Effects of calcium ions on pyruvate kinase in pigeon liver (original) (raw)
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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 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,
Difference in the Effect of Glucagon and Starvation upon l-Type Pyruvate Kinase from Rat Liver
European Journal of Biochemistry, 1978
1. 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 pM 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.
Two interconvertible forms of L-type pyruvate kinase from rat liver
Biochimica et biophysica acta, 1973
I. Reduced L-type pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.4o ) from rat liver can be converted into an oxidized form by incubation with oxidized mercaptoethanol and oxidized glutathione. This interconversion can be completely reversed by incubation with reduced mercaptoethanol.
On the regulation and allosteric model of L-type pyruvate kinase from rat liver
Biochimica et biophysica acta, 1974
1 The influence of P1 and the phosphorylated hexoses Fru-l,6-P2, Fru-6-P, Fru-l-P, GIc-I,6-P2, Glc-6-P, Glc-l-P and Gal-I-P on L-type pyruvate klnase from rat liver has been Investigated at physiological ADP, P-enolpyruvate, GSH, ATP and alanme concentrations At 0 1 mM P-enolpyruvate and m the presence of 2 mM MgATP + 1 mM alanlne the enzyme is reactive and the only effective activator in a physiological concentration range is Fru-l,6-P2 2 The phosphorylated hexoses were used as a probe for the conformation of the enzyme A comparison of the effects of MgATP and alanme on the "affimt~es*" for the hexoses reveals the following differences (a) MgATP (2 mM) and alanlne (1 mM) increase the concentration of GIc-I,6-P2 and Glc-6-P necessary for halfmaximal activation to the same extent, however, MgATP and alanme introduce a different cooperative interaction towards Glc-l,6-Pz and Glc-6-P at low P-enolpyruvate concentrations (b) MgATP (2 mM) has less Influence on the "affinities*" for Fru-l,6-P2 and Fru-6-P as compared to alanme (1 mM) (c) Enzyme mo&ficatlon by oxldatmn of the thlol groups increases the effect of MgATP on the "affinity" for Glc-I,6-P2, whereas the influence of alanine is decreased These results reveal the conclusion that MgATP and alanlne introduce a different conformatmn, probably by binding at &fferent sites The results are &fficult to explain by the R ~ T eqmhbrmm model of Monod, Wyman and Changeux and indicate that sequentml conformation changes are revolved in the alloster~c transitions of the enzyme * With "affinity" is meant the relative ablhty of a compound to activate the enzyme, this ability ~s concluded from the concentration of activator necessary for half maximal activation