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The binding affinity of (alpha-P-borano) and other NTP analogs to rabbit muscle pyruvate kinase (PK) was investigated using a fluorescence quenching approach to obtain structure-activity relationships for substrate specificity of nucleotide analogs.
Intermediates have been synthesized that are rapidly utilized by triose phosphate isomerase, yeast aldolase and pyruvate kinase. In each case the compounds have the properties of an enol expected for a stepwise proton transfer mechanism. Apparently the apparatus required for doing this chemistry is sufficiently unique for a large measure of structural homology to have been imposed upon the enzymes of this class during evolution.
The initial velocity of the reaction catalysed by rabbit muscle pyruvate kinase was studied as a function of the concentrations of the modifiers phenylalanine and fructose 1,6-bisphosphate under conditions where the relationships between the initial velocities and the concentrations of substrates are non-hyperbolic. It is shown that these data can be represented by the exponential model for a regulatory enzyme.
The synteny of human mannose phosphate isomerase and pyruvate kinase and the assignment of the genes for these two enzymes to chromosome 15 were confirmed by analysis of 43 independently derived human-mouse hybrid clones. Hybrids between mouse cells deficient in hypoxanthine-guanine phosphoribosyltransferase and human fibroblasts carrying an X/15 chromosome translocation were also included in this study.
Pyruvate kinase catalyzes the phosphorylation of 3-nitro-2-hydroxypropionate by ATP. The product (3-nitro-2-phosphopropionate) decomposes nonenzymatically and inactivates the enzyme. The inactivation process is irreversible and the enzyme is completely protected against inactivation by mercaptoethanol or the competitive inhibitor oxalate. The Km for 3-nitro-2-hydroxypropionate (DL) is 40 mM and kcat = 5 s-1 at pH 8.6. In terms of kcat/km, 3-nitro-2-hydroxypropionate is a fourfold better substrate than glycolate.
A method based on isothermal calorimetry is described for the direct kinetic assay of pyruvate kinase. In agreement with earlier findings based on the standard coupled assay system for this enzyme in the presence of a fixed ADP concentration, the essentially rectangular hyperbolic dependence of initial velocity upon phosphoenolpyruvate concentration is rendered sigmoidal by the allosteric inhibitor phenylalanine. This effect of phenylalanine can be countered by including a high concentration of a space-filling osmolyte such as proline in the reaction mixtures. This investigation thus affords a dramatic example that illustrates the need to consider potential consequences of thermodynamic nonideality on the kinetics of enzyme reactions in crowded molecular environments such as the cell cytoplasm.
An electrophoretic study was made of the isozymes of pyruvate kinase (PK) in various adult and foetal tissues, normal or pathological (hypothyroid or dysmature as the result of retardation of intra-uterine growth) rats. The simultaneous kinetic study of PK with and without fructose diphosphate (FDP) showed that retardation of the maturation of the tissues revealed the presence of the M2 isozyme (PK III) longer than the normal foetal age. The increase of negative charges, observed in relation to age, may be the result of structural changes in the enzyme molecule which are yet to be resolved. The variations of relative electrophoretic mobility, and the reactivation of the isozyme by FDP suggest that there is a repression of the synthesis of certain isozymes in normal adult organs.
In liver, L-type pyruvate kinase (LPK) was considered to be the major isozyme. Using an immunocytometric method, it was demonstrated that labeled LPK (LPK+) hepatocytes were largely influenced by the nutritional status of the Rat, as well as addition of glucagon in vitro. In the fed Rats, the percentage of LPK-hepatocytes remained stable for at least 90 min. This decreased to one half value in the fasted Rats. Addition of glucagon (114 nM) had no effect in fasted Rats, but decreased the percentage of LPK-hepatocytes to a half in the fed Rats. It was concluded that neoglucogenesis status (fasting or addition of glucagon in fed Rats liver cells) induced a fall in the percentage of LPK-hepatocytes.
It was shown that pyruvate kinase (PK) in the supernatant fraction from Calicophoron ijimai is able to regulate the direction of metabolic flow at glucose break down from phosphoenolpyruvate (PEP) level. The enzyme for activity required substrate, dinucleotides, cations K+ and Mn++. The activity with Mg++ as divalent cation is low. The addition of fructose-1.6-diphosphate (FDP) did not affect the enzyme activity with Mn++, however, increased the affinity for PEP. The velocity of Mg++ activated reaction increased by 8.2 times in the presence of FDP. PK in C. ijimai is sensitive to ATP inhibition, being weakly inhibited by malate. L-alanine did not influence on the enzyme activity. The effect of some anthelminthic preparations on the PK activity was shown.
The expression of the pyruvate kinase (PK) isoenzymes L and M2 was analysed in the livers of rats treated with the hepatocarcinogenic agent N-nitrosomorpholine (NNM) in the drinking water. In control animals L-PK expression was restricted to liver parenchymal cells, whereas M2-PK was detected in bile duct epithelial, blood vessel wall, endothelial and Kupffer cells. In rats treated with NNM proliferating oval cells were consistently L-PK negative and M2-PK positive, while the ductal cells of cholangiofibroses were clearly L-PK positive and coexpressed M2-PK. However, no morphological differentiation of ductal cells into hepatocyte-like cells was observed. In the clear and acidophilic cell foci storing glycogen in excess strong staining for L-PK was observed. In glycogen-poor foci induced by NNM a shift from L-PK to M2-PK expression takes place.
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By ammonium-sulphate fractionation, phosphocellulose column chromatography, and isoelectric focusing, pyruvate kinase (EC 2.7.1.40) was purified from pig dental pulps to yield a homogeneous preparation with a single protein band of approx. 63,000 daltons upon SDS-polyacrylamide gel electrophoresis. As the molecular weight of the enzyme was estimated to be 250,000 by Sephadex G-200 gel filtration, a tetrameric structure is indicated. The isoelectric point of the purified enzyme was 8.0; the optimal pH, pH 7.2. At this pH, there was no substrate inhibition of the enzyme by either phosphoenolpyruvate (PEP) or ADP, and the relationship between reaction velocity and each substrate concentration was well-explained by the Michaelis-Menten equation. The Km values were 53 and 286 microM for PEP and ADP, respectively. The enzyme had different catalytic properties at pH 8.0: with 0.4 mM ADP, the kinetic behaviour gave a sigmoidal curve with respect to PEP and fructose-1,6-diphosphate acted as an allosteric activator. With 2.0 mM ADP, a hyperbolic curve resulted with PEP.
The influence of temperature, K+, Mg2+ and fructose 1,6-bisphosphate on human red cell pyruvate kinase was investigated. Kinetic measurements between 4 degrees C and 43 degrees C revealed a remarkable influence of the temperature on the allosteric behaviour of the enzyme. Below a transition region between 15 degrees C and 20 degrees C (as obtained from an Arrhenius plot) the enzyme shows non-cooperative behaviour, as can be deduced from Michaelis-Menten, Hill and Scatchard plots. At temperatures above 20 degrees C cooperativity increases with rising temperature. This effect becomes even more pronounced at higher temperatures upon addition of increasing amounts of K+ and Mg2+ accompanied by a slight decrease of the reaction velocity. Fructose 1,6-bisphosphate, however, abolishes cooperativity at every temperature and salt concentration measured. Difficulties which arise in evaluating the correct values of V, Km and the Hill coefficient nH with cooperative systems are met by using a computer program of Wieker, Johannes and Hess, especially designed for the determination of kinetic parameters obtained from sigmoidal steady-state kinetics.
We describe the cellular and molecular biologic studies of the erythrocyte pyruvate kinase (PK) deficiency of the Amish deme in Pennsylvania. Nucleotide sequencing of the patient's PK gene showed a point mutation, CGC to CAC, corresponding to no. 1436 from the translational initiation site of the R-type PK (R-PK) mRNA, and it caused a single amino acid substitution from Arg to His at the 479th amino acid residue of the R-PK. The substituted Arg residue is located in the C domain of PK subunit, that is essential for both the intersubunit contact and the allosteric regulation. Because this enzyme shows the catalytic activity only as a dimer or tetramer, it is rational that the structural alteration would result in severe PK deficiency. To elucidate the effect of the PK deficiency on red blood cell (RBC) membrane, we performed the cellular studies of the patients' RBCs. Ouabain-insensitive K+ efflux was increased to 142% to 145% of normal controls and not inhibited by furosemide, as previously observed in HbSC disease RBCs.
Elevated glucose concentrations stimulate L-pyruvate kinase (L-PK) gene transcription in liver and islet beta-cells. A glucose response element termed the L4 box (two noncanonical E-boxes located -165 and -154 base pairs upstream of the transcriptional start point) has previously been defined within the proximal promoter region of the gene. However, the identity of the transacting factor(s) which binds to this site remains unclear. We have used photon counting digital imaging of firefly luciferase activity to monitor promoter activity continuously in single living islet beta and derived INS-1 cells, and to analyze the molecular basis of the regulation by glucose. L-PK promoter activity, normalized to cytomegalovirus promoter activity using the distinct Renilla reniformis luciferase, was >/=6-fold higher in cells cultured at 16 mM glucose or above compared with cells cultured at 3 mM glucose. Microinjection of antibodies against the ubiquitous transcription factor USF2 inhibited L-PK promoter activity in beta- and INS-1 cells incubated at 30 mM glucose by 71-87%. Anti-USF2 antibodies had a much smaller effect on promoter activity in INS-1 cells cultured at 3 mM glucose, and on the activity of a modified promoter construct lacking an L4 box. These data support the view that glucose enhances L-PK gene transcription in beta-cells by modifying the transactivational capacity of USF2 bound to the upstream L4 box.
BACKGROUND: Recently, a high validity correlation of the tumor M2 pyruvate kinase (Tu M2-PK) isoenzyme in comparison to standard tumor markers has been demonstrated in solid tumors. We investigated this marker in 67 patients with advanced breast cancer (ABC) in comparison to healthy controls. MATERIALS AND METHODS: Plasma Tu M2-PK was measured using an ELISA assay (ScheBo Tech, Giessen, Germany) while serum CA27.29 was determined using a chemiluminescent immunoassay (Bayer Diagnostics, Tarrytown, USA). RESULTS: In a ROC analysis, the cut-off to discriminate patients from controls was established at 15 U/ml for Tu M2-PK (specificity 85%; positive predictive value 81%) and 30 U/ml for CA27.29 (specificity 91%; positive predictive value 92%). Median ABC baseline levels (ranges) in patients with ABC for Tu M2-PK and CA27.29 were 12.8 U/ml (4.8-252,495) and 130 U/ml (13.3-8130), respectively. Response assessment was done in 45 chemotherapy courses of 38 pts. In 13 out of 19 blocks (68.4%) with PD (progressive disease), an elevated level of Tu M2-PK at baseline or in the follow-up was found. In 17 out of 20 blocks (85%) with SD (stable disease), the Tu M2-PK level was normal at baseline or normalised within 4 weeks of treatment. All 6 patients with disease remission had a normal baseline Tu M2-PK level or the levels decreased promptly. CONCLUSION: Tu M2-PK gives additional information about ABC, indicating disease activity and sensitivity to chemotherapy while CA27.29 reflects tumor burden.
Proliferating cells express the pyruvate kinase isoenzyme type M2 (M2-PK). This enzyme exists as an active tetramer and an inactive dimer. The dimeric form is predominantly found in tumor cells and is therefore termed Tumor M2-PK (TuM2-PK). TuM2-PK molecules are released into the peripheral blood and may hereby function as a marker of tumor load in cancer patients. Our study was aimed to investigate TuM2-PK as a potential plasma marker in melanoma patients compared to the well-established serum marker S100beta. We measured the concentration of TuM2-PK in plasma and S100beta in corresponding serum samples from 300 melanoma patients and 53 healthy controls using a sandwich ELISA and an immunoluminometric assay, respectively. Plasma concentrations of TuM2-PK were significantly increased in melanoma patients compared to healthy controls (9.30 U/ml vs. 7.20 U/ml; p = 0.0036) and correlated with tumor load (p < 0.0005) and disease stage (p < 0.0005). Patients with elevated plasma TuM2-PK (cut-off = 15 U/ml) presented a reduced overall (p < 0.000005) and progression-free (p = 0.023) survival. Multivariate analysis revealed plasma TuM2-PK and serum S100beta as independent predictors of overall survival in metastasized patients. Neither plasma TuM2-PK nor serum S100beta showed prognostic relevance for tumor-free patients. Although the sensitivity and specificity to predict disease progression or death was higher for serum S100beta compared to plasma TuM2-PK, the combination of both markers improved the estimation of prognosis compared to that of serum S100beta alone.
Expression of L-type pyruvate kinase (L-PK) is upregulated in the liver by dietary carbohydrate. Previously, 3 carbohydrate/insulin response elements were identified in the 5'-flanking region of the L-PK gene up to bp -170. Studies of the 5'-flanking region beyond bp -183 in transgenic mice suggested that other regulatory elements may be present upstream of bp -183, but the positions of these elements were uncertain. In the present study, the existence of regulatory regions of the L-PK gene responding to stimulation by feeding was examined using in vivo hydrodynamics-based gene transfection (HT) in mouse liver. The firefly-luciferase (FL) gene, fused with various lengths of the 5'-flanking region of the L-PK gene, was introduced into mouse liver by HT. The mice had free access to a high-carbohydrate diet. In liver homogenate, luciferase activity of pL-PK(-1467)-FL (which included the 5'-flanking region from bp -1467 to +17), was markedly stimulated by feeding. 5'-Deletion up to bp -1065 caused only minor changes in luciferase activity, but further deletion up to bp -690 and bp -203 caused significant, gradual decreases in activity. Further analyses utilizing 5'-deletion mutants indicated the existence of positive regulatory regions that respond to stimulation by feeding between bp -1065 and -945, and between -300 and -203 on the L-PK gene. These results suggest that unidentified cis-acting DNA elements exist in the upstream region of the L-PK gene, and that HT is a useful approach for detecting regulatory regions of genes expressed in the liver.