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PubMed1982

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.

Adenosine Diphosphate↗
PubMed1989

On the regulatory properties of the pyruvate kinase from Trypanosoma cruzi epimastigotes.

The pyruvate kinase from Trypanosoma cruzi epimastigotes was activated by fructose 2,6-diphosphate ((A) 0.5 = 0.17 microM), through a decrease in (S) 0.5 and an increase in Vmax for both substrates. The enzyme was 50% inhibited by 0.9 mM ATP or 0.5 mM Pi in the presence of 30 mM MgCl2; these inhibitions were completely counteracted by 1.5 microM fructose 2,6-diphosphate. Both facts suggest that the effects are allosteric, and not due to chelation.

Adenosine Triphosphate↗
PubMed1975

Increased activity of pyruvate kinase in plasma of vitamin E-deficient rats.

The effect of dietary vitamin E on activities of pyruvate kinase and lactate dehydrogenase was studied in tissues of rats. The activity of pyruvate kinase in plasma of 1-month-old male rats fed a vitamin E-deficient diet for 4 months increased 8.5-fold over that of 45 ppm vitamin E-supplemented animals. Relative to the supplemented group, the enzyme activity increased 23% (P less than 0.001) in red blood cells, was unchanged in liver and lung, and decreased 20% (P less than 0.001) in muscle of vitamin E-deficient rats. The activity of lactate dehydrogenase was not significantly altered by dietary vitamin E in all tissues measured. Similar results were obtained when 2-month-old rats were fed the respective diets for 3 months. The results suggest that vitamine E deficiency in rats may cause muscular damage and release of pyruvate kinase into blood circulation.

Age Factors↗
PubMed1975

Rapid micromethod for the preparation of leucocyte-free haemolysates for the determinaton of pyruvate kinase and other erythrocyte enzymes.

In pyruvate kinase deficiency the high activity of the leucocyte isoenzyme may mask the erythrocyte defect. Separation of leucocytes from erythrocytes bythe commonly used sedimentation or filtration procedures requires rather large volumes of blood, is time-consuming, and sometimes leaves an unsatisfactorily large proportion of leucocytes. In the present study at least 95 % of leucocytes, measured with the lysosomal enzyme beta-N-acetylhexosaminidase as a very sensitive marker for leucocyte lysis, were consistently removed by differential lysis of erythrocytes in hypotonic saline. Haemolysates suitable for erythrocyte enzyme assays can be prepared from very small quantities of capillary blood (10-100 micro1) very rapidly, cheaply, and reproducibly by this method.

Biomarkers↗
PubMed1971

Effect of endotoxin on pyruvate kinase activity in mouse liver.

The pyruvate kinase (PK) activity of mouse liver increases after injection of endotoxin. It decreases in animals given cortisone alone and remains essentially unchanged in those given cortisone and endotoxin at the same time. CCl(4) causes an increase in liver PK activity, but neither it nor endotoxin changes the activity of muscle PK. Addition of octonoate to liver homogenates inhibits the activity of liver PK. These results suggest that the rapid depletion in liver glycogen after administration of endotoxin or CCl(4) may be related to increased PK activity. Induction of tolerance does not prevent the increase in liver PK activity in challenged animals.

Animals↗
PubMed1983

The refolding of denatured rabbit muscle pyruvate kinase.

The refolding of rabbit muscle pyruvate kinase after denaturation by guanidine hydrochloride was studied. On dilution of the denaturing agent, enzyme activity is only partially regained. The extent of regain of activity is dependent on protein concentration, showing a marked decrease at higher concentrations. The failure to regain complete activity appears to be related to the formation of inactive aggregates, which can be separated from active enzyme by gel filtration. Insoluble aggregates can be partially re-activated after solubilization in guanidine hydrochloride. Changes in the circular-dichroism and fluorescence spectra during refolding suggest that a partially folded, inactive species is formed rapidly; this differs from native enzyme in being more susceptible to proteolysis by trypsin.

Animals↗
PubMed1983

Hormone dependence of the L and M isozymes of pyruvate kinase in isolated rat hepatocytes.

L Pyruvate kinase (LPK) is considered to be the major form in the liver. Two isozymes, LPK and MPK, have been localized in the isolated rat hepatocyte in vitro with an immunocytometric method. MPK is induced by insulin, which also creates a slight stimulation of LPK (at physiological doses) in both fed and fasted animals. Glucagon inhibits LPK in fed animals (the fasting rat is already in a situation of gluconeogenesis and this hormone is ineffective). MPK is insensitive to glucagon, regardless of the nutritional state of the animals. Each PK isozyme is thus controlled predominantly by one of the two hormones, corresponding to a sophisticated regulation of hepatic glycolysis and gluconeogenesis.

Animals↗
PubMedDate not supplied

[2 interconvertible forms of L type pyruvate kinase from the rabbit adrenal cortex].

Pyruvate kinase (PK-II) isoenzyme from rabbit adrenal cortex was isolated by chromatography on DEAE-cellulose during long-term incubation in the cold. The enzyme is resistant to fructose-1,6-diphosphate (FDP) and L-alanine. Dithiotreitol and preincubation for 10 min. at 30 degrees C did not change the resistance of the enzyme to these factors. Preincubation for 30 min. at 30 degrees C in the presence of EDTA (3.3 mM) converted the enzyme into the FDP-sensitive form, the dependency curve of the initial reaction rate on the concentration of phosphoenolpyruvate being S-form. Sucrose and dithiotreitol contributed the isolation of PK-II from rabbit adrenal cortex in the form having a higher degree of the interaction of active sites.

Adrenal Cortex↗
PubMed2001

Purification and characterization of pyruvate kinase from lamprey (Entosphenus japonicus) muscle.

Pyruvate kinase from skeletal muscle of lamprey (Entosphenus japonicus), which is one of the most primitive living vertebrates, has been purified by approxImately 110-fold. The isolation procedure includes chromatography on Phosphocellulose, Phenyl-5PW, and Sephacryl S-300. Sodium dodecyl sulfate gel electrophoresis shows 59000 as the deduced subunit molecular weight and gel filtration shows 232000 as the tetramer of the subunits. The apparent Km for phosphoenolpyruvate and ADP are 0.41 mM and 0.31 mM at pH 7.4, respectively, when the purified enzyme is saturated with the second substrate. When the enzyme is activated in the presence of fructose-1,6-diphosphate, the Km for PEP changes to 0.087 mM, and the Hill coefficient changes from 1.3 to 0.98.

Acrylic Resins↗
PubMed1987

Human liver type pyruvate kinase: cDNA cloning and chromosomal assignment.

Pyruvate kinase (PK) has four isozymes (L,R,M1,M2) that are encoded mainly by two different genes. We isolated a cDNA clone from a Japanese adult liver lambda gt10 cDNA library by using a rat liver(L)-type PK cDNA probe. One positively hybridizing clone, hlPK-1, which contained a 1,049-base pair cDNA insert, was subjected to DNA sequence analysis. Comparisons of the sequence data with the rat PK cDNAs indicated that the cDNA encoded information for the carboxyl terminal 105 amino acids of a human L-type PK and a 3' untranslated region of 734 nucleotides. Furthermore, the karyotype analysis of several human-mouse hybrid cells and Southern blot analysis of DNAs of the hybrids with a hlPK-1 indicated that the human L-type PK gene is located on chromosome 1.

Base Sequence↗
PubMed1989

Phylogeny congruence analysis and isozyme classification: the pyruvate kinase system.

As the isozymes of pyruvate kinase (PK) are best known in rats, the characteristics of the rat isozymes are generally used to classify the PK isozymes in other species. Given the discrepancies generated by this classification by analogy, we evaluated a classification using a phylogeny congruence analysis of the compositional relatedness of vertebrate PK's. While our phylogenetic analysis confirmed the well established separation of the L and R isozymes from the K and M isozymes, its power became most evident in the identification of non-orthologous (or variant) forms of PK. Our analysis emphasized the uniqueness of chicken liver PK which cannot be classified either as a K or an L isozyme, confirmed that tumors express a variety of forms of PK, and indicated that lungs systematically express PK's which are not orthologous with PK's from other tissues. The determination of orthology by the phylogeny congruence analysis assumes that the structural data from different sources are subject to similar methodological error. However, we cannot reject the possibility that an apparent lack of orthology be due to artifacts during purification and analysis.

Amino Acids↗
PubMed1995

Upstream stimulatory factor proteins are major components of the glucose response complex of the L-type pyruvate kinase gene promoter.

L-type pyruvate kinase (L-PK) gene transcription is induced by glucose through its glucose response element (GlRE) composed of two degenerated E boxes able to bind in vitro ubiquitous upstream stimulator factor (USF) proteins. Here we demonstrate in vivo, by transient transfections in hepatoma cells, that (i) native USF proteins synthesized from expression vectors can act as transactivators of the L-PK promoter via the GlRE, stimulating transcription without glucose and, therefore, decreasing the glucose responsiveness of the promoter; (ii) expression of the truncated USF proteins, able to bind the GlRE but devoid of the NH2-terminal activation domain, represses the activation of the L-PK promoter by glucose; and (iii) a similar repression of the glucose effect is observed upon expression of mutant USF proteins devoid of the basic DNA binding domain, able to dimerize with endogenous USF but not to bind the GlRE. We conclude that USF proteins are components of the transcriptional glucose response complex assembled on the L-PK gene promoter.

Base Sequence↗
PubMed2003

Effects of metabolites on the structural dynamics of rabbit muscle pyruvate kinase.

The activity of rabbit muscle pyruvate kinase (PK) is regulated by metabolites. Besides requiring the presence of its substrates, PEP and ADP, the enzyme requires Mg(2+) and K(+) for activity. PK is allosterically inhibited by Phe for activity. The presence of PEP or Phe has opposing effects on the hydrodynamic properties of the enzyme without an apparent change in secondary structure. In this study, the structural perturbation induced by ligand binding was investigated by Fourier transform infrared (FT-IR) spectroscopy. Furthermore, the structural dynamics of PK was probed by H/D exchange monitored by FT-IR. Substrates and activating metal ions induce PK to assume a more dynamic structure while Phe exerts an opposite effect. In all cases there is no significant interconversion of secondary structures. PEP is the most efficient ligand in inducing a change in the microenvironments of both helices and sheets so much so that they can be detected spectroscopically as separate bands. These results provide the first evidence for a differential effect of ligand binding on the dynamics of structural elements in PK. Furthermore, the data support the model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the observed change in hydrodynamic properties represent the two extreme end states.

Adenosine Diphosphate↗
PubMed1989

Characterization of pyruvate kinase from human rhabdomyosarcoma in relation to immunohistochemical and morphological criteria.

We have compared the pyruvate kinase (PK) isoenzyme pattern of three rhabdomyosarcomas with foetal skeletal muscle tissue of 19 and 23 weeks of gestation, together with adult muscle in relation to immunohistochemical and morphological criteria. In foetal tissue of 19 weeks of gestation a focal immunopositivity for desmin and myoglobin was observed, whereas in tissue of 23 weeks an overall positivity for these proteins was present. Two of the three neoplasms were poorly differentiated and of the alveolar subtype. They were desmin immunoreactive. Some large spindle-shaped cells expressed myoglobin. The third one was more differentiated in microscopic characteristics and all cells showed immunoreactivity for desmin and myoglobin. In the foetal tissues five forms of pyruvate kinase isoenzymes were present with K2M2 as the predominant form. In adult muscle tissue only M4 was present. The tumors were characterized by a profound shift to the K-type, whereas the M4-type was not expressed at all. A difference in isoenzyme composition of pyruvate kinase was found between the morphologically less differentiated tumors and the more differentiated tumor; in the latter more M-subunits were expressed.

Adolescent↗
PubMed1993

Transcriptional regulation of the pyruvate kinase erythroid-specific promoter.

Mammal pyruvate kinases are encoded by two genes. The L gene produces the erythroid (R-PK) or the hepatic (L-PK) isozymes by the alternative use of two promoters. We report the characterization of the cis- and trans-acting elements involved in the tissue-specific activity of the L gene erythroid promoter. A R-PK DNA fragment extending from -870 to +54 relative to the cap site confers erythroid specificity to a reporter gene. Within this region, we define a minimal promoter (-62 to +54) that displays erythroid-specific activity and contains two DNA binding sites. One, located at -50, binds members of the CCACC/Sp1 family and the other, located at -20, binds the erythroid factor GATA-1. Although the -20 GATA binding site (AGATAA) is also a potential TFIID binding site, it does not bind TFIID. Furthermore, the substitution of this GATA binding site by a canonical TFIID binding site suppresses the promoter activity. Mutations and deletions of both sites indicate that only the association of CCACC/Sp1 and GATA binding sites can drive efficient and tissue-specific expression of this R-PK minimal promoter. Finally, by co-transfection experiments, we study the elements involved in the hGATA-1 transactivation of the R-PK promoter in HeLa cells.

Adult↗
PubMed1980

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.

Adenosine Diphosphate↗
PubMed1973

Kinetic studies on the regulation of rabbit liver pyruvate kinase.

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(-)/ADP(2-) 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 Mg(2+) 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 (n(H)=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 K(m) 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 (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).

Adenosine Diphosphate↗