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PubMed2002

Determination of pyruvate kinase type tumor M2 in human renal cell carcinoma: a suitable tumor marker?

Renal cell carcinoma (RCC) expresses an isoform of the glycolytic enzyme pyruvate kinase (type M2). The dimeric form (TuM2-PK) is over expressed in tumor cells and is detectable in blood with a sensitive enzyme-linked immunosorbent assay (ELISA). The aim of the present study was to evaluate the clinical value of TuM2-PK as a tumor marker for RCC. The TuM2-PK concentration in EDTA-plasma was determined quantitatively and immunologically using an ELISA. We measured the TuM2-PK plasma levels of 83 patients before and after surgery. Ninety-seven patients with various non-malignant diseases were also recruited as a control group. The control group displayed mean levels of 11.37 U/ml of TuM2-PK. Values were elevated in patients with RCC prior to surgery (mean 21.88 U/ml). The plasma levels increased after surgery until day 5 (mean 53.97 U/ml). At day 10, marker levels started to decrease without reaching preoperative values (mean 43.5 U/ml). Plasma levels in the renal vein (obtained during surgery) were not different from those in the peripheral blood. Follow-ups after 2-6 months showed a decrease to below preoperative levels (mean 16.3 U/ml). A significant difference was obtained by comparing the patients according to their Robson score. We found a significant difference (P < 0.01, Wilcoxon's two-sample test) in TuM2-PK levels between patients with RCC and the control group. Nevertheless, using the manufacturer's recommended cut-off value (15 U/ml), sensitivity was only 50.6% and specificity was 80.4%. Our results suggest that TuM2-PK is not a suitable tumor marker for RCC.

Biomarkers, Tumor↗
PubMed2003

Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus, Aeropyrum pernix, and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: unusual regulatory properties in hyperthermophilic archaea.

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.

Adenosine Diphosphate↗
PubMed1993

Carbohydrate regulation of the rat L-type pyruvate kinase gene requires two nuclear factors: LF-A1 and a member of the c-myc family.

Transcription of the L-type pyruvate kinase (L-PK) gene is induced in response to increased carbohydrate metabolism in the liver. We have demonstrated previously that a segment of the 5'-flanking region of the L-PK gene between -183 and -96 is necessary and sufficient for the glucose response in primary hepatocytes. To explore the protein factors that are involved in carbohydrate regulation, we have performed mutational analyses and in vitro binding studies of this segment. Sequences critical for the glucose response were mapped from -171 to -124. This segment contains the consensus binding sites for two nuclear transcription factors: LF-A1 and MLTF. Both factors are capable of binding to the corresponding L-PK sites in vitro. Mutational and functional analyses indicated that LF-A1 is indeed involved in glucose induction of the L-PK gene. The PK MLTF-like site consists of two imperfect CACGTG motifs, the core binding site for a family of transcription factors related to c-myc. Unexpectedly, mutations in either motif that resulted in defective glucose stimulation retained in vitro binding to MLTF. Furthermore, an authentic MLTF binding site from the adenovirus major late promoter was not functionally interchangeable with the natural sequence. These data indicate that binding of MLTF, in presence of LF-A1, is not capable of supporting the glucose response. Conversion of either imperfect motif to CACGTG within the context of the mutations disrupting the opposite site restored the response to elevated glucose. Thus, the factor that recognizes the PK MLTF-like site and participates in mediating the carbohydrate response of the L-PK gene appears to be a member of the c-myc family distinct from MLTF.

Animals↗
PubMed1997

Ligand-induced domain movement in pyruvate kinase: structure of the enzyme from rabbit muscle with Mg2+, K+, and L-phospholactate at 2.7 A resolution.

The structure of rabbit muscle pyruvate kinase crystallized as a complex with Mg2+, K+, and L-phospholactate (L-P-lactate) has been solved and refined to 2.7 A resolution. The crystals, grown from solutions of polyethylene glycol 8000 at pH 7.5, belong to the space group P2(1) and have unit cell parameters a = 144.4 A, b = 112.6 A, c = 171.2 A, and beta = 93.7 degrees. The asymmetric unit contains two tetramers. The crystal structure reveals that the eight subunits within the asymmetric unit adopt several different conformations. These conformations are characterized by differences in the relative positions of protein domains A and B, resulting in different degrees of closure of the active site cleft that occupies the interface between these two domains. The global conformational differences may be described as rotations of the B domain with respect to the (beta/alpha)8-barrel of the A domain. Carbon atoms of the backbone in domain B rotate >20 degrees from the most open to the most closed subunit. The different conformations among subunits within the asymmetric unit are accompanied by 3-3.8 A shifts in the position of Mg2+ and a significant change in the orientation of the phenyl ring of Phe 243. In all of the subunits, Mg2+ coordinates to the protein through the carboxylate side chains of Glu 271 and Asp 295. In the subunit having the most closed conformation, Mg2+ also coordinates to the carboxylate oxygen, the bridging ester oxygen, and a nonbridging phosphoryl oxygen of L-P-lactate. Mg2+ to L-P-lactate coordination is missing in subunits exhibiting a more open conformation. K+ coordinates to four protein ligands and to a phosphoryl oxygen of the L-P-lactate. The position and liganding of K+ are unaffected by the different conformations of the subunits. The side chain of Arg 72, Mg2+, and K+ provides a locus of positive charge for the phosphate moiety of the analog in the closed subunit.

Animals↗
PubMed1997

Members of the nuclear factor 1 family and hepatocyte nuclear factor 4 bind to overlapping sequences of the L-II element on the rat pyruvate kinase L gene promoter and regulate its expression.

The L-II element (-149 to -126 bp) in the enhancer unit of the rat pyruvate kinase L (PKL) gene is required for cell-type-specific transcription and induction by carbohydrates. This element was found to bind multiple nuclear proteins with different heat stabilities. A heat-labile factor was shown to be hepatocyte nuclear factor (HNF) 4 by the electrophoretic mobility-shift assay (EMSA) using various competitor DNAs and anti-HNF4 serum. A heat-stable factor was purified from rat liver nuclear extract and was resolved as two protein bands migrating at about 33 kDa on SDS/polyacrylamide gels. Peptide sequence analysis revealed that these proteins were nuclear factor (NF) 1-L and NF1/Red1. The heat-stable factor was also identified as a member of the NF1 family by using various competitor DNAs and anti-NF1 serum in an EMSA. In addition, we found that a factor bound to the accessory site of the rat S14 gene, which is necessary for carbohydrate responsiveness of this gene, was also a member of the NF1 family, raising the possibility that the NF1 family is involved in the carbohydrate regulation of gene transcription by interactions with other proteins. The NF1 family members and HNF4 interacted with overlapping sequences of the L-II element, wherein the 5' half-site was more critical for NF1 binding, and the 3' site was more important for HNF4 binding. Co-transfection of a vector expressing either NF1-L or NF1/Red1 repressed the transcription of the PKL enhancer unit-chloramphenicol acetyltransferase (CAT) fusion gene in HepG2 cells, whereas co-transfection of a vector expressing HNF4 activated the transcription of the same reporter gene. Furthermore NF1 family members antagonized the effect of HNF4 on PKL enhancer unit-CAT fusion gene expression when both expression plasmids were co-transfected. We conclude that NF1 family members and HNF4 regulate transcription of the PKL gene in an opposing manner by binding overlapping sequences of the L-II element.

Amino Acid Sequence↗
PubMed2002

ChREBP rather than USF2 regulates glucose stimulation of endogenous L-pyruvate kinase expression in insulin-secreting cells.

There is controversy whether or not upstream stimulatory factors (USF) regulate the glucose responsiveness of L-pyruvate kinase (L-PK) promoter activity in hepatocytes. It has been suggested that USF-2 is required for glucose stimulation of L-PK promoter activity in single islet beta-cells and INS-1 cells (Kennedy, H. J., Viollet, B., Rafiq, I., Kahn, A., and Rutter, G. A. (1997) J. Biol. Chem. 272, 20636-20640). In the present study, the tet-on system has been employed to achieve tightly controlled and inducible expression of USF-1 and -2 and their dominant-negative mutants DN-USF-1 (DeltabTDU1) and -2 (TDU2) in INS-1 cells. Quantitative Northern blot analysis shows that neither basal level nor glucose responsiveness of endogenous L-PK mRNA is affected by overexpression of USF-1 and -2. Likewise, the L-PK expression is unaltered by dominant-negative suppression of USF function. Western blotting demonstrates that USF-1 and -2 and DN-USF-1 and -2 proteins are stably expressed in nuclear fractions of INS-1 cells. Immunofluorescence staining indicates the uniform induction of these transgene-encoded proteins in the cell nuclei. Electrophoretic mobility shift assays using the L-PK promoter segment reveal that induction of USF-1 and -2 dramatically enhances the USF binding activity, whereas DN-USF-1 and -2 abolish binding. DN-USF-1 and -2 exert their dominant-negative effect by forming non-functional heterodimers with endogenous USF proteins. Carbohydrate response element-binding protein (ChREBP) was recently shown to regulate the glucose responsiveness of the L-PK promoter activity in hepatocytes. We now report the presence of this transcription factor in rat islets and INS-1 cells. Glucose stimulates ChREBP transcription in INS-1 cells, as shown by nuclear run-on experiments. Overexpression of ChREBP in INS-1 cells using the tet-on system results in a left shift of glucose responsiveness of L-PK expression and an enhanced L-PK promoter activity. Both endogenous and doxycycline-induced ChREBP proteins bind to the L-PK promoter in a glucose-dependent manner. These unprecedented results suggest that ChREBP rather than USF mediates glucose-promoted L-PK expression in insulin-secreting cells.

Animals↗
PubMed2005

Cytosolic pyruvate kinase: subunit composition, activity, and amount in developing castor and soybean seeds, and biochemical characterization of the purified castor seed enzyme.

Antibodies against Brassica napus cytosolic pyruvate kinase (PKc) (EC 2.7.1.40) were employed to examine PKc subunit composition and developmental profiles in castor and soybean seeds. A 56-kDa immunoreactive polypeptide was uniformly detected on immunoblots of clarified extracts from developing castor endosperm or soybean embryos. Maximal PKc activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (micromol of pyruvate produced/min) g(-1) FW, respectively) and were up to 25-fold greater than those of fully mature seeds. Time-course studies revealed a close correlation between extractable PKc activity and the relative amount of the immunoreactive 56-kDa PKc polypeptide. PKc from developing COS was purified 1,874-fold to homogeneity and a final specific activity of 73.1 (micromol of pyruvate produced/min) mg(-1) protein. Gel filtration and SDS-PAGE indicated that this PKc exists as a 230-kDa homotetramer composed of 56-kDa subunits. The mass fingerprint of tryptic peptides of the 56-kDa COS PKc subunit best matched three putative PK(c)s from Arabidopsis thaliana. The purified enzyme was relatively heat-stable and displayed a broad pH optimum of 6.4. However, more efficient substrate utilization (in terms of Vmax /Km for phosphoenolpyruvate or ADP) was observed at pH 7.4. Glutamate was the most effective inhibitor, whereas aspartate functioned as an activator by partially relieving glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PKc and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PKc exists as tissue-specific isozymes that exhibit substantial differences in their respective physical and regulatory properties.

Allosteric Regulation↗
PubMed1994

Developmental variations in the interactions of pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase with subcellular structure in cavian tissues.

The activities and interactions with cellular structure of glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase have been studied in the major tissues of the guinea pig during development. The extent of activity variation in these tissues is described along with the putative physiological determinants of these alterations in activity. As to binding, overall the present data provide a firm indication that the extent of enzyme-structure interactions is appreciable at all ontogenic stages, and when viewed in conjunction with other parallel studies on other enzymes and other animals, serve to confirm the broad biological significance of enzyme-structure associations in the compartmentation of glycolysis. The existence and significance of genetic and epigenetic modifications of these enzymes during development is also discussed.

Aging↗
PubMed1972

A kinetic study of Baker's-yeast pyruvate kinase activated by fructose 1,6-diphosphate.

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by yeast pyruvate kinase when activated by fructose 1,6-diphosphate and K(+). The experimental results indicate that the reaction mechanism is of the Ordered Tri Bi type with the substrates binding in the order phosphoenolpyruvate, ADP and Mg(2+). Direct phosphoryl transfer takes place in the quaternary complex, with pyruvate released before MgATP. A dead-end enzyme-pyruvate complex is also indicated. Values have been determined for the Michaelis, dissociation and inhibition constants of the reaction. Several of the rate constants involved have also been evaluated.

Adenosine Diphosphate↗
PubMed1987

Erythrocyte pyruvate kinase (PK): the variable significance of "nucleotide specificity" in the characterization of mutant variants.

The half-saturation constant (K0.5s) phosphoenolpyruvate (PEP) for red cell pyruvate kinase (PK) with co-factors UDP and GDP is less than one-half that with ADP with or without additions of the allosteric modifier, fructose-1, 6-dephosphate (F-1, 6-P2) to the assay. The Vmax is markedly greater with ADP than with UDP or GDP, but with (PEP) at 0.5 mM, activity with all co-factors is about equal and at lower concentrations greater with UDP and GDP. With high K0.5s (PEP) mutant enzymes, and at the usual test concentration (lmM) for PEP when nucleotide specificity is assessed, the abnormally low saturation of variant enzymes may result in higher activity with UDP and GDP than with ADP--the opposite of the "normal situation." The apparent aberration in nucleotide specificity may thus be illusory and secondary to the abnormal K0.5s (PEP) of the mutant. Example data are recorded. Variations in K0.5s (PEP) may also be introduced during enzyme preparation for assay, particularly when partial purification is employed.

Erythrocytes↗
PubMedDate not supplied

Measurement of fecal pyruvate kinase type M2 (tumor M2-PK) concentrations in patients with gastric cancer, colorectal cancer, colorectal adenomas and controls.

BACKGROUND: The determination of Tumor M2 Pyruvate Kinase (Tumor M2-PK) in EDTA plasma has been described as a tumor marker in a variety of different tumor types. Since most GI tumors grow intraluminally, it appeared interesting to determine whether Tumor M2-PK is detectable in the feces of tumor patients. MATERIALS AND METHODS: Measurements were performed with a commercially available ELISA (ScheBo Biotech AG, Giessen, Germany) modified for fecal analysis. Samples of controls, GI cancer and adenoma patients were tested. RESULTS: Fecal Tumor M2-PK concentrations could be quantified. A significant difference between cancer patients and controls was found. The highest concentrations were observed in colorectal cancer. However, fecal Tumor M2-PK was not detectable in 5 tumor patients. CONCLUSION: The measurement of fecal Tumor M2-PK concentrations might provide an interesting screening tool for colorectal cancer. Further studies comparing the determination of Tumor M2-PK in stool to occult blood in stool are in progress.

Adenoma↗
PubMed1983

Analysis of progress curves. Interaction of pyruvate kinase from Escherichia coli with fructose 1,6-bisphosphate and calcium ions.

The influence of fructose 1,6-bisphosphate and Ca2+ on the kinetics of pyruvate kinase from Escherichia coli K12 was studied (at pH 7.0 and 25 degrees C) by using the pH-stat method for the measurement of the reaction progress as well as initial-rate analysis. The data were analysed on the basis of a concerted model with three conformational states [Markus, Plesser, Boiteux, Hess & Malcovati (1980) Biochem. J. 189, 421-433] by using a novel procedure for a computer-directed treatment of progress curves [Markus & Plesser (1976) Biochem. Soc. Trans. 4, 361-364]. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phosphoenolpyruvate and Mg2+ is abolished and the activity of the enzyme is described by classical saturation kinetics. This is explained by exclusive binding of fructose 1,6-bisphosphate at an allosteric site of the conformational state that forms the active complex. We observe that Ca2+ is an activator of the enzyme at low Mg2+ and Ca2+ concentrations; otherwise it is an inhibitor. These effects can be understood by assuming that Ca2+ has the same binding properties as Mg2+, although it does not allow a catalytic turnover.

Calcium↗
PubMed1986

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis.

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.

Gluconeogenesis↗
PubMed1975

Pyruvate kinase isozyme patterns of human neoplastic, fetal and adult tissues.

A method of starch gel electrophoresis is described which enables detection of 6 isozymes of human pyruvate kinase (PK, E.C.2.7.1.40). Comparing the PK-isozyme patterns of 53 diverse human tumors with those of normal adult and fetal organ tissues, it was found that the isozyme PK I is predominant in all malignant tumors as well as in almost all fetal organs.

Adult↗
PubMed1999

[Molecular study of red cell pyruvate kinase deficiency in 15 patients with chronic hemolytic anemia. Group of Erythropathology of Hematology and hemotherapy Association of Spain (HHAS)].

BACKGROUND: Identification of RBC pyruvate-kinase (PK) gene mutations by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) followed by PK gene sequencing in positive cases has been assessed and the results obtained with a preliminary study of 15 unrelated patients of Spanish origin are presented. PATIENTS AND METHODS: Patients have been classified into two different groups: group 1, propositus (15 cases), and group 2, relatives of the patients included in group 1 (10 males and 5 females). In group 1, a PCR was followed by SSCP and sequencing, and in group 2, the PCR was followed by digestion with specific restriction endonucleases (PCR-ER). RESULTS: Group 1: from 15 patients included in the study 2 were identified as homozygous, 4 as heterozygous and 9 as compound heterozygous. In this group, were identify 26 affected alleles with 11 different mutations: T1456 10 alleles (38.6%), T721 3 alleles (11.6%), A1010, C514, C1015 and T1223 2 alleles (7.7%), and C1070, A1291, T1508, A1595 y T1675 one allele. Relatives from 8 out of 15 patients from group 1 showed the following pattern: homozygous (one case), heterozygous (10 cases), compound heterozygous (2 cases) and normal (2 cases). CONCLUSIONS: SSCP procedure followed by direct gene sequencing in positive cases is fast and simple enough to allow the identification of PK deficient variants, avoiding the need of biochemical characterisation of semipurified deficient enzyme, which is more cumbersome and time consuming. In addition, the PCR-ER method is a very useful tool for screening of the most frequent molecular variants, as well as, for the detection of the carrier condition of this enzymopathy (family studies).

Amino Acids↗
PubMed1990

ATP inhibition of Phycomyces pyruvate kinase: a kinetic study of the inhibitory effects on the allosteric kinetics shown by the enzyme.

Studies on ATP effects on the allosteric kinetics shown by pyruvate kinase from Phycomyces blakesleeanus NRRL 1555 (-) are reported. Phosphoenolpyruvate showed an allosteric ATP-dependent substrate inhibition. The results supported the existence of spatially distinct catalytic binding sites and the inhibitory binding sites for phosphoenolpyruvate, and ATP showed opposite heterotropic effects with respect to these two types of binding site. With respect to Mg2+ ions, ATP caused a negative heterotropic effect. The global inhibitory effect of ATP was in agreement with the predictions postulated by the two-state concerted-symmetry model of Monod, Wyman and Changeux.

Adenosine Triphosphate↗