Effects of calcium ions on pyruvate kinase in pigeon liver (original) (raw)
Biochemical Journal, 1968
1. Preincubation of partially purified rat liver L-type pyruvate kinase at 25° for 10min. causes a marked increase in co-operativity with respect to both the substrate, phosphoenolpyruvate, and the allosteric activator, fructose 1,6-diphosphate. 2. The results are consistent with the existence of two forms of liver L-type pyruvate kinase, designated forms LA and LB. It is postulated that form LA has a low Km for phosphoenolpyruvate (about 0·1mm) and is not allosterically activated, whereas form LB is allosterically activated by fructose 1,6-diphosphate, exhibiting in the absence of the activator sigmoidal kinetics with half-maximal activity at about 1mm-phosphoenolpyruvate. In the presence of fructose 1,6-diphosphate, form LB gives Michaelis–Menten kinetics with Km less than 0·1mm. It is further postulated that preincubation converts form LA into form LB. 3. The influence of pH on the preincubation effect was studied. 4. The inhibition of pyruvate kinase by Cu2+ was studied in detai...
Biochemical and Biophysical Research Communications, 1976
Incubation of isolated rat hepatocytes with glucagon (10 -6 M), db-cAMP (0.I mM) and db-cGMP (0.I mM) causes a decrease in pyruvate kinase activity of 46, 49 and 34% respectively, when measured at 1 mM Mg~ + and suboptimal substrate (P-enolpyruvate) • ree concentrations, wh1~e the Vma x is unlnfluenced. An increase in activity (25%) is noticed when the cells are incubated with 1 mM pyruvate. The glucagon inactivated enzyme (Lb) shows a decreased affinity for the substrate P-enolpyruvate and for the allosteric activator Fru-l,6-P 2 as compared to the activated form (La). The nature of the hormone and cyclic nucleotide-induced changes in pyruvate kinase is discussed. It is concluded that the P-enolpyruvate cycle is under comparable acute hormonal control as the FDPase-PFK cycle. Both cycles are linked by the common effector Fru-l,6-P 2 making not only direct but also indirect hormonal control of pyruvate kinase flux possible. Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) catalyses the last step of glycolysis. Its activity is important in the regulation of the balance between gluconeogenesis and glycolysis. Although liver contains two types of pyruvate kinase (I), isolated hepatocytes only contain the L-type (2). This enzyme possesses many regulatory properties but the mechanism to inactivate the enzyme under gluconeogenic conditions is still uncertain (3-6). From kinetic studies in vitro it was concluded that the known properties of L-type pyruvate kinase could not lead to the presence of an inactive enzyme during gluconeogenesis. This was mainly the consequence of the high affinity of the enzyme for its allosteric acti vator Fru-l,6-P 2 (6). It was suggested that "A change in the enzyme itself can influence its affinity for Fru-l,6-P2" and the use of isolated parenchymal cells to test this possibility was therefore proposed. Taunton et al. (7,8) showed recently that short term changes in Vma x of pyruvate kinase may be induced by alteration of 917
Biochemical Journal, 1983
The kinetics of rabbit muscle pyruvate kinase were studied in assays at pH 7.4, where the relationships between the initial velocities of the catalysed reaction and the concentrations of substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The data were used to test the applicability of the exponential model for a regulatory enzyme, which has been here extended to describe the behaviour of a three-substrate enzyme. It appears that the data can be represented by the model and as a result permit the conclusion that the substrates influence one another's binding by the same type of charge interactions that are evident in the Michaelis-Menten kinetics of the enzyme observed at pH 6.2. Evidence is also presented indicating that MgADP acts as a dead-end inhibitor of the enzyme at pH 7.4.
Pyruvate kinase type M2 is phosphorylated in the intact chicken liver cell
Biochemical and Biophysical Research Communications, 1983
Isolated hepatocytes from 24 h starved chicks were depleted of phosphate and incubated in the presence of 32p-orthophosphate. Pyruvate kinase type M 2 from crude cell extracts was partially purified by chromatography on DEAE-Sephacel and hydroxylapatite. SDS slab gel electrophoresis of the fractions containing the enzyme and immunopreeipitation with antisera showed the phosphorylation of pyruvate kinase type M 2. Phosphoamino acid analysis identified serine as phosphate acceptor. Pyruvate kinase (EC 2.7.1.40) isoenzyme type L from mammalian liver is inactivated in vitro via phosphorylation by a cAMP dependent protein kinase (for rev. see ref. I). This has been shown principally relevant also in vivo (2-4). In contrast to type L, pyruvate kinase type M 2 from chicken liver, where it represents the predominant amount of pyruvate kinase activity (5,6), is inactivated and phosphorylated in vitro by a cAMP independent protein kinase (7), probably at serine (8). In this paper we demonstrate for the first time (a) the phosphorylation of chicken liver pyruvate kinase type M2 in the intact cell and (b) that this phosphorylation is found at serine. MATERIALS and METHODS Materials: Biochemicals were from Boehringer Mannheim (Mannheim, FRG), Nonidet NP40 from Fluka (Neu Ulm), DEAE-Sephacel and protein A-Sepharose from Pharmacia (Freiburg), hydroxylapatite (DNA grade) from Bio-Rad Laboratories (MOnchen), 32p i from Amersham-Buchler (Braunschweig). All other reagents were of analytical grade and purchased from E. Merck (Darmstadt) and Serva Feinbiochemica (Heidelberg). Animals were male Lohmann-White Leghorn chicks from a cockerel farm near Giessen, fed on 13-B-Junghennenalleinfutter,
The immunological and catalytically active form of L-type pyruvate kinase in rat liver cytosol
The International journal of biochemistry, 1983
The protein species precipitated from rat liver cytosol by rabbit antisera raised to pure L-type pyruvate kinase were investigated by sodium dodecyl sulphate gel electrophoresis. The primary antisera (anti-L-type pyruvate kinase) precipitated protein species with mol. wts 56,000, 41,000 and 39,000. The 41,000 mol. wt protein was identified as fructose-bis-phosphatase. Double diffusion and immunotitration experiments established that L-type pyruvate kinase and fructose-bis-phosphatase shared common antigenic determinants. This information enabled an improved antiserum (anti-LPK) to be obtained. The use of anti-LPK showed that the 56,000 mol. wt subunit was the only catalytically and immunologically active form of L-type pyruvate kinase in liver. This was confirmed by biosynthetic experiments with cultured hepatocytes. The specific activity of the enzyme in liver extracts was also determined by quantitative immunotitration with anti-LPK. Despite changes in dietary status which varied ...
Catalyic and regulatory properties of muscle pyruvate kinase fromCancer magister
Journal of Experimental Zoology, 1980
Despite the marked changes that crustacean muscle undergoes during the molt cycle, pyruvate kinase is present as the same form throughout the molt cycle. This pyruvate kinase was subject to feed-forward activation by fructose-1, 6 bisphosphate (FBP) as well a s feed-back inhibition by MgATP. The enzyme showed a high affinity for phosphoenolpyruvate (K, = 0.1 mM) but showed no cooperativity in substrate binding. The addition of 0.05 mM FBP reduced the PEP K, to 0.05 mM. MgATP inhibition showed a Ki of 1.8 mM versus PEP. The inhibition due to MgATP could be reversed by FBP. Various other compounds inhibited the enzyme, including citrate, m-ketoglutarate, tryptophan, and malate, although at rather high levels. Measurements of the reversal of this pyruvate kinase, taken together with the low levels of phosphoenolpyruvate carboxykinase and pyruvate carboxylase, predict only minimal levels of gluconeogenic flux in crustacean muscle.
Biosystems, 2002
Potassium pyrophosphate was used instead of ATP as a model ligand for magnesium cation for the study of effector influence on the kinetics of pyruvate kinase muscle isozyme M 1 . The pyruvate kinase activation by low concentration of pyrophosphate and inhibition by high concentration of pyrophosphate was considered to be the result of reversible reactions of magnesium cation with pyrophosphate, ADP, ATP, and PEP. The apparent K m and V m or in some cases the pseudo-first order reaction rate constant (instead of K m and V m ) of pyruvate kinase at any given pyrophosphate concentration were analysed as a function of concentration of free magnesium cation and its complexes with all ligands present in an assay mixture. The functions of reaction parameters with respect to concentration of magnesium complexes indicate the coexistence in the reaction mixture of simple and mixed complexes of magnesium cation with substrates, pyrophosphate, and an enzyme Á/substrate complex. The parameters of the simulated reaction for the proposed interactions fit the measured experimental data. A simple model with nonallosteric feedback has been proposed. According to this model, mutual and simultaneous interactions of reaction products with substrates and with an enzyme result in the coexistence of simple and mixed, labile and inert complexes. #
Purification and regulatory properties of pigeon erythrocyte pyruvate kinase
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1989
1. Pigeon erythrocyte pyruvate kinase (PK) was purified 22,000 fold by successive column chromatography on Sephadex DEAE A-50 and Red Agarose. The resulting enzyme preparation had a specific activity of 815.3 U/mg protein and an overall yield of 18.5%. 2. The molecular weight, as determined by gel filtration on Sephadex G-200 was 152,000. 3. Isoelectric focusing in the pH range of 3-10 showed that pigeon erythrocyte contained at least 3 PK isozymes with isoelectric points of 5, 5.7 and 6. 4. The variation of activity of PK at various ADP and phosphoenolpyruvate (PEP) concentrations was studied. The Km values for ADP and PEP were 0.40 and 0.46 mM respectively. 5. The enzyme was activated by FDP, and inhibited by ATP, highly phosphorylated inositol derivatives and 2,3-DPG: 6. It was activated by K+ and Mg2+ ions. 7. Phosphorylated hexoses and Pi stimulated the activity of PK. 8. The regulatory role of PK of pigeon erythrocytes, which lack the typical 2,3-DPG bypass, is discussed.
Effect of Glucagon and Starvation on L-Type Pyruvate Kinase from Rat Liver
Biochemical Society Transactions, 1978
Recent studies have indicated that addition of glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to isolated hepatocytes or perfused liver causes a decrease in pyruvate kinase activity, when measured at suboptimal substrate (phosphoenolpyruvate) concentrations, whereas the V