM fructose 2,6-bisphosphate, with a marked increase in Vmax, while the high-phosphate enzyme is not. Neither fraction is activated appreciably by the addition of G-actin or the chymotrypsin-resistant actin "core". The covalently cross-linked trimer of actin stimulates the activity of both the low- and high-phosphate enzyme fractions. However, the previously mentioned synergistic activation characteristic of fraction I fails to occur in solutions containing the trimer plus fructose 2,6-bisphosphate. Phosphorylation of fraction I in an in vitro reaction catalyzed by the cAMP-dependent protein kinase causes its properties to become more like those of fraction II. The total amount of covalent phosphate present after in vitro phosphorylation approaches 2 mol of P/82 000 g of enzyme for both fractions.
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PMID:Factors affecting the activation of rabbit muscle phosphofructokinase by actin. 293 27

Calmodulin has been shown to interact with high affinity with muscle phosphofructokinase (Mayr, G. W. (1984) Eur. J. Biochem. 143, 513-520, 521-529). In this study, direct binding measurements indicated that each of the two subunits of dimeric phosphofructokinase bound two calmodulins with Kd values of about 3 nM and 1 microM, respectively, in a strictly Ca2+-dependent way. To get more detailed information about this interaction, calmodulin-binding fragments were isolated from a CNBr digest of phosphofructokinase using affinity chromatography on calmodulin-agarose. Two fragments, M11 (Mr 3080) and M22 (Mr 8060), formed a 1:1 stoichiometric complex with Ca2+-calmodulin. The amino acid sequences of these fragments were determined, and their positions in the three-dimensional structure-model of phosphofructokinase are proposed. Fragment M11, which binds to calmodulin with the higher affinity (Kd 11.4 nM), is located in a region of the subunit where two dimers have been proposed to make contacts if associating to active tetrameric enzyme. A stabilization of the dimeric form of the enzyme by binding of calmodulin supports this location of M11. The weaker binding fragment M22 (Kd 198 nM) corresponds to the C-terminal part of the polypeptide and contains the site which is phosphorylated by cAMP-dependent protein kinase. Both fragments have structural properties in common with the isolated calmodulin-binding domains of myosin light chain kinase: two cationic segments rich in hydrophobic residues, one constantly possessing a tryptophan, and the other exhibiting an amino acid sequence resembling sites phosphorylated by cAMP-dependent protein kinase.
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PMID:Characterization of the calmodulin-binding sites of muscle phosphofructokinase and comparison with known calmodulin-binding domains. 295 60

Phosphofructokinase from rat heart perfused with epinephrine was purified to homogeneity and various allosteric properties were determined under conditions which approximate physiological concentrations of the substrates, effectors, and pH. The molecular weights of the protomer of the enzyme isolated from the hormone-stimulated and the control hearts are both approximately 83,000. The epinephrine-stimulated and the control enzymes contain 1.1 and 0.66 mol of phosphate/mol of protomer, respectively. Both enzymes can be fully phosphorylated by cAMP-dependent protein kinase indicating that the phosphorylation site is new and distinct from the known phosphorylation site of skeletal muscle phosphofructokinase. Pure phosphofructokinase isolated from the epinephrine-stimulated heart is significantly less sensitive to inhibition by ATP and citrate, and the K0.5 values for Fru-6-P (0.18 mM) and Fru-2,6-P2 (3 microM) are one-half those for the enzyme from control hearts. In the presence of in vivo concentrations of ATP, citrate, and Fru-6-P at pH 7.1, both enzymes are inactive in the absence of Fru-2,6-P2. Moreover, the K0.5 values for Fru-2,6-P2 of the hormone-stimulated and untreated enzymes are 3 and 6 microM, respectively. These differences in the allosteric properties of phosphofructokinases from the hormone-treated and the control hearts disappear when the enzymes are dephosphorylated by alkaline phosphatase. Determination of the glycolytic intermediates showed a 2-fold increase in Fru-6-P, Fru-2,6-P2, and AMP and 13-fold increase in Fru-1,6-P2. Partially purified Fru-6-P,2-kinase from epinephrine-stimulated and control hearts show KFru-6-P0.5 = 4 and 15 microM, respectively. These results indicate that rat heart phosphofructokinase in vivo requires Fru-2,6-P2 for its activity. Epinephrine stimulates phosphorylation of phosphofructokinase which results in a more active form. The hormone also increases Fru-2,6-P2 which appears to be the result of an activation of Fru-6-P,2-kinase by a covalent modification.
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PMID:Regulation of phosphofructokinase in perfused rat heart. Requirement for fructose 2,6-bisphosphate and a covalent modification. 316 Jul 3

Low phosphate and high phosphate forms of phosphofructokinase (Furuya, E., and Uyeda, K. (1980) J. Biol. Chem. 255, 11656-11659) from rat liver were purified to homogeneity and various properties were compared. The specific activities of these enzymes and their electrophoretic mobilities on polyacrylamide in sodium dodecyl sulfate are the same. A limited tryptic digestion yields products with no change in the enzyme activity but with a reduction in the molecular weight of about 2000. Both low and high phosphate enzymes can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, and approximately twice as much [32P]phosphate is incorporated into the low phosphate than the high phosphate enzyme. A comparison of their allosteric kinetic properties reveal that the high phosphate enzyme is much more sensitive to inhibition by ATP and citrate and shows a higher K0.5 for fructose 6-phosphate than the low phosphate enzyme, and the difference in the K0.5 values becomes greater at lower pH values. Furthermore, the high phosphate phosphofructokinase is less sensitive to activation by AMP and fructose 2,6-bisphosphate. Moreover, when the low phosphate enzyme is phosphorylated by protein kinase, the resulting phosphorylated enzyme exhibits a higher K0.5 for fructose 2,6-bisphosphate than does the untreated enzyme. These results demonstrate that the phosphorylation affects the allosteric kinetic properties of the enzyme and results in a less active form of phosphofructokinase.
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PMID:Differences in the allosteric properties of pure low and high phosphate forms of phosphofructokinase from rat liver. 622 34


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