Regulatory Properties of Pyruvate Kinase from Liver of the Summer-Active Arctic Ground Squirrel

1974 ◽  
Vol 52 (6) ◽  
pp. 547-559 ◽  
Author(s):  
Hans W. Behrisch ◽  
Craig E. Johnson
Keyword(s):  
Pyruvate Kinase ◽  
Ground Squirrel ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Effect Of Temperature ◽  
Arctic Ground Squirrel ◽  
Physiological Importance ◽  
Atp Inhibition ◽  
High Concentrations ◽  
Regulatory Properties ◽  
Regulation Of Enzyme Activity

The properties of liver pyruvate kinase (PyK) from summer-active Arctic ground squirrel (Citellus undulatus) were examined over the physiological temperature range of the animal. One form of the enzyme with a pI (isoelectric point) value of 5.3 was observed and exhibited kinetics similar to those of L-type PyK. This form has a molecular weight of 243 000, similar to that of M-type PyK. Enzyme–phosphoenolpyruvate (PEP) affinity falls with decreasing temperature while affinity for ADP increases under these conditions. The effect of temperature upon extent of negative cooperativity of PEP binding and its possible physiological importance are briefly discussed. Fructose-1,6-phosphate (FDP) increases the affinity of PyK for PEP but has no effect upon enzyme-ADP interaction. However, Vmax in the presence of saturating concentrations of both substrates is increased two to threefold by FDP, the allosteric activator. Ground squirrel PyK is inhibited by ATP. Inhibition by ATP of PyK activity is diminished by decreasing the temperature or by increasing the concentrations of substrate. FDP reverses this inhibition but the degree of reversal is sensitive to substrate concentration and temperature. Inhibition by ATP is competitive for the PEP site and appears to be of the "mixed-competitive" variety when carried out at varying concentrations of ADP. Inhibition by alanine is noncompetitive for both substrate sites. As a result of the very high concentrations of amino acids needed to obtain appreciable inhibition of live PyK, this does not appear to be a physiological means of regulation of enzyme activity. The effect of intermediates of the glycolytic, pentose shunt, and Krebs acid cycle pathways upon PyK activity is briefly discussed. Inosine nucleotides, involved in the reversal of the PyK reaction by phosphoenolpyruvate carboxykinase, have a strong modulating effect upon PyK activity. These data suggest formal mechanisms for the regulation of liver PyK in the ground squirrel.

Regulation of enzyme activity in the hibernator. A kinetic and spectroscopic study of muscle pyruvate kinase from the Arctic ground squirrel

1981 ◽  
Vol 59 (9) ◽  
pp. 762-769 ◽  
Author(s):  
George A. Morse ◽  
Hans W. Behrisch
Keyword(s):  
Pyruvate Kinase ◽  
Ground Squirrel ◽  
Arrhenius Plot ◽  
Gel Filtration ◽  
The Arctic ◽  
Subunit Interaction ◽  
Arctic Ground Squirrel ◽  
Derivative Spectroscopy ◽  
Muscle Pyruvate Kinase ◽  
Regulation Of Enzyme Activity

Pyruvate kinase skeletal muscle of the Arctic ground squirrel was purified to homogeneity. The purified enzyme variants from the summer-active and winter hibernating squirrel appear to be identical with a near-neutral pI of 6.9 and a molecular weight of 234 000 as determined by gel filtration chromatography on Bio-Gel A-0.5m. Evidence for subunit interaction during inhibition by L-phenylalanine is demonstrated with ultraviolet derivative spectroscopy. A model for this interaction and its importance for a regulatory role are discussed. The absence of a temperature break in the Arrhenius plot for the pyruvate kinase reaction, the kinetic and physical data, and the near-neutral pI, suggest an amino acid composition that conserves the overall geometry and resultant kinetic behavior which render regulation of the enzyme insensitive to temperature.

Phosphoenolpyruvate Carboxykinase in C4 Plants: Its Role and Regulation

1997 ◽  
Vol 24 (4) ◽  
pp. 459 ◽  
Author(s):  
Robert P. Walker ◽  
Richard M. Acheson ◽  
László I. Técsi ◽  
Richard C. Leegood
Keyword(s):  
Molecular Mass ◽  
Malic Enzyme ◽  
Phosphoenolpyruvate Carboxykinase ◽  
C4 Plants ◽  
Panicum Maximum ◽  
C4 Plant ◽  
Cam Plants ◽  
Crude Extracts ◽  
High Concentrations ◽  
Regulatory Properties

Some of the recent findings which revise our view of the role and regulation of phosphoenolpyruvate carboxykinase (PEPCK) in C4 plants are discussed. Evidence is presented that PEPCK is present at appreciable activities in the bundle-sheath of some NADP-malic enzyme-type C4 plants, such as maize, but it was not detectable in NAD-malic enzyme-type C4 plants. PEPCK is rapidly inactivated in crude extracts of leaves of the C4 plant, Panicum maximum. This inactivation could be prevented by high concentrations of dithiothreitol or by the inclusion of ADP or ATP, suggesting the involvement of thiols at the active site. PEPCK is also subject to rapid proteolysis in crude extracts of a range of C4 plants, resulting in cleavage to a smaller (62 kDa) form. This can be reduced by extraction at high pH and by the inclusion of SDS, but it means that intact PEPCK has never been purified from a C4 plant. The molecular mass of PEPCK varies considerably in C4 plants, unlike C3 and CAM plants in which it is usually 74 kDa. PEPCK is phosphorylated during darkness (and reversed by light) in some C4 plants with PEPCK of a larger molecular mass, such as Panicum maximum (71 kDa), but it was not phosphorylated in the PEPCK-type C4 plant, Sporobolus pyramidalis (69 kDa). The known regulatory properties of PEPCK are discussed in relation to its role in C4 photosynthesis, in particular its sensitivity to regulation by adenylates and by Mn2+.

Temperature and the Regulation of Enzyme Activity in the Hibernator. Isoenzymes of Liver Pyruvate Kinase from the Hibernating and Non-hibernating Arctic Ground Squirrel

1974 ◽  
Vol 52 (10) ◽  
pp. 894-902 ◽  
Author(s):  
Hans W. Behrisch
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Wide Temperature Range ◽  
Ground Squirrel ◽  
Energy Charge ◽  
High Energy ◽  
Ground Squirrels ◽  
The Arctic ◽  
Arctic Ground Squirrel ◽  
Temperature Range

Liver of the hibernating (H) Arctic ground squirrel (Citellus undulatus) contains a single species of pyruvate kinase (PyK) that is distinct from the single isoenzyme of pyK observed in the non-hibernating (NH) ground squirrel, which has been previously described (Behrisch &Johnson (1974) Can. J. Biochem. 52, 547–559). The H-PyK has a pI value of 5.7 and a molecular weight of 241 000 – 243 000. Affinity of the H-PyK for the substrates phosphoenolpyruvate (PEP) and ADP is not affected by changing temperature. It is argued that this stability of the apparent Km's for substrate over a wide temperature range permits the hibernator to take advantage of the Q10 effect in maintaining a low rate of the PyK reaction. Similarly, affinity of H-PyK for the allosteric activator fructose-1,6-phosphate (FDP) and the inhibitor ATP is also conspicuously independent of temperature, suggesting a fine stoichiometry in the relative concentrations of the regulatory ligands in control of H-PyK over a wide temperature range. Further, affinity of H-PyK for the inhibitor ATP is about three- to fourfold lower than that of the NH-PyK, a condition that would favor the maintenance of a high energy charge in the hibernating liver cell. ATP apparently inhibits PyK by causing a dissociation of the enzyme molecule into two "halves" of about 110 000 molecular weight each. This dissociation is offset and reversed by FDP. Removal of the ATP by dialysis does not of itself result in a reassociation of the PyK "halves"; FDP and/or the substrates are required for the two subunits of PyK to reassociate. As the apparent Ki of H-PyK for ATP is higher than that of NH-PyK, substantially higher concentrations of ATP are required to effect the dissociation of H-PyK. Similarly, elevated concentrations of FDP are required to offset the ATP-caused dissociation of the H-PyK.Hibernating Arctic ground squirrels that are preparing to emerge finally from the hibernating state already possess substantial activities of the NH-PyK isoenzyme. This suggests that the animal "anticipates" its transition from one metabolic state from another. On the basis of these data a formal mechanism is proposed for the regulation of liver PyK in the Arctic ground squirrel in both the non-hibernating and hibernating states.

scholarly journals Structural and regulatory properties of pyruvate kinase from Pseudomonas citronellolis.

1979 ◽  
Vol 254 (17) ◽  
pp. 8434-8441 ◽  
Author(s):  
D.T. Chuang ◽  
M.F. Utter
Keyword(s):  
Pyruvate Kinase ◽  
Regulatory Properties

scholarly journals Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates

1978 ◽  
Vol 174 (3) ◽  
pp. 979-987 ◽  
Author(s):  
Victor A. Zammit ◽  
Eric A. Newsholme
Keyword(s):  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Marine Invertebrates ◽  
Adductor Muscle ◽  
Sea Anemone ◽  
Anaerobic Conditions ◽  
Fructose Bisphosphate ◽  
Low Concentrations ◽  
Optimum Ph ◽  
Hill Coefficients

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13μm produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the Km values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn2+ in addition to Mg2+ for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.

scholarly journals Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor oil seeds

1991 ◽  
Vol 279 (2) ◽  
pp. 495-501 ◽  
Author(s):  
F E Podestá ◽  
W C Plaxton
Keyword(s):  
Pyruvate Kinase ◽  
Castor Oil ◽  
Mixed Type ◽  
Competitive Inhibition ◽  
Ricinus Communis ◽  
Cytosolic Ph ◽  
Oil Seeds ◽  
Saturation Kinetics ◽  
Regulatory Properties

The kinetic and regulatory properties of cytosolic pyruvate kinase (PKc) isolated from endosperm of germinating castor oil seeds (Ricinus communis L.) have been studied. Optimal efficiency in substrate utilization (in terms of Vmax/Km for phosphoenolpyruvate or ADP) occurred between pH 6.7 and 7.4. Enzyme activity was absolutely dependent on the presence of a bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Mg2+ binding showed positive and negative co-operativity at pH 6.5 (h = 1.6) and pH 7.2 (h = 0.69) respectively. Hyperbolic saturation kinetics were observed with phosphoenolpyruvate (PEP) and K+, whereas ADP acted as a mixed-type inhibitor over 1 mM. Glycerol (10%, v/v) increased the S0.5(ADP) 2.3-fold and altered the pattern of nucleotide binding from hyperbolic (h = 1.0) to sigmoidal (h = 1.79) without modifying PEP saturation kinetics. No activators were identified. ATP, AMP, isocitrate, 2-oxoglutarate, malate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-phosphoglycerate, glycerol 3-phosphate and phosphoglycolate were the most effective inhibitors. These metabolites yielded additive inhibition when tested in pairs. ATP and 3-phosphoglycerate were mixed-type inhibitors with respect to PEP, whereas competitive inhibition was observed for other inhibitors. Inhibition by malate, 2-oxoglutarate, phosphorylated triose sugars or phosphoglycolate was far more pronounced at pH 7.2 than at pH 6.5. Although 32P-labelling studies revealed that extensive phosphorylation in vivo of soluble endosperm proteins occurred between days 3 and 5 of seed germination, no alteration in the 32P-labelling pattern of 5-day-germinated endosperm was observed after 30 min of anaerobiosis. Moreover, no evidence was obtained that PKc was a phosphoprotein in aerobic or anoxic endosperms. It is proposed that endosperm PKc activity of germinating castor seeds is enhanced after anaerobiosis through concerted decreases in ATP levels, cytosolic pH and concentrations of several key inhibitors.

Pyruvate kinase from the liver and kidney of Arapaima gigas

1978 ◽  
Vol 56 (4) ◽  
pp. 852-859 ◽  
Author(s):  
H. Guderley ◽  
J. H. A. Fields ◽  
J. M. Cardenas ◽  
P. W. Hochachka
Keyword(s):  
Pyruvate Kinase ◽  
Arapaima Gigas ◽  
Liver And Kidney ◽  
Low Levels ◽  
Pyruvate Kinases ◽  
Regulatory Properties ◽  
Partial Reversal

Pyruvate kinases from the kidney and liver of the osteoglossid Arapaima gigas were partially purified and characterized kinetically. The two enzymes have different elect rophoretic mobilities at pH 7.0, and while they share some qualitative similarities they show quantitative differences in their catalytic and regulatory properties. Both enzymes are activated by fructose 1.6-bisphosphate and inhibited by low levels of alanine and MgATP. The liver isozyme shows hyperbolic phosphoenolpyruvate binding, with a K1 for alanine inhibition of 0.7 mM and a K1 for MgATP inhibition of 1.0 mM. In contrast, the kidney isozyme shows cooperative phosphoenolpyruvate binding, which is accentuated at low levels of ADP. MgATP inhibition does not increase the cooperativity and shows an apparent K1 of 1.68 mM. The inhibition of alanine leads to considerable increases in the cooperativity and is effective at 1 mM and lower levels. Fructose 1.6-bisphosphate completely reverses the inhibition by alanine for both isozymes, while only leading to a partial reversal of the MgATP inhibition. These regulatory properties of both the kidney and the liver isozymes suit them for function in tissues which undergo both glycolysis and gluconeogenesis.

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