Purification Of Pyruvate Kinase Research Articles

Functional Role of HSP90 Complexes with Endothelial Nitric-oxide Synthase (eNOS) and Calpain on Nitric Oxide Generation in Endothelial Cells

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.

Direct and Novel Regulation of cAMP-dependent Protein Kinase by Mck1p, a Yeast Glycogen Synthase Kinase-3

The MCK1 gene of Saccharomyces cerevisiae encodes a protein kinase homologous to metazoan glycogen synthase kinase-3. Previous studies implicated Mck1p in negative regulation of pyruvate kinase. In this study we find that purified Mck1p does not phosphorylate pyruvate kinase, suggesting that the link is indirect. We find that purified Tpk1p, a cAMP-dependent protein kinase catalytic subunit, phosphorylates purified pyruvate kinase in vitro, and that loss of the cAMP-dependent protein kinase regulatory subunit, Bcy1p, increases pyruvate kinase activity in vivo. We find that purified Mck1p inhibits purified Tpk1p in vitro, in the presence or absence of Bcy1p. Mck1p must be catalytically active to inhibit Tpk1p, but Mck1p does not phosphorylate this target. We find that abolition of Mck1p autophosphorylation on tyrosine prevents the kinase from efficiently phosphorylating exogenous substrates, but does not block its ability to inhibit Tpk1p in vitro. We find that this mutant form of Mck1p appears to retain the ability to negatively regulate cAMP-dependent protein kinase in vivo. We propose that Mck1p, in addition to phosphorylating some target proteins, also acts by a separate, novel mechanism: autophosphorylated Mck1p binds to and directly inhibits, but does not phosphorylate, the catalytic subunits of cAMP-dependent protein kinase.

Studies on metabolic properties in the Northern Krill, Meganyctiphanes norvegica (Crustacea, Euphausiacea): influence of nutrition and season on pyruvate kinase

The specific activity and the kinetic properties of partly purified pyruvate kinase (PK) (EC 2.7.1.40) from the Northern Krill, Meganyctiphanes norvegica, were investigated in relation to varying food resources. In order to evaluate the effect of starvation on the total energy metabolism, the respiration rates of fed and unfed krill were determined. The FPLC–elution profile of PK displayed two distinct peaks — PK I and II. The first isoform represented 80% of the total PK activity in the organism, and 20% was contributed by the second isoform. PK I was inhibited by ATP but was not influenced by fructose–1,6–bisphosphate (FBP). In contrast, PK II showed ATP inhibition and up to 2.5-fold increased activity by addition of 17 μmol·l −1 FBP. The Michaelis–Menten constants of both isoforms were 2–10-fold higher for ADP than for phosphoenolpyruvate (PEP). Alanine showed no regulatory effect on PK I and II. In specimens starved for 7 days oxygen consumption decreased by 20%. Neither the feeding experiments nor the animals captured in the field during low and high productive seasons indicate that PK properties of M. norvegica are modified in relation to food supply. Accordingly, alternative mechanisms are involved in the depression of the metabolic rate in terms of oxygen consumption.

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica.

The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50,805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica.

Simultaneous separation and purification of pyruvate kinase and lactate dehydrogenase by dye-ligand chromatography

This work describes an improved downstream processing protocol for the simultaneous separation and purification of two diagnostic enzymes, lactate dehydrogenase (LDH) and pyruvate kinase (PK), from rabbit muscles. The purification scheme was specifically designed so that both purified enzymes were comparable, in terms of specific activity and impurity content, to commercial high-purity analytical grade. The proposed protocol comprises two dye-columns (dye-sorbents), Cibacron blue 3GA and Procion yellow MX-4G both immobilised on CL-Sepharose 6B, which were integrated in the following six-step scheme: (i) muscle extract preparation, (ii) 0–45% ammonium sulphate fractionation, (iii) blue-column chromatography where the two enzymes were separated and LDH was purified, (iv) blue-column chromatography, (v) thermal treatment, and (vi) yellow-column chromatography where PK was purified. This protocol affords LDH of specific activity 470 units/mg (no detectable PK) at 65% overall yield, and PK of specific activity 251 units/mg (no detectable LDH and enolase) at 40% overall yield.

Antagonizing effects of phorbol 12-myristate 13-acetate on hormonally stimulated gluconeogenesis in isolated rat hepatocytes involve activity changes of pyruvate kinase

The tumor-promoting phorbol ester phorbol 12-myristate 13-acetate partially neutralized the stimulatory effects of epinephrine ( α 1-adrenergic actions), glucagon, and dibutyryl-cAMP on gluconeogenesis in isolated hepatocytes of fasted rats, when lactate or dihydroxyacetone was used as the substrate. By constructing metabolic crossover plots and by comparing rates of lactate production from dihydroxyacetone with K 0.5 values of extracted pyruvate kinase for phospho enolpyruvate, we obtained evidence that phorbol ester actions on hormonally stimulated gluconeogenesis were accompanied by proportionate increases in activity of pyruvate kinase. Although purified pyruvate kinase from rat liver was a substrate for protein kinase C in vitro, phosphorylation was not accompanied by modulation of kinetic parameters. Furthermore, incubation of pyruvate kinase extracted from hormone-treated hepatocytes with protein kinase C revealed no activation of the prephosphorylated enzyme. This and the absence of effects of the phorbol ester on basal rates of gluconeogenesis and lactate production suggest that effects of protein kinase C on pyruvate kinase activity in hepatocytes may result from impairment of steps at the level of hormone-induced signal transduction.

Purification of Pyruvate Kinase from Germinating Castor Bean Endosperm

Cytosolic pyruvate kinase from endosperm of germinating castor beans (Ricinus communis L.; cv Hale) has been purified 3100-fold to apparent homogeneity and a final specific activity of 203 micromole pyruvate produced/minute per milligram protein. Purification steps included: heat treatment, polyethylene glycol fractionation, Q-Sepharose, ADP-agarose, Mono-Q and Phenyl Superose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the final sample resulted in a single protein staining band which co-migrated with pyruvate kinase activity. Two protein staining bands of 57 and 56 kilodaltons were observed following SDS polyacrylamide gel electrophoresis of the final preparation. The native molecular mass was found to be about 240 kilodaltons. This enzyme appears to be a tetramer composed of two different subunits. The presence of dithioerythritol (2 millimolar) was required for optimal activity of the purified enzyme.

Purified pyruvate kinases type M2 from unfertilized hen's egg are substrates of protein kinase C.

To characterize pyruvate kinase isoenzymes from cells with the capability to proliferate, this enzyme was purified from yolk and vitelline membrane of unfertilized hen's egg. Pyruvate kinase type M2 from vitelline membrane was obtained in a homogeneous form after a 1150-fold purification to a specific enzymatic activity of 450 mumol X min-1 X mg-1. It was saturated half-maximally with phosphoenolpyruvate at KPPrv0.5 = 0.36 mM phosphoenolpyruvate and was activity by fructose 1,6-bisphosphate and L-serine at suboptimal substrate concentrations. After 11 000-fold purification to a specific enzymatic activity of 60 mumol X min-1 X mg-1, the pyruvate kinase isoenzymes type M2 (KPPrv0.5 = 0.32 mM) and M1 (KPPrv0.5 = 0.04 mM) were obtained from the yolk substance. Kinetic differences were noted between the pyruvate kinase type-M2 isoenzymes from vitelline membrane and yolk. A comparison of the amino acid composition of the purified pyruvate kinase isoenzymes from hen's egg revealed that all isoenzymes were related to pyruvate kinase type M1 from chicken breast muscle. The M2-type isoenzyme from vitelline membrane was related to the M2-type isoenzyme from chicken tumors, but was not related to the M2-type pyruvate kinase from chicken lung or liver. Protein kinase C from chicken oviduct phosphorylated in vitro both pyruvate kinase M2 isoenzymes from the unfertilized hen's egg preferably at serine and less at threonine residues. Pyruvate kinase type M1 from egg yolk was a weak substrate of protein kinase C. An activation of pyruvate kinase type M2 from vitelline membrane was observed at suboptimal concentrations of phosphoenolpyruvate under the conditions of phosphorylation, in the presence of phosphatidylserine.

Purification of pyruvate kinase from pig dental pulp.

Purification of pyruvate kinase from pig dental pulp.

Regulation by phosphorylation-dephosphorylation of pyruvate kinase in Venus gallina and Scapharca inaequivalvis

1. 1. Pyruvate kinase from the posterior adductor muscle of bivalve molluscs Venus gallina and Scapharca inaequivalvis can be converted into a more active form by treatment with a cyclic AMP-dependent protein kinase. 2. 2. The enzyme from Scapharca is inhibited through incubation with a non-specific phosphatase. 3. 3. Purified pyruvate kinase from Venus treated with protein kinase is affected by alanine in a way similar to the untreated enzyme. 4. 4. Phosphorylation is thought to be an additional mechanism of regulation of pyruvate kinase in anaerobiosis and under gluconeogenetic conditions.

Pyruvate kinase: is the mechanism of phospho transfer associative or dissociative?

To test for the possibility that pyruvate kinase proceeds via a dissociative path, we have investigated whether the complex enzyme . ADP . metaphosphate is transiently formed from the complex enzyme . ATP. It is shown that when highly purified pyruvate kinase is used, the rate of positional oxygen isotope exchange in ATP (beta, gamma bridge and beta nonbridge) is about 10(4) times slower in the absence of the cosubstrate pyruvate than it is in the presence of pyruvate. Further, the rate of racemization of the gamma-phospho group of [gamma (S)-16O,17O,18O]-ATP is undetectable, being at least 30 times slower even than the rate of positional isotope exchange. These tests thus provide no evidence that pyruvate kinase follows a dissociative mechanism. Indeed, it is argued that the available data are more consistent with an associative path. Evidence is presented that the single, associative, transition state is symmetrical, in which bond making and bond breaking processes are rather precisely balanced.

The effect of fructose on pyruvate kinase activity in isolated hepatocytes. Inhibition by allantoin and alanine.

1. Incubation of isolated hepatocytes with fructose at concentrations above 3 mM resulted in an apparent inhibition of pyruvate kinase assayed in crude extracts at sub-optimal phosphoenolpyruvate concentrations. 2. Fructose at concentrations below 3 mM caused an activation of the enzyme. 3. Increases in the hepatocyte contents of the positive effectors fructose 1.6-bisphosphate and fructose 1-phosphate were found at all concentrations of fructose up to 10mM. 4. Removal of the extrahepatocyte medium from the hepatocytes by washing resulted in an activation of the enzyme at all concentrations of fructose examined. 5. Inhibitors of the enzyme were shown to accumulate in the hepatocytes despite the depletion of ATP (a known negative effector) caused by higher concentrations of fructose. Indeed the inhibition of pyruvate kinase appeared to be correlated to the depletion of ATP. 6. Alanine (a known inhibitor) was shown to accumulate in hepatocytes as a consequence of incubation with fructose. 7. Allantoin and uric acid were shown to be inhibitors of a partially purified pyruvate kinase preparation assayed both in the presence and in the absence of fructose 1.6-bisphosphate. 8. Allantoin, but not uric acid, accumulated in the extrahepatocyte medium as a result of incubation of the cells with 10 mM-fructose.

Some kinetic properties of pyruvate kinase from Trypanosoma brucei: Influence of pH and fructose-1,6-diphosphate

The influence of pH on the activity of purified pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been studied. The K m for the coenzyme ADP is pH-dependent and shows the involvement of a dissociable group on the free enzyme with a p K a of 6.5–6.7. The cooperative interaction of the multiple phospho enolpyruvate (PEP) binding sites is independent of pH in the range 5.7–7.8. Variation of the V max value with pH indicates the presence of a dissociated group (p K a 6.2–6.3) and of an undissociated group (p K a 7.5–7.6) in the enzyme-substrate complex. A doubly dissociated phosphate group on PEP is shown to be essential by the effects of pH on the S 0.5 value for this substrate, as is an undissociated enzyme group with a p K a in the range 6.7–7.0. It is shown that PEP and fructose-1,6-diphosphate (FDP) act entirely in conjunction in allosterically activating the enzyme. FDP, the heterotropic effector, decreases the interaction between PEP binding sites at low concentration, and decreases the S 0.5 value for PEP at higher concentration. A model for the interaction of the enzyme with its substrates is discussed.

Pyruvate kinase phosphatase

1. Introduction Liver pyruvate kinase can be phosphorylated and simultaneously inactivated by CAMP-dependent pro- tein kinase; the inactivated enzyme can be reactivated by a multifunctional protein phosphatase (review [ 13). The inactivation of pyruvate kinase has been observed in isolated hepatocytes incubated in the presence of glucagon and in this case the decrease in pyruvate kinase activity was correlated with the stimulation of gluconeogenesis [2]. The glucagon induced inactiva- tion of pyruvate kinase is however transient and reac- tivation occurred within 20 min when sub-optimal doses of glucagon were added. This reactivation was more rapid in the presence of insulin [2,3]. The purpose of this work was to investigate the nature of the enzyme that reactivates pyruvate kinase and its relationship with the well-known protein plios- phatases that act on glycogen phosphorylase a. 2. Methods The inactive form of pyruvate kinase was partially purified from livers of anesthetized rats given glucagon (i.v., 1 mg/kg) 5 min prior to sacrifice. Livers were homogenized at 0°C in 4vol. 0.1 M KF, 15 mM EGTA and 50 mM glycyl glycine, at pH 7.4. Following cen- trifugation at 18 800 X g for 10 min, pyruvate kinase was purified essentially as in [4] by treatment at pH 5.2, ammonium sulfate precipitation and a single chromatography on DEAE-cellulose. The enzyme was eluted from the DEAE-column (1.5 cm X 10 cm) with 100 mM potassium phosphate (pH 7.2) at a rate of 5-6 ml/min. The peak fraction was dialyzed over- night against a buffer containing 30% glycerol, 20 mM mercaptoethanol, and 100 mM Tris at pH 7.2 and stored at 5°C. When the final pyruvate kinase prepara- tion was assayed as described below at 0.15 mM PEP (1.‘6.1s), the activity was consistently <IO% of the maximal velocity measured at 5 mM PEP (V,). This value of the v,.,s/V’s ratio is characteristic of the inac- tive form of pyruvate kinase [2,5]. When purified pyruvate kinase was incubated in the presence of 10 mM MgC12, no reactivation occurred indicating that no magnesium-dependent phosphatase activity was present in this preparation. The addition of exog genous pyruvate kinase phosphatase caused a complete reactivation of pyruvate kinase as evidenced by an increase in the v,.,,/Vs ratio from 0.1-0.45. The liver extract, used as a source of pyruvate kinase phosphatase was prepared as follows. Livers were homogenized in 2 vol. 50 mM imidazole (pH 7.4), 250 mM sucrose and 0.5 dithiothreitol the homogenate was centrifuged at 18 800 X g for 10 min at 0°C. The high-speed supernatant was prepared by centrifuging the extract at 106 500 X g for 30 min. Additional treatment is noted in the legend of table 1. The reactivation of pyruvate kinase was measured at 25°C in the presence of 50 mM Tris (pH 7.2), 5% glycerol, bovine serum albumin (1 mgiml), 25 mM KCI, 10 mM MgCI,, 20 mM mercaptoethanol and non- saturating levels of pyruvate kinase (8 units/ml), unless noted otherwise. The reaction was initiated by the addition of the pyruvate kinase phosphatase prep-

A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas.

1. An investigation of the reaction mechanism of the fructose 1,6-bisphosphate-activated pyruvate kinase isolated from the hepatopancreas of the crab Carcinus maenas was conducted. The enzyme was assayed in the presence of 500 microns-fructose 1,6-bisphosphate, 75 mM-KCl and 8 mM-Mg2+free at 25 degrees C. The results are consistent with a rapid-equilibrium random mechanism. 2. Evidence is presented that suggests the formation of two mixed-substrate-product dead-end complexes, enzyme-ADP-pyruvate and enzyme-ADP-ATP. 3. Competitive substrate inhibition was observed for both substrates, ADP and phosphoenolpyruvate, suggesting the formation of the complexes enzyme-ADP-ADP and enzyme-phosphoenolpyruvate-phosphoenolpyruvate in the suggested mechanism. 4. Data from the ATP product-inhibition studies indicate the formation of the complex enzyme-ATP-ATP. This suggests that in the reverse reaction ATP also will show substrate inhibition. 5. The presence of a saturating concentration of fructose 1,6-bisphosphate does not cause full activation of the purified preparations of the enzyme. 6. Pyruvate kinase activity in the supernatant of a hepatopancreas homogenate was completely activated by fructose 1,6-bisphosphate, suggesting that the binding of this ligand to the purified pyruvate kinase was impaired.

Modulation of the phosphorylation state of rat liver pyruvate kinase by allosteric effectors and insulin.

The regulation of pyruvate kinase in isolated hepatocytes from fasted rats was studied where the intracellular level of fructose 1,6-bisphosphate was elevated 5-fold by the addition of 5 mM dihydroxyacetone. In this case, flux through pyruvate kinase was increased. The increase in flux correlated with an elevation in fructose bisphosphate levels but not with P-enolpyruvate levels which were unchanged. Pyruvate kinase was activated and its affinity for P-enolpyruvate was increased 7-fold in hepatocyte homogenates. Precipitation of the enzyme from homogenates with ammonium sulfate removed fructose 1,6-bisphosphate and activation was no longer observed. These results indicate that flux through and activity of pyruvate kinase can be controlled by the intracellular level of fructose 1,6-bisphosphate. The effect of elevated fructose 1,6-bisphosphate levels on the ability of glucagon to inactivate pyruvate kinase was also studied where only covalent enzyme modification is observed. Inactivation by maximally effective hormone concentrations was unaffected by elevated levels of fructose 1,6-bisphosphate, but the half-maximally effective concentration was increased from 0.3 to 0.8 nM. Activation of the cyclic AMP-dependent protein kinase by 0.3 nM glucagon was unaffected, but the initial rate of pyruvate kinase inactivation was suppressed. These results suggest that alterations in the level of fructose 1,6-bisphosphate can affect the ability of physiological concentrations of glucagon to inactivate pyruvate kinase by opposing phosphorylation of the enzyme. Consistent with this view was the finding that physiological concentrations of fructose 1,6-bisphosphate inhibited in vitro phosphorylation of purified pyruvate kinase. Inactivation of pyruvate kinase by 0.3 nM glucagon or 1 microM phenylephrine was also suppressed by 10 nM insulin. Insulin did not act by increasing fructose 1,6-bisphosphate levels. The antagonism to glucagon correlated well with the ability of insulin to suppress activation of the cyclic AMP-dependent protein kinase. However, no such correlation was observed with phenylephrine in the absence or presence of insulin. Thus, insulin can enhance pyruvate kinase activity by both cyclic AMP-dependent and independent mechanisms.

Rat-brain pyruvate kinase: Purification and effects of lithium

Purified pyruvate kinase was prepared from pooled brains obtained from untreated rats. Its properties suggest that it is similar to type 'M' pyruvate kinase. Lithium inhibition was demonstrated at pharmacologically significant lithium concentrations (7%–12% at 2 mmol 1 −1 Li) and this was similar in character to that previously seen in rabbit muscle pyruvate kinase, namely noncompetitive with respect to phosphoenol pyruvate, K +, and Mg 2+ but competitive with ADP.

Mutants of Saccharomyces cerevisiae having structurally altered pyruvate kinase

Two mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC.2.7.1.40) are described. The mutations are recessive, segregate 2+:2- in tetrads and do not complement each other. Single-step spontaneous revertants, isolated on glucose plates, get back pyruvate kinase activity. The enzymes from various revertants display a wide spectrum of specific activity, thermolability and altered affinity for ligands such as P-enol pyruvate, ADP and fructose 1,6-diphosphate. The mutants produce materials crossreacting to the rabbit antibody raised against purified pyruvate kinase from the wild type yeast. These mutations thus define the structural gene of pyruvate kinase.

Pyruvate kinase from muscle and fat body of the house cricketAcheta domesticus L.: Purification and catalytic studies

1. A procedure for the purification of pyruvate kinase (E.C. 2.7.1.40) from the house cricket,Acheta domesticus, is described. 2. The 1114-fold purified enzyme exhibits a specific activity of 235 units/mg of protein (Table 1). 3. Cricket PK is subject to allosteric regulation, being activated by fructose-1,6-diphosphate and inhibited by L-alanine and ATP (Figs. 3 and 6, Table 2b). 4. 1–2 mM ADP are required for maximal activity (Fig. 5). The enzyme exhibits a pH optimum of 6.0 to 7.0 (Fig. 4). 5. Bivalent cation requirement is shown by magnesium activation (Fig. 7). Potassium salts stimulate PK, with a maximum of activation at 75 mM for KCl and KBr and about 35 mM for K2SO4. KI, KSCN, NaCl and CsCl are strong inhibitors in presence of potassium chloride (Table 2). 6. The reaction rate of cricket PK is temperature dependent with a Q10 of 1.7–2.7 at saturating substrate concentrations (Fig. 8). 7. FDP and PEP stabilize the enzyme against inactivation by elevated temperature.

Some Properties of Partially Purified Pyruvate Kinase from Euglena gracilis Klebs var. bacillaris

A method of purification of pyruvate kinase (EC 2.7.1.40) from light-grown Euglena gracilis var. bacillaris was developed which yielded an enzyme preparation purified 115-fold over crude extracts. During organelle formation, levels of pyruvate kinase in extracts prepared from cells engaged in light-induced chloroplast development do not change significantly. The enzyme has a molecular weight of approximately 240,000 and a requirement for both K(+) and Mg(2+). Fructose 1,6-diphosphate activates the enzyme when the concentration of phosphoenol-pyruvate is limiting; it does not activate when the concentration of ADP is limiting. ATP, citrate, and Ca(2+) are inhibitors of the enzyme and inhibit the fructose 1,6-diphosphate stimulation of the enzyme activity. ATP inhibition is only partially reversed by high concentrations of fructose 1,6-diphosphate. Further reversal of inhibition can be achieved by dialysis. Ca(2+)-dependent inhibition can be reversed by a chelating agent but not by increased concentrations of Mg(2+).The significance of the properties of pyruvate kinase in the regulation of photosynthetic carbohydrate metabolism, especially in connection with the inability of fructose 1,6-diphosphate to reverse Ca(2+) and ATP inhibitions, is emphasized.