scholarly journals Kinetic studies on the regulation of rabbit liver pyruvate kinase

1973 ◽  
Vol 131 (2) ◽  
pp. 287-301 ◽  
Author(s):  
M. G. Irving ◽  
J. F. Williams
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Rabbit Liver ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Low Concentrations ◽  
Ammonium Sulphate Fractionation ◽  
The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

scholarly journals Factors affecting the activity of pyruvate kinase of Acetobacter xylinum

1969 ◽  
Vol 112 (5) ◽  
pp. 631-636 ◽  
Author(s):  
Moshe Benziman
Keyword(s):  
Pyruvate Kinase ◽  
Tricarboxylic Acid Cycle ◽  
Relative Activity ◽  
Acetobacter Xylinum ◽  
Hill Coefficient ◽  
Factors Affecting ◽  
Phosphofructokinase Activity ◽  
Ammonium Sulphate Fractionation ◽  
Relationship Of ◽  
The Hill

1. Extracts of Acetobacter xylinum were found to contain the glycolytic enzymes involved in the conversion of triose phosphate into pyruvate. Pyruvate kinase had the lowest relative activity. Phosphofructokinase activity was not detected in the extracts. 2. Only slight differences in the activity of pyruvate kinase were observed between cells grown on glucose and those grown on intermediates of the tricarboxylic acid cycle. 3. Pyruvate kinase, partially purified from ultrasonic extracts by ammonium sulphate fractionation, required Mg2+ ions for activity. It was not activated by K+ or NH4+ ions. 4. The plots representing the relationship between initial velocity and phosphoenolpyruvate concentration were sigmoidal, suggesting a co-operative effect for phosphoenolpyruvate. The Hill coefficient (n) for phosphoenolpyruvate was 2. The rate of the reaction changed with increasing ADP concentrations according to normal Michaelis–Menten kinetics. 5. The enzyme was inhibited by ATP (Ki0·9×10−3m). The inhibition was competitive with regard to ADP but not with regard to phosphoenolpyruvate. It was not relieved by excess of Mg2+ ions. 6. The possible relationship of the properties of pyruvate kinase to regulatory mechanisms for controlling gluconeogenesis and carbohydrate oxidation in A. xylinum is discussed.

Partial Characterization of Pyruvate Decarboxylase Isolated From Germinating Pea Seeds

1980 ◽  
Vol 7 (1) ◽  
pp. 35 ◽  
Author(s):  
S Leblova ◽  
J Valik
Keyword(s):  
Sodium Phosphate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Pyruvate Decarboxylase ◽  
Temperature Stability ◽  
Hill Coefficient ◽  
Ph Optimum ◽  
The Hill

Pyruvate decarboxylase (EC 4.1.1.1), isolated from 4-day-old germinating peas, was precipitated from a sodium phosphate extract when (NH4)2SO4 was increased from 15 to 30% saturation, desalted on Sephadex G-25 or by dialysis for 24 h, and then chromatographed on a DEAE-cellulose column. This procedure increased the specific activity of the enzyme 120-fold compared with the sodium phosphate extract. The behaviour of the enzyme during gel filtration indicates that it has a high molecular weight. The pea enzyme exhibits a sigmoid dependence on the pyruvate concentration; reaction velocity is half-maximal at a substrate concentration of 1.8 mM and the Hill coefficient is 1.8. Thiamin pyrophosphate (TPP) is the coenzyme, which is relatively firmly bound to the apoenzyme, but can be removed by dialysis for 48 h. The apoenzyme is activated optimally at 2 mM TPP and inhibited by concentrations above 4 mM. The pH optimum for pea pyruvate decarboxylase is 5.8 and maximal temperature stability occurs at 48°C.

Regulation of pyruvate kinase by 6-phosphogluconate in isolated hepatocytes

1981 ◽  
Vol 240 (3) ◽  
pp. E279-E285
Author(s):  
S. B. Smith ◽  
R. A. Freedland
Keyword(s):  
Pyruvate Kinase ◽  
Parenchymal Cell ◽  
Isolated Hepatocytes ◽  
Hill Coefficient ◽  
Liver Parenchymal Cell ◽  
Liver Parenchymal ◽  
Highly Correlated ◽  
Parenchymal Cells ◽  
The Hill ◽  
Isolated Liver

Isolated liver parenchymal cells from rats fed a 65% sucrose diet for 14 days were incubated in the presence and absence of 10(-6) M glucagon. The pyruvate kinase obtained from homogenates of the glucagon-treated cells displayed and increased Ks 0.5 for phosphoenolpyruvate (P-enolpyruvate), as well as an increased Ka 0.5 for 6-phosphogluconate (6-P-gluconate), compared to pyruvate kinase from untreated cells. Additionally, glucagon treatment decreased the maximal stimulation of pyruvate kinase by 6-P-gluconate by approximately two-thirds and decreased the Hill coefficient value of pyruvate kinase for 6-P-gluconate from 1.76 to 1.56. 6-Aminonicotinamide, an inhibitor of 6-P-gluconate dehydrogenase, increased 6-P-gluconate levels in isolated liver parenchymal cells three- to sevenfold, depending on the substrates present. The flux of P-enolpyruvate through pyruvate kinase was increased from 18 to 40% in these preparations and was highly correlated with the increase in 6-P-gluconate levels. The results suggest that 6-P-gluconate could regulate pyruvate kinase activity in the intact liver parenchymal cell. Furthermore, the activator would be of greatest importance in the lipogenic animal.

scholarly journals Analysis of Hill slopes predicted by the four-ligand exponential model for a regulatory enzyme

1986 ◽  
Vol 234 (3) ◽  
pp. 717-726 ◽  
Author(s):  
S Ainsworth
Keyword(s):  
Pyruvate Kinase ◽  
Kinetic Studies ◽  
Exponential Model ◽  
Hill Slope ◽  
Observed Behaviour ◽  
The Individual ◽  
The Hill

The four-ligand exponential model for a regulatory enzyme is described as it is applied to kinetic studies of yeast pyruvate kinase in which the concentrations of four ligands are systematically varied. The Hill slopes predicted by this model are calculated for the two situations in which the fourth ligand is either a substrate or an effector. It is shown that the individual terms that make up the expression for the Hill slope assist the interpretation of the observed behaviour in terms of the constants employed by the model.

Phosphorylation is switch of L-type pyruvate kinase allostery

2010 ◽  
Vol 5 (2) ◽  
pp. 135-142 ◽  
Author(s):  
Ilona Faustova ◽  
Aleksei Kuznetsov ◽  
Erkki Juronen ◽  
Mart Loog ◽  
Jaak Järv
Keyword(s):  
Pyruvate Kinase ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Allosteric Enzyme ◽  
E Coli ◽  
Glycolysis Regulation ◽  
Regulatory Phosphorylation ◽  
Pyruvate Kinase Isoenzymes ◽  
Dependent Protein ◽  
The Hill

AbstractAmong four pyruvate kinase isoenzymes, M1, M2, R and L, only M1 is considered as a nonallosteric enzyme. However, here we show that the non-phosphorylated L-type pyruvate kinase (L-PK) is also a non-allosteric enzyme with respect to its substrate phosphoenolpyruvate (PEP). The allosteric catalytic properties of L-PK are switched on through phosphorylation by cAMP-dependent protein kinase. The non-phosphorylated enzyme was produced by expressing the rat L-PK in E. coli, as the bacterium does not have mammalian-type protein kinases. The resulting tetrameric protein was phosphorylated with a stoichiometric ratio of one mole of phosphate per one L-PK monomer. Activity of the phosphorylated enzyme was allosterically regulated by PEP with the Hill coefficient n=2.5. It was observed that allostery was engaged by phosphorylation of the first subunit in the tetrameric enzyme, while further phosphorylation only modulated this effect. The discovered switching between non-allosteric and allosteric forms of L-PK and the possibility of modulating the allostery by phosphorylation are important for understanding of the interrelationship between allostery and the regulatory phosphorylation in general, and may have implication for further analysis of glycolysis regulation in the liver.

scholarly journals Glucose 6-phosphate activation of pyruvate kinase from Mycobacterium smegmatis

1981 ◽  
Vol 193 (2) ◽  
pp. 435-440 ◽  
Author(s):  
R Kapoor ◽  
T A Venkitasubramanian
Keyword(s):  
Pyruvate Kinase ◽  
Mycobacterium Smegmatis ◽  
Kinase Activity ◽  
Hill Coefficient ◽  
Saturation Curve ◽  
Pyruvate Kinase Activity ◽  
Physiological Conditions ◽  
Sigmoidal Kinetics ◽  
High Degree ◽  
Variable Substrate

1. Activation of glucose 6-phosphate is one of the unique properties of pyruvate kinase from Mycobacterium smegmatis. 2. Pyruvate kinase, partially purified from ultrasonic extracts of the mycobacteria by (NH4)2SO4 fractionation, exhibited sigmoidal kinetics at various concentrations of phosphoenolpyruvate, with a high degree of co-operativity (Hill coefficient, h = 3.7) and S0.5 value of 1.0 mM. 3. In the presence of glucose 6-phosphate, the degree of co-operativity shown by the phosphoenolpyruvate saturation curve was decreased to h = 2.33 and the S0.5 value was lowered to 0.47 mM. 4. The enzyme was activated by AMP and ribose 5-phosphate also, but the activation constant was lowest with glucose 6-phosphate (0.24 mM). 5. The enzyme was strongly inhibited by ATP at all phosphoenolpyruvate concentrations. The concentrations of ATP required to produce half-maximal inhibition of enzyme activity at non-saturating (0.2 mM) and saturating (2 mM) phosphoenolpyruvate concentrations were 1.1 mM and 3 mM respectively. 6. The inhibition of ATP was partially relieved by glucose 6-phosphate. 7. The enzyme exhibited Michaelis-Menten kinetics with ADP as the variable substrate, with an apparent Km of 0.66 mM. 8. The enzyme required Mg2+ or Mn2+ ions for activity. It was not activated by univalent cations. 9. The kinetic data indicate that under physiological conditions glucose 6-phosphate probably plays a significant role in the regulation of pyruvate kinase activity.

scholarly journals Studies on the biosynthesis of hepatic pyruvate kinase and its correlation with enhanced hepatic lipogenesis in meal-trained rats

1979 ◽  
Vol 182 (2) ◽  
pp. 383-397 ◽  
Author(s):  
T J Hopkirk ◽  
D P Bloxham
Keyword(s):  
Fatty Acid ◽  
Pyruvate Kinase ◽  
Fatty Acid Synthesis ◽  
Specific Activity ◽  
Acid Synthesis ◽  
High Carbohydrate Diet ◽  
Hill Coefficient ◽  
Carbohydrate Diet ◽  
High Carbohydrate ◽  
Maximum Activation

Metabolic and enzymic changes were measured in meal-trained rats fed on high-carbohydrate diet. Rates of hepatic fatty acid synthesis are probably greater than rates of gluconeogenesis throughout the 24 h day provided that animals are fed. The daily enhancement of fatty acid synthesis on meal feeding coincided with the maximum activation of hepatic pyruvate kinase. Maximum activation of this enzyme was reflected in increased total catalytic activity (Vmax.), increased activity at 0.5 MM-phosphoenolpyruvate (V0.5), decreased Vmax./V0.5 ratio and a decrease in co-operativity of phosphoenolpyruvate binding as measured by the Hill coefficient (h). The latter changes are consistent with a decrease in enzyme phosphorylation during activation of the enzyme. To estimate changes in enzyme protein, quantitative enzyme precipitation with rabbit antisera was used. Giving a high-carbohydrate diet to meal-trained animals induced enzyme synthesis within a few hours. Adaptations in diet that enhanced fatty acid synthesis (chow to high carbohydrate; starved to high carbohydrate) led to an increased steady-state concentration of pyruvate kinase protein. An approximate estimate of the half-life of hepatic pyruvate kinase was 56 h. Whenever pyruvate kinase specific activity was measured in liver tissue extracts it was always considerably less (20–100 mumol/min per mg of protein, depending on dietary status) than the specific activity of pure pyruvate kinase (200 mumol/min per mg of protein). Antigenically active, catalytically inactive protein was removed during enzyme purification from cytosol at the stage of (NH4)2SO4 fractionation. The fraction precipitated by 30–45%-satd. (NH4)2SO4 was enzymically active, antigenically reacting protein was identified in the remaining (NH4)2SO4 fractions (0–30%- and 45–85%-satd.) and this contained no enzyme activity. These may correspond to inactive proteolytic fragments of pyruvate kinase. The rate-determining step in adjusting enzyme concentration seems to be proteolysis.

Kinetic studies on the biological activity of calmodulin in canine heart and liver during endotoxin shock

1991 ◽  
Vol 69 (11) ◽  
pp. 1670-1676 ◽  
Author(s):  
B. Ruth Clark ◽  
Maw-Shung Liu
Keyword(s):  
Biological Activity ◽  
Calcium Transport ◽  
Myocardial Dysfunction ◽  
Kinetic Studies ◽  
Endotoxin Shock ◽  
Hill Coefficient ◽  
Transport Systems ◽  
Canine Heart ◽  
Endotoxin Administration ◽  
The Hill

Effects of endotoxin administration on the biological activity of calmodulin isolated from canine heart and liver were studied. Calmodulin was isolated and purified to homogeneity. The biological activity of calmodulin was determined by its ability to activate Ca2+-dependent phosphodiesterase. Results obtained 4 h after endotoxin administration show that the Vmax and A0.5 for calmodulin, the Vmax and Km for cAMP, and the Vmax and the Hill coefficient for Ca2+ were unchanged, while the S0.5 for Ca2+ for the activation of phosphodiesterase were significantly increased in the heart. The kinetic parameters as described above were not significantly altered in the liver. These data indicate that the biological activity of calmodulin is inhibited in the heart during endotoxin shock and that the nature of inhibition is associated with a mechanism involving a decrease in the affinity (1/S0.5) towards Ca2+ binding. Since calmodulin plays an important role in the regulation of cardiac function through calmodulin-dependent calcium transport systems, our findings may have a pathophysiological significance in contributing to the understanding of myocardial dysfunction in endotoxin shock.Key words: endotoxin shock, calmodulin biological activity, phosphodiesterase, heart, liver.

Oxygen transport, tissue glycogen stores, and tissue pyruvate kinase activity in muskrats

1985 ◽  
Vol 63 (6) ◽  
pp. 1440-1444 ◽  
Author(s):  
Gregory K. Snyder ◽  
Edward L. Binkley
Keyword(s):  
Pyruvate Kinase ◽  
Gastrocnemius Muscle ◽  
Saturation Pressure ◽  
Anaerobic Capacity ◽  
Hemoglobin Concentration ◽  
Hill Coefficient ◽  
Terrestrial Mammals ◽  
Oxygen Stores ◽  
Glycogen Stores ◽  
The Hill

Oxygen stores, respiratory properties of blood, tissue glycogen concentrations, and the rate-limiting enzyme for glycolysis, pyruvate kinase, were examined in muskrats (Ondatra zibethicus). Of the potential oxygen stores, lung volume (19.6 ± 0.68 mL/kg), and blood hemoglobin concentration (13.0 ± 0.34 g Hb/100 mL blood) were typical of terrestrial mammals, while concentrations of myoglobin in heart (7.4 ± 0.1 mg Mb/g tissue) and gastrocnemius muscle (13.3 ± 0.5 mg Mb/g tissue) were significantly higher than values from the same tissues in the laboratory rat. Blood P50 (oxygen half-saturation pressure) at pH 7.4, 24.4 ± 1.36 Torr (1 Torr = 133.322 Pa), and Bohr effect, −0.64 ± 0.04, also were significantly different, while the Hill coefficient and buffering capacity were comparable for both species. Of our measures of anaerobic capacity, glycogen concentrations and pyruvate kinase activities in heart, brain, and gastrocnemius muscle of O. zibethicus were all comparable to values obtained for terrestrial mammals. We conclude that muskrats tolerate submersion by adaptations associated with aerobic metabolism, although a review of the literature shows that these adaptations are fully developed only in animals freshly captured during winter.

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