Kinetic Properties Of Pyruvate Kinase Research Articles

Dissimilar properties of pyruvate kinase from rabbit and hare muscle

Some catalytic and kinetic properties of pyruvate kinase (PK, EC 2.7.1.40) isolated from rabbit and hare heart and skeletal muscle with a 9–16-fold degree of purification were studied. The initial specific activity of the enzyme in hare heart homogenates was by 66% while in hare skeletal muscle by 25% higher than in respective rabbit tissues. Temperature optimums and thermostability of PK from hare tissues were higher as compared with those in rabbits. A comparison of KM (S0.5) values shows that PK from hare skeletal muscle exhibits a highest affinity to phosphoenol pyruvate, but lowest to ADP, as compared with PK from rabbit skeletal muscle. Moreover, PK from both hare tissues exhibits a positive kinetic cooperativity (Hill coefficient > 1.35) of the phosphoenol pyruvate and ADP binding sites. In contrast to PK from rabbit tissues, the enzyme from hare heart and skeletal muscle is more sensitive to the inhibiting effect of ATP. It is suggested that in hare skeletal muscle PK is presented by its allosteric isoform which might be advantageous under extreme conditions of the hare’s habitation.

Design of a Coupled Bioluminescent Assay for a Recombinant Pyruvate Kinase from a Thermophilic Geobacillus

A simple and rapid method using coupled bioluminescent assay was developed to determine level of ADP. ADP is involved in many biological reactions and ADP assay can be used for assaying some reactions universally by monitoring ADP formation or depletion. ADP analysis involves incubation of ADP or extracts containing ADP with pyruvate kinase (PK) and PEP. The ATP formed by this reaction is determined by measuring the intensity of the initial light flash produced when luciferin-luciferase preparation injected into the reaction mixture. In regard to the main role of the PK in this assay, the gene of PK from a Geobacillus species has been cloned in expression vector pET28a (+), sequenced and overexpressed in Escherichia coli. Recombinant protein was purified using Ni-NTA column and then the purified PK was used in a coupled bioluminescent assay for ADP measurement. Kinetic properties of PK are determined according to a bioluminescent assay using firefly luciferase.

Effect of insecticide injection on pyruvate kinase of the insect Tenebrio molitor (Coleopteran)

In the present study, we report on the changes observed in the kinetic properties of pyruvate kinase (PK) from whole body homogenate in the three developmental stages of the insect Tenebrio molitor after insecticide or acetone injection, and after injury and 24 h starvation. The apparent K m value of the enzyme towards phosphoenolpyruvate was higher in the pupae stage than in the rest developmental stages although the same enzyme type appears to be present throughout the life span of the insect as judged by its V max and sensitivity towards ATP and Alanine. The highest specific activity, twice as high as that of larvae and pupae, was observed in adults. Starvation resulted to activation of the enzyme by increasing the K m value in the two feeding stages, namely larvae and adult, while injury had the opposite effect. Acetone injection did not have any significant effect at any stage. Insecticide injection had varying effects, depending upon the developmental stage, the dose and the type of the insecticide. In larvae, low doses of methyl paraoxon and methyl parathion activated the enzyme but as the dose increased the K m value returned to normal levels. Low dose of decamethrin activated the enzyme also but at high dose it caused a severe inactivation as judged by a fivefold increase of the observed K m. In pupae, decamethrin had no effect, methyl parathion had similar effects to those observed in larvae, and methyl paraoxon caused activation of the enzyme at any dose given. In the adult stage none of the injected insecticides had any effect on the K m value although V max and specific activity were significantly altered.

Effects of anoxia on the extra- and intracellular acid-base status in the land snail helix lucorum (L.): lack of evidence for a relationship between pyruvate kinase down-regulation and acid-base status

The aims of the present study were to describe a possible correlation between the regulation of the key glycolytic enzyme pyruvate kinase and the acid-base status in the haemolymph and in several other tissues of land snails during anoxia. To illustrate whether such a relationship exists, we determined (i) the acid-base variables in the haemolymph and tissues of the land snail Helix lucorum, (ii) the kinetic properties of pyruvate kinase from several tissues and (iii) the levels of the anaerobic end-products d-lactate and succinate in the haemolymph and tissues of aerobic and anoxic Helix lucorum. The results showed that the pH of haemolymph (pHe) decreased significantly over the first 20 h of anoxia and then recovered slowly towards control values. A similar pattern was observed for intracellular pH (pHi), which decreased significantly over the first 16 h of anoxia and slowly returned towards control levels. The reduction and recovery of pHi and pHe seem to reflect the rate of anaerobic metabolism. The main anaerobic end-products, d-lactate and succinate, accumulated rapidly during the initial stages of anoxia and more slowly as anoxia progressed. The decrease in the rate of accumulation of anaerobic end-products during prolonged anoxia was due to the conversion of tissue pyruvate kinase to a less active form. The results demonstrate a correlation between pyruvate kinase down-regulation and the recovery of acid-base status in the haemolymph and the tissues of land snails during anoxia.

Regulation of pyruvate kinase (PK) from the ventricle of the land snail Helix lucorum L. during early and prolonged estivation and hibernation

The kinetic properties of pyruvate kinase (PK) from the ventricles of early and prolonged estivated and hibernated land snails, Helix lucorum, were studied. The kinetic properties of PK from the ventricles of snails acclimated to cold for 4 days and 2 months were also determined. Short estivation (4 days) converted the PK to a less active form, possibly via enzyme phosphorylation. After 2 months of estivation, PK exhibited kinetic properties similar to control enzyme, except that the V max increased about 2-fold, indicating metabolic reorganization. During early and prolonged hibernation, the kinetic properties of PK did not change significantly. The results indicate that low temperature and low pH may be the main modulators of enzyme activity in the ventricle of H. lucorum during hibernation.

Some kinetic properties of pyruvate kinase from Trypanosoma brucei

We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg 2+ had the highest catalytic activity, but also the highest S 0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn 2+. The reaction rates of Mg 2+ -pyruvate kinase and Mn 2+ -pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co 2+ -pyruvate kinase were hyperbolic. However, Co 2+ -pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg 2+ and Mn 2+ as the activating metal ions. Moreover, with Co 2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K 0.5 values for the former 3 compounds increased when Mg 2+ was replaced by Co 2+, while the K 0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased. The effect of pH on the cooperativity of the reaction rate of pyruvate kinase from T. brucei towards phosphoenolpyruvate and fructose 2,6-bisphosphate was unique. At alkaline pH the positive cooperativity in the conversion of phosphoenolpyruvate was abolished while the apparent affinity for phosphoenolpyruvate was not altered and the enzyme was still sensitive to heterotropic activation. Optimal catalysis occurred at pH 6.0. At acidic pH the velocity versus fructose 2,6-bisphosphate concentration curve became sigmoidal and the activation constant increased. Between 15′C and 37′C, temperature did not influence the kinetic properties of Mg 2+ -pyruvate kinase with respect to phosphoenolpyruvate.

Alteration of kinetic properties of pyruvate kinase in Phascolosoma arcuatum (Sipunculida) exposed to environmental anoxia

Abstract After 48 h of exposure to environmental anoxia, the kinetic properties of pyruvate kinase (PK) in the local sipunculid worm Phascolosoma arcuatum were altered. However, no change in the kinetic properties of phosphoenolpyruvate car☐ykinase was observed. The modified PK extracted from the whole body of the anoxic worm had a lower apparent affinity for phosphoenolpyruvate compared to the PK extracted from the aerobic worms. Results of cellulose acetate electrophoreses and isoelectric focusing revealed the presence of three distinct PK isozymes in P. arcuatum. The kinetic and physical properties of the PK isozymes in the body wall and the proboscis cum attched retractor muscle but not those of the PK in the digestive tract, were altered during anoxia. Results obtained from treating the PK in the extracts from the anoxic worms with alkaline phosphatase in vitro indicate that anoxia-induced phosphorylation of PK occurs in P. arcuatum.

Time-course of covalent modification of pyruvate kinase during anaerobiosis in the mantle muscle and the hepatopancreas of the limpet Patella caerulea (L)

The changes in some kinetic properties of pyruvate kinase from the mantle muscle and the hepatopancreas of Patella caerulea, over a time-course of 16 or 8 h of acute hypoxia were studied. Up to the second hour of anaerobiosis PK exhibited changes of the kinetic properties in the direction of activation, whereas in the late stages of anaerobiosis in both tissues examined these changes occurred in the direction of inactivation. Although PKs from these tissues seemed to be regulated in a similar pattern during the early stages of anaerobiosis, they exhibited different kinetic properties under both normoxic and hypoxic conditions. Two forms of PK were found to be present in both the mantle muscle and the hepatopancreas of P. caerulea with different kinetic properties. The ratio of the enzyme units distributed between the two forms of PK (designated peak I and peak II) was varied during anaerobiosis indicating that the two forms of PK are interconvertible.

Anaerobic energy metabolism in the oligochaeteLumbriculus variegatus Müller

Anoxia tolerance, glycogen degradation, free amino acid pool, adenylate energy charge and the accumulation and excretion of end products were monitored inLumbriculus variegatus Muller throughout 48 h of anoxia. A transition period lasting about 4 h could be distinguished from subsequent events during which malate, present in high amounts in the resting animals, is utilized, probably by conversion to succinate. Up to the 12th hour of anoxia there is an increase in concentration of free amino acids, except aspartate. Glutamate increases rapidly during the first half hour but decreases thereafter. Beginning with the second hour of anoxia the alanine concentration increases at the same rate glutamate concentration decreases, but the source of nitrogen during the first hour is unknown. It is argued that the nitrogen required for the synthesis of some of the amino acids is ultimately derived from proteolysis. After about 3 h of anoxia propionate and acetate are synthesized. At first these acids accumulate in the tissues, but after 4–6 h they are excreted into the surrounding medium. Acetate is excreted over the whole experimental period at a constant rate, whereas the excretion rate of propionate decreases slowly with time. The propionate/acetate ratio is in excess of 2. Classic malate dismutation is by far the most important mechanism in the maintenance of redox balance. Depletion of glycogen stores appears to play an important role in determining anoxic survival time. Due to extremely low activity of PEPCK the ratio of the specific activities of PK and PEPCK is very high. Further, the kinetic properties of pyruvate kinase do not support the assumption of a shift of the glycolytic carbon flow at the PEP level.

Interactions of temperature and pH on the regulatory properties of pyruvate kinase from organs of a marine mollusc

The effects of low temperature assay (5 °C) on the properties of the aerobic (low phosphate) vs. anoxic (high phosphate) forms of pyruvate kinase (PK) from foot muscle and gill of the whelk Busycon canaliculatum (L.) were assessed at two pH values, pH 7.00 and 7.25, and compared to control conditions of 20 °C and pH 7.00 (all assayed in imidazole buffer). When pH was held constant at 7.00, the decrease in assay temperature to 5 °C had large effects on the measured kinetic parameters of all PK forms, as compared to 20 °C and pH 7.00. However, when assay pH was allowed to rise, from 7.00 to 7.25, with the temperature decrease to 5 °C there were fewer alterations of kinetic parameters and quantitatively smaller changes to enzyme properties. It appears, then, that when pH rises with decreasing temperature following alphastat predictions, kinetic properties of PK are largely conserved. Low temperature, at either pH value, had several significant effects on PK properties. For example, low temperature raised the S 0.5 for phosphoenolpyruvate of PK-anoxic from gill by 3–6 fold and decreased the I 50 Mg · ATP for PK-anoxic from foot by the same amount. Arrhenius plots of PK activity for the gill PK forms showed a distinct break at 10 °C; > 10 ° C Q 10 was 2.5 whereas < 10 ° C Q 10 was 8.4. Temperature-dependent changes in all cases affected enzyme properties in a manner that would restrict enzyme function at low temperature.

Effect of anoxia on the kinetic properties of pyruvate kinase and phosphofructokinase, and on glycogen phosphorylase activity in marine worms and earth worms

The kinetic properties of PK and PFK were studied in aerobic versus 12-hours anoxic marine worms Hedistae(=Nereis) diversicolor and Diopatra neapolitana and earth worms Allolobophora calliginosa and Eisenia foetida. The total glycogen phosphorylase (a+b) activity and the percentage of active a form were also measured in the marine and earth worms under the same conditions. Anoxia exposure did not result in any significant changes of kinetic parameters of PK and total activities of glycogen phosphorylase from marine worms, but it altered the kinetic characteristics of PFK from H. diversicolor. Chromatographical studies showed that PK from both aerobic and anoxic marine worms is eluted from DEAE-cellulose as a single peak at 50 mM KCl. In contrast to marine worms, however, anoxia caused a marked change in kinetic properties of PK from both earth worms, resulting in a reduction of enzyme affinity for its substrate PEP. In addition, the enzyme existed in both earth worms in two distinct variants eluted from DEAE-cellulose column as peak I and peak II at 50 mM and 150 mM KCl, respectively. The ratio of enzyme units (peak I/peak II) was reduced significantly after 12 h of anoxia, indicating that these two peaks are interconvertible. Anoxia also caused a reduction of total glycogen phosphorylase activity in E. foetida and lowered the percentage of active a form of the enzyme by approximately 50% in both earth worms. Kinetic properties of PFK from both earth worms were not significantly affected by anoxia. However, their low Ka values for F-2,6-P2 imply that this effector may play an important role in PFK control in earth worms under anoxia.

Influence of pH on the regulatory properties of aerobic and anoxic forms of pyruvate kinase in a marine whelk

AbstractThe effects of pH on the kinetic properties of pyruvate kinase were determined for both the aerobic and anoxic (16 hours exposure to N2‐bubbled seawater) enzyme forms from foot muscle and gill of the marine whelk, Busycotypus canaliculatum. The two enzyme forms from each tissue had distinctly different properties; compared to the aerobic enzyme, the anoxic enzyme form had significantly increased values for S0.5 phosphoenolpyruvate and Ka fructose‐1,6‐bisphosphate and greatly reduced I50 values for L‐alanine. Effects of pH on kinetic constants were assessed at pH 6.6, 7.0, and 7.5. Both the aerobic and anoxic forms of pyruvate kinase in whelk foot showed minimal values for S0.5 PEP and Ka FBP at pH 7.0; these rose by up to twofold at the higher or lower pH values. I50 values for alanine and Mg.ATP were strongly affected by low pH; I50 Mg.ATP fell by tenfold at pH 6.6 compared to pH 7.0, whereas I50 ALA dropped eightfold for the aerobic and 2.5‐fold for the anoxic enzyme form over the same pH change. The qualitative effects of pH change were similar for the anoxic enzyme form from gill. PK‐aerobic from gill behaved differently, however; as pH decreased, S0.5 PEP increased, whereas Ka FBP and I50 ALA both decreased. Furthermore, inhibition by Mg‐ATP occurred only at low pH. Although anoxia‐induced phosphorylation of PK is clearly the primary mechanism for changing the activity state of PK during anaerobiosis, the results suggest that the progressive fall in intracellular pH that occurs during anoxia would enhance the inactivation of PK activity in whelk organs. In particular, the effect of low pH in increasing the sensitivity of the anoxic enzyme forms to inhibitors could facilitate the anoxia‐induced changes in carbon flow at the phosphoenolpyruvate branchpoint.

Influence of Hormones, Second Messengers and pH on the Expression of Metabolic Responses to Anoxia in a Marine Whelk

ABSTRACT The roles of hormones, second messengers and pH in triggering or potentiating biochemical responses to anaerobiosis were evaluated using in vitro incubations of isolated muscle tissues (foot, radular retractor, ventricle) from the marine whelk Busycon canaliculatum (L.). Incubating tissues in vitro under anoxic conditions stimulated changes in muscle fructose-2,6-bisphosphate levels and pyruvate kinase kinetics (Km values for phosphoenolpyruvate, values for L-alanine) that were virtually equivalent to those that occur in vivo. Additions of hormones (epinephrine, norepinephrine, octopamine, serotonin, glucagon, insulin) or inhibitors of prostaglandin synthesis (dexamethasone, aspirin) had no effect on these metabolic responses to anoxia. The second messenger compounds, dibutyryl cyclic AMP and Ca2+ + ionophore A23187 + phorbol myristate acetate, produced isolated and tissue-specific responses in muscles incubated under aerobic conditions, but the magnitude and pattern of these responses differed from those seen in anoxia. Second messengers also had no effect on the development of biochemical responses in anoxic muscles. Tissue pH was artificially altered in order to evaluate the role of pH change (acidification occurs during anoxia in vivo) in the control of metabolic responses to anoxia. In all cases, the changes in the kinetic properties of pyruvate kinase (PK) correlated with the state of oxygenation of the tissue and not with the measured tissue pH value; higher tissue pH did not prevent anoxia-induced phosphorylation of PK and lower tissue pH did not alter the kinetic patterns of the aerobic enzyme. Overall, the study indicates that cells and tissues of the whelk respond individually to anoxia and that coordination of the action of protein kinases during anoxia is not mediated by pH or by common second-messenger mechanisms.

Kinetic properties of pyruvate kinase from the epaxial muscle of the marine fishes Mugil lisa and Chaetoditerus faber

1. 1. Kinetic studies were carried out on the reaction catalyzed by pyruvate kinase (ATP: pyruvate phosphotransferase, E.C.2.7.1.40) purified from white striated (epaxial) muscle of two marine fish Mugil lisa (Brazilian mullet) and Chaetoditerus faber (harvest fish). 2. 2. This included the establishment of kinetic parameters. 3. 3. Attention was given to the effect of fructose 1,6-bisphosphate (Fru-P 2) on PK activity. 4. 4. Effects of ATP, alanine and the divalent ions, Mg 2+, Mn 2+, Cu 2+, Be 2+ and Co 2+ on the fish muscle enzyme were also studied.

Some kinetic properties of pyruvate kinase from Phycomyces blakesleeanus

1. 1. Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555(−) has been partially purified and some kinetic properties has been investigated at pH 7.5. 2. 2. Positive homotropic interactions were observed with phosphoenolpyruvate and Mg 2+, showing Hill coefficient values of 2.8 and 2.5, respectively, whereas hyperbolic kinetics are found when ADP was the variable substrate. 3. 3. Fructose 1,6-bisphosphate acts as a heterotropic allosteric activator, markedly decreasing the S 0.5 value for phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form. 4. 4. ATP inhibits pyruvate kinase from mycelium of Phycomyces blakesleeanus. ATP appears to be a non-competitive inhibitor with respect PEP and competitive inhibitor with respect ADP.

The in situ assay of Candida albicans enzymes during yeast growth and germ-tube formation.

Conditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0.2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the in situ assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal in situ activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and N-acetylglucosamine kinase) and wall enzymes (beta-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1.9 and a S0.5 of 0.6 mM, whereas in cell extracts, it had a Hill coefficient of 1.9 and a S0.5 of 1.0 mM. The Km for ATP was 1.6 mM in cell extracts and 1.8 mM in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (S0.5 of 2.3 mM, Hill coefficient of 4.0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming cells.

Immunological and kinetic properties of pyruvate kinase in rat pancreatic islets.

Pyruvate kinase in rat pancreatic islets was characterized immunologically and kinetically. It is concluded that this activity is predominantly if not totally of the M(2) type.

Kinetic properties of pyruvate kinase of rabbit brain.

The kinetic properties of rabbit brain pyruvate kinase have been studied to determine its role in the regulation of glycolysis. One of the substrates of the enzyme, phosphoenolpyruvate, exhibits homotropic cooperativity (Hill coeff. of 1.45); thus, it is a moderate activator of the enzyme. The other substrate, ADP, shows normal Michaelis-Menton kinetics. Fructose-6-phosphate and glucose-6-phosphate activate the enzyme only slightly at the 1mm level and inhibit slightly at higher levels, and hence have no metabolic influence on the enzyme activity. Fructose-1, 6-diphosphate also has a slight activation up to 0.5 mm but no inhibition at higher level; therefore, it has no influence either. ATP, 2-phosphoglycerate, and phenylalanine are inhibitors of the enzyme. ATP, being the energy reservoir derived from glycolysis as well as a product of the reaction catalyzed by the enzyme, is a significant feedback inhibitor of the enzyme. These kinetic properties suggest a key role for pyruvate kinase in the regulation of glycolysis. Phenylalanine inhibition of the enzyme has been reported to be a possible mechanism of damage to the developing brain in phenylketonuria. The inhibition by phenylalanine at 10 mm in the assay mixture is reversed by alanine, cysteine, or serine at 0.2 mm level. Furthermore, the effect of these amino acids in reversing the phenylalanine inhibition are mutually enhancing. Consequently phenylalanine cannot have a significant inhibition on the activity of pyruvate kinase in brain.

Difference in the Effect of Glucagon and Starvation upon l‐Type Pyruvate Kinase from Rat Liver

The mechanism of the glucagon action upon pyruvate kinase in isolated hepatocytes from fed or starved rats has been investigated. Glucagon incubation of hepatocytes increases the reaction responsible for pyruvate kinase inactivation. This inactivation reaction is completely dependent upon the presence of adenosine 3′: 5′‐monophosphate (cAMP), (half maximal activation at 0.1 μM cAMP) with cells incubated without glucagon, while with cells incubated with glucagon cAMP addition only has a marginal effect. It is concluded that glucagon acts by activation of the protein kinase responsible for pyruvate kinase inactivation. Glucagon addition to isolated hepatocytes from fed and starved rats causes a similar decrease in pyruvate kinase activity of 40.7 ± 2.5% and 36.6 ± 3.6%, respectively, when measured at suboptimal phosphoenolpyruvate concentrations. Although optimal concentrations. of glucagon (1 μM) are used during the cell incubations, the pyruvate kinase activity can be inactivated further in vitro by incubation under conditions for optimal protein kinase activity. From the kinetic curves for the substrate phosphoenolpyruvate, obtained before and after further incubation in vitro, it can be concluded that pyruvate kinase by its presence inside the cell is protected against a complete inactivation by glucagon. The difference in apparent affinity of pyruvate kinase for phosphoenolpyruvate from glucagonincubated cells versus cells incubated without glucagon disappears after further incubation of the supernatants of these cells in vitro under optimal conditions for protein kinase activity (plus 10 μM cAMP) and an enzyme with a low affinity for phosphoenolpyruvate is obtained (K0.5= 4.4 mM). These results indicate that the glucagon‐induced change in kinetic properties of pyruvate kinase can be adequately explained by an increased phosphorylation state of pyruvate kinase. The difference in apparent affinity of pyruvate kinase for phosphoenolpyruvate from fed liver versus starved liver (P &lt; 0.001) still persists after further incubation of the liver supernatants under optimal conditions for protein kinase activity in vitro (P &lt; 0.001). For the completely inactivated enzyme from livers of fed and starved rats K0.5 values for phosphoenolpyruvate of respectively 2.8 and 4.4 mM are obtained. These results indicate that the nature of the effect of starvation differs from that of glucagon. It is suggested that the effect of starvation might be caused by partial proteolysis of the enzyme or oxidation of essential thiol groups.

Some kinetic properties of skeletal muscle pyruvate kinase from air-breathing and water-breathing fish of the Amazon

The kinetic properties of pyruvate kinase from skeletal muscle were studied in two species of air-breathing fish, Hoplerythrinus unitaeniatus and Arapaima gigas, and two species of water-breathing fish, Hoplias malabaricus and Osteoglossum bicirrhosum. It was found that the enzymes from Hoplias and Hoplerythrinus showed hyperbolic saturation kinetics for all substrates, were activated slightly by fructose 1,6-diphosphate, and were inhibited by phosphocreatine and citrate. The enzyme from Hoplias was inhibited by alanine, whereas the enzyme from Hoplerythrinus was not. The enzymes from Arapaima and Osteoglossum showed hyperbolic saturation kinetics for adenosine diphosphate, but the saturation kinetics for phusphoenol-pyruvate were sigmoidal. These enzymes were strongly activated by fructose 1,6-diphosphate and strongly inhibited by alanine, the former completely reversing the inhibition by the latter. Phosphocreatine and citrate were also found to be inhibitors of these enzymes, but the inhibition by phosphocreatine was not reversed by additions of fructose 1,6-diphosphate. The enzymes from the water-breathing fish were more sensitive to inhibition by alanine than were those from the air-breathing fish, but in other respects the enzymes were very similar.