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PubMed1995

Diet differentially regulates glucokinase and L-type pyruvate kinase gene expression in rat liver.

The regulation of gene expression of glucokinase (GK) and L-type pyruvate kinase (L-PK) in rat liver was investigated and compared with the previously reported regulation of lipogenic enzymes. Experiments were conducted in which the time courses and responses to diet quantity of mRNA concentrations and enzyme activities after refeeding a carbohydrate/protein diet (CP) to food-deprived rats were measured. The effects of dietary nutrients on the gene expression were investigated in rats refed either the CP diet, a carbohydrate diet without protein (C), a protein diet without carbohydrate (P), or a carbohydrate/protein/corn oil diet (CPF). The effects of the CPF diet on the gene expression after insulin treatment to diabetic rats were also investigated. After refeeding the CP diet, GK mRNA concentration and enzyme activity reached maximum levels in 2 h and 16-24 h, respectively, whereas those of L-PK peaked in 16 h and 48 h, respectively, similar timecourse to lipogenic enzymes. Moreover, GK mRNA concentrations were maximal in rats fed 20% of the ad libitum diet intake, and L-PK mRNA concentrations, like lipogenic enzyme mRNA, were maximal in rats fed approximately 50% of ad libitum intake. GK mRNA concentrations were significantly increased in parallel with an increase in plasma insulin and glucose concentrations. GK and L-PK mRNA and enzyme levels in rats fed the C diet were comparably induced to the levels in those fed the CP diet. L-PK mRNA induction by the CP diet was significantly reduced by dietary polyunsaturated fatty acids (CPF diet), whereas the GK mRNA induction was not significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Identification of a 6 base pair insertion in West Highland White Terriers with erythrocyte pyruvate kinase deficiency.

OBJECTIVE: To define the disease-causing mutation in West Highland White Terriers (WHWT) with erythrocyte pyruvate kinase (R-PK) deficiency and to design a genetic test capable of recognizing affected (homozygous) and carrier (heterozygous) dogs. ANIMALS: 3 anemic WHWT littermates and 1 unaffected littermate; 16 dogs from the same kennel, including 4 unrelated, phenotypically normal dogs (control dogs), and 12 for which PK activity was not known; 2 PK-deficient Basenjis; 2 PK-deficient Beagles; 4 unaffected English Springer Spaniels; and 1 mixed-breed dog. PROCEDURES: cDNA was cloned and sequenced, and cDNA sequences were compared with the published sequence for canine R-PK cDNA to identify the putative disease-causing mutation. Genomic DNA spanning the affected region was cloned and sequenced to verify the mutation. Subsequently, polymerase chain reaction primers were designed to amplify the section of the gene containing the mutation from DNA in blood or buccal swab samples. Gel electrophoresis allowed assignment of genotypes on the basis of allele separation. RESULTS: 4 single base polymorphisms attributable to sequencing errors in the published sequence were identified, along with a 6 base pair (bp) insertion in exon 10 that was recognized as a putative disease-causing mutation. An identical insertion was found in genomic DNA. Amplification of genomic DNA yielded a 117 bp product for genotypically normal dogs and a 123 bp product for WHWT homozygous for PK deficiency. Carriers had 1 copy of each allele and variable heteroduplex structures. CONCLUSIONS AND CLINICAL RELEVANCE: A 6 bp insertion in the C domain of R-PK was identified in WHWT with PK deficiency. Affected and carrier dogs could be distinguished with a genetic test.

Animals↗
PubMed2000

Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures: implications for the integration of glycolysis with nitrogen assimilation.

Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201-fold to electrophoretic homogeneity and a final specific activity of 51 micromol phosphoenolpyruvate utilized per min per mg protein. SDS/PAGE and gel filtration analyses of the final preparation indicated that this PKc is a 220-kDa homotetramer composed of 56-kDa subunits. The enzyme was relatively heat-stable and displayed a broad pH optimum of pH 6.8. PKc activity was absolutely dependent upon the simultaneous presence of a bivalent and univalent cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed for phosphoenolpyruvate, ADP, Mg2+ and K+ (apparent Km values = 0.12, 0.075, 0.21 and 0.48 mM, respectively). Although the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate, oxalate, and the flavonoids rutin and quercetin were the most effective inhibitors (I50 values = 4, 0.3, 0.07, and 0.10 mM, respectively). L-Aspartate functioned as an activator (Ka = 0.31 mM) by causing a 40% increase in Vmax while completely reversing the inhibition of PKc by L-glutamate. Reciprocal control by L-aspartate and L-glutamate is specific for these amino acids and provides a rationale for the in vivo activation of PKc that occurs during periods of enhanced NH +4-assimilation. Allosteric features of B. napus PKc are compared with those of B. napus phosphoenolpyruvate carboxylase. A model is presented that highlights the pivotal role of L-aspartate and L-glutamate in the coordinate regulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.

Adenosine Diphosphate↗
PubMed1994

Expression of the L-type pyruvate kinase gene and the hepatocyte nuclear factor 4 transcription factor in exocrine and endocrine pancreas.

The L-pyruvate kinase (L-PK) gene is slightly active in normal and tumoral endocrine pancreatic tissues while, in vivo, this gene is not transcribed in the exocrine pancreas. Nevertheless, the L-PK gene is re-expressed at a very low level in cultured 266.6 cells derived from an exocrine pancreas carcinoma. The L-PK gene is early activated in endodermal tissues, e.g. yolk sac and primitive intestine; it remains transcribed in fetal pancreas. In adult, L-PK gene expression is restricted to some endocrine cells. Hepatocyte nuclear factor (HNF) 1 and HNF4 are the main tissue-restricted transcription factors involved in tissue-specific expression of the L-PK gene. HNF1 concentration is similar in liver and all pancreatic cells. HNF4 concentration is high in liver, much lower in islets of Langerhans, endocrine pancreatic tumors, and cultured insulinoma cells, and is scarcely detectable in adult exocrine pancreas. This distribution of HNF4 parallels the expression of the L-PK gene. In vivo footprinting experiments show that the HNF1 binding site is similarly occupied in both adult liver and adult pancreas, in which this gene is practically inactive. In this latter tissue, however, the HNF4 binding site is differently occupied with respect to the liver. Since the chromatin structure remains open around the L-PK promoter in pancreas, the L-PK gene can probably be re-expressed under certain circumstances, for instance in cancerous pancreatic cells.

Animals↗
PubMed1997

Frame shift mutation, exon skipping, and a two-codon deletion caused by splice site mutations account for pyruvate kinase deficiency.

Three novel splice site mutations and two novel missense mutations were identified by molecular analysis of pyruvate kinase (PK) deficiency associated with hereditary nonspherocytic hemolytic anemia. A Nepalese PK variant, PK Kowloon, was found to have a homozygous transversion at the 5'-splice site of the seventh intervening sequence (IVS) of the L-type PK gene (Ivs7[+1]gt --> tt). Using a reverse transcription polymerase chain reaction (RT-PCR) assay, we showed that the R-type PK mRNA in the proband's reticulocytes included the seventh IVS between the seventh and eighth exon, introducing a stop codon 3 nucleotides downstream of the mutated site. Consequently, the translational product may lack 44% of the R-PK polypeptide. A transition at the last nucleotide of exon 9 (1269GCG --> GCA) was found in a Japanese PK variant, PK 'Kamata.' The mutation did not alter the amino acid sequence, but caused skipping of the ninth exonic sequence in the R-PK transcripts. As a result, the affected R-type PK lost 51 amino acid residues (373Met-423Ala del). A transversion at the splice acceptor site of the third IVS (Ivs 3[-2]ag --> tg) was identified in PK 'Aomori.' The mutation resulted in aberrant splicing at a cryptic splice site within exon 4, causing deletion of two codons in the aberrant R-PK transcript (95 Gly-96 Pro --> del). Both PK 'Kamata' and PK 'Aomori' had a missense mutation on the other allele, 1044AAG --> AAT (348Lys --> Asn) and 1075CGC --> TGC (359Arg --> Cys), respectively. Although both 348Lys and 359Arg were located in the sixth loop of A domain (beta/alpha)8 barrel, which has been shown to contain the substrate and cation binding sites, the degree of anemia was much more severe in PK 'Kamata' than PK 'Aomori,' possibly because the 51 amino acid deletion of PK 'Kamata' but the 2 amino-acid deletion of PK 'Aomori' may abolish PK catalytic activity.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1996

Tissue specificity of L-pyruvate kinase transgenes results from the combinatorial effect of proximal promoter and distal activator regions.

The L-type pyruvate kinase (L-PK) gene is regulated by diet and hormones and expressed at high levels in the hepatocytes, enterocytes, and proximal tubular cells of the kidney and at low levels in the endocrine pancreatic cells. Two regulatory regions have been shown to be important in transgenic mice to confer on a reporter gene a similar tissue-specific and diet-responsive expression: a proximal promoter fragment, with binding sites for the tissue-specific hepatocyte nuclear factors 1 and 4, and presence of the glucose-response element (GIRE) and a distal activator corresponding to a liver-specific hypersensitive site at -3000 bp with respect to the cap site. Although the proximal promoter is able to confer by itself tissue-specific expression on a reporter gene, its activity in vivo is strongly stimulated by the distal activator. To determine the possible role of the distal region on diet responsiveness and tissue specificity of the L-PK gene expression, we have created lines of transgenic mice in which the gene for SV40 T antigen (Tag) was directed by composite regulatory sequences consisting of the L-PK promoter and different enhancers: either the SV40 early enhancer (SV) or the H enhancer of the aldolase A gene (H). The induction of the composite H-PK/Tag and SV-PK/Tag transgenes by a carbohydrate-rich diet in the liver was similar to that of the endogenous L-PK gene. This suggests that in fasted mice the L-PK promoter, and especially the GIRE, is able to silence the activating influence of a strong viral enhancer such as the SV40 enhancer. The H-PK/Tag mice expressed the transgene similarly to the endogenous gene, except in the pancreas, where expression was practically undetectable. Consistently, whereas L-PK/Tag mice develop insulinomas, H-PK/Tag mice develop only hepatomas. In contrast, the transgene expression was partly aberrant in SV-PK/Tag mice. In addition to a normal activation of the transgene in the liver, a strong expression was also detected in the kidney medulla, whereas the transgene was practically silent in enterocytes. Finally, the effect of the distal region (-2070 to -3200) on an ubiquitous promoter was tested by ligating the distal L-PK gene fragment in front of a thymidine kinase/CAT transgene. Such a transgene was constantly expressed in the pancreas and, strikingly, in the brain. It appears, therefore, that the L-PK distal activator exhibits, by itself, a certain neuropancreatic specificity required in combination with the proximal promoter for L-PK gene expression in pancreas endocrine cells.

Animals↗
PubMed2004

Stimulation of Na+/H+ antiport and pyruvate kinase activities by high glucose concentration in human erythrocytes.

This study aims to demonstrate the effect of high glucose concentrations on NHE-1 and PK activities and investigate the implicated signal transduction pathways. Erythrocytes drawn from healthy volunteers were incubated in the presence of 5 or 50 mM of glucose, fructose, galactose or mannitol. When appropriate, specific inhibitors of NHE-1, PKC or p42/44 MAPK were used. Erythrocyte NHE-1 activity has been estimated by fluorometrical determination of the intracellular pH and quantification of sodium uptake using 22Na. Pyruvate kinase activity was measured by a NADH-lactate dehydrogenase enzymatic assay. p42/44 MAPK activity was assessed with a specific enzyme linked immunosorbent assay (ELISA). Increased concentrations of glucose but not galactose, fructose or mannitol enhanced erythrocyte NHE-1, PK and p42/44 MAPK activity. Inhibition of PKC, counteracted these effects of glucose. Similarly, inhibition of NHE 1 abolished the effect of high glucose on PK and p42/44 MAPK as well. Finally, inhibition of p42/44 MAPK also hindered the effect of glucose on NHE-1 and PK activities. The data of the present study indicate an acute effect of glucose on signal transduction pathways in human erythrocytes. This pathway involves NHE-1, PKC, and p42/44 MAPK. A positive feedback between NHE 1 and p42/44 MAPK is suggested.

Blood Glucose↗
PubMed1978

Relationship of density distribution and pyruvate kinase electrophoretic pattern of erythrocytes in sickle cell diseases and other disorders.

The density distributions of red cells from sickle cell disorders and other hematological diseases were determined. In sickle cell anemia there was an increase in the proportion of cells in both the heaviest and lightest fractions. In hemoglobin SC disease, a small fraction was shifted to heavier cells. Sickle cell trait blood had a normal density pattern. In hereditary spherocytosis an increase in the number of heavy cells was observed. Thin-layer polyacrylamide gel electrophoresis of red cell pyruvate kinase demonstrated that the pyruvate electrophoretic pattern was related to density distribution.

Anemia↗
PubMed1979

Changes in pyruvate kinase isozymes of rat small intestine during development and the synergistic effect on them of thyroid and glucocorticoid hormones.

Pyruvate kinase isozymes in rat small intestine changed markedly during the postnatal period. The activities of type M2 subunits rapidly increased before weaning, while those of type L subunits decreased slightly. The administration of hydrocortisone induced normal changes prematurely, whereas the administration of thyroxine increased their extent. On simultaneous administration, the two hormones had synergistic effects.

Aging↗
PubMed1996

The genes for phosphofructokinase and pyruvate kinase of Lactobacillus delbrueckii subsp. bulgaricus constitute an operon.

In Lactobacillus delbrueckii subsp. bulgaricus, the pyk gene coding for pyruvate kinase and the pfk gene coding for phosphofructokinase formed a bicistronic operon transcribed into a 2.9-kb RNA. The nucleotide sequence of the pyk gene indicated that the encoded protein possessed an extra C-terminal domain with a potential phosphoenolpyruvate-dependent autophosphorylation site.

Amino Acid Sequence↗
PubMed1977

Red cell pyruvate kinase deficiency: adverse effect of oral contraceptives.

Erythrocyte enzymopathies are being detected with increasing frequency. The homozygous state for pyruvate kinase (PK) deficiency usually causes chronic non-spherocytic haemolytic anaemia. Aggravation of the condition by a variety of extrinsic factors has been reported, but drugs have rarely been incriminated. A case of severe PK deficiency is presented in which an anovulant drug upsets the usual haematologic balance. This strengthens the suspicion that contraceptive pills may be hazardous in such patients.

Adult↗
PubMed1977

An isozyme of erythrocyte pyruvate kinase (PK-Los Angeles) with impaired kinetics corrected by fructose-1, 6-diphosphate.

A mutant isozyme of erythrocyte pyruvate kinase was found in a family of French-Canadian ancestry in association with hemolytic anemia. The isozyme was characterized by normal maximal activity, pH optimum, heat stability, and fructosediphosphate activation constants, but had markedly reduced affinity for the substrate, phosphoenolpyruvate. This kinetic defect was corrected almost entirely in vitro by low concentrations of fructosediphosphate.

Anemia, Hemolytic, Congenital↗
PubMed1992

Hereditary nonspherocytic hemolytic anemia due to pyruvate kinase deficiency: a prevalence study in Quebec (Canada).

Fifty-eight cases of hereditary nonspherocytic hemolytic anemia due to pyruvate kinase deficiency were collected from both laboratories that have performed the assay since 1972 and from attending physicians in the province of Quebec. Using the postal addresses of the probands, a prevalence map was constructed according to the administrative regions of the province of Quebec. The prevalence was found to be higher in eastern Quebec (1/81,838) than in western Quebec (1/139,086). Fifty probands were of French Canadian origin whereas the remaining 6 were recent immigrants.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1993

Isolation of two pyruvate kinase activities in the parasitic protozoan Leishmania mexicana amazonensis.

Using phosphocellulose affinity chromatography we were able to separate two pyruvate kinase (EC 2.7.1.40) activities in the parasitic protozoan Leishmania mexicana amazonesis. One activity (PYK1) showed hyperbolic kinetics and was decreased by fructose 2,6-bisphosphate, whereas the second activity (PYK2) showed sigmoidal kinetics for the substrate phosphoenolpyruvate and was activated by fructose 2,6-bisphosphate. Molecular sieve chromatography (Sephacryl S-400) of PYK1 produced a single peak of apparent molecular mass around 200,000, while PYK2 eluted at a position corresponding to M(r) 55,000.

Animals↗
PubMed1975

Activity of some enzymes of carbohydrate metabolism in human subcutaneous adipose tissue in newborns, infants and adults. I. Activity of pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase.

The activity of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, pyruvate kinase (PK) and lactate dehydrogenase (LDH) was measured in human adipose tissue (AT) of newborns aged 0-2 and 2-48 h, of 3- to 5-month-old infants and of adults. AT was sampled by means of a special needle from the gluteal region without anesthesia. We observed significantly higher activity of the two pentose phosphate shunt enzymes when calculated on protein content and wet weight basis in newborns as compared to infants and adults. There were no differences for PK and LDH between newborns and adults when the values were calculated for the soluble protein. The protein concentration of the AT decreased significantly during the development.

Adipose Tissue↗
PubMed1984

Change of pyruvate kinase (PK) isozymes in classical type PK deficiency and other PK deficiency cases during red cell maturation.

Conversion of pyruvate kinase (PK) isozymes during the maturation of erythroblasts in five cases of PK deficiency was compared with that in normal subjects using immunofluorescent antibody techniques. In normal erythroblasts, M2-type PK was clearly seen at the proerythroblast stage, then markedly declined with cell maturation whereas L-type PK continued to increase. In two patients with classical type PK deficiency, M2-type PK was still clearly seen in orthochromatic erythroblast, whereas L-type PK was hardly detected during maturation. In three patients with other types of PK deficiency, change of L-type PK was similar to that of normal subjects whereas M2-type PK was clearly seen even at the later stage of maturation as in classical-type PK deficiency. The present studies indicate that compensatory M2-type PK production occurs in the erythroblasts of patients with PK deficiency.

Adolescent↗
PubMed1988

Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver.

A new method for the histochemical demonstration of pyruvate kinase (PK) activity was developed using a semi-permeable membrane and ATP-dependent phosphorylation of glucose coupled with tetrazolium reduction via glucose-6-phosphate dehydrogenase (G6PD) in order to investigate normal liver tissue and neoplastic hepatic nodules induced by thioacetamide (TAA). A series of control reactions and comparison with microbiochemical analysis of microdissected lyophilised material were used to determine the specificity of the reaction. In agreement with earlier reports, an activity gradient in control liver decreasing from zone 3 to zone 1 was apparent both histochemically and after biochemical analysis. Liver neoplastic nodules induced by 25 weeks dietary thioacetamide administration and characterized by increased G6PD demonstrated a clear decrease in PK activity. In contrast, epithelial cells within areas of cholangiocellular tumour development were characterized by a strong increase. Comparison of the results with immunohistochemical and biochemical data from the literature indicate that the specific histochemical method described will be of great assistance in future assessment of disease and physiological alteration in activity of this key enzyme of glycolysis.

Acetamides↗
PubMed2004

Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer.

Proliferating cells, especially tumour cells, express a special isoenzyme of pyruvate kinase, termed M2-PK, which can occur in a tetrameric form with a high affinity to its substrate, phosphoenolpyruvate (PEP), and in a dimeric form with a low PEP affinity. In tumour cells, the dimeric form is usually predominant and is therefore termed Tumour M2-PK. The levels of Tumour M2-PK within tumours and in EDTA-plasma correlate with staging and the ability of the tumour cells to metastasise. Since most colorectal tumours grow intraluminally, it appeared interesting to determine whether Tumour M2-PK is detectable in the faeces of tumour patients. Stool samples were tested by ELISA from controls without colorectal cancer and colorectal cancer patients. Whereas Tumour M2-PK levels were low in the control group (mean value+/-s.e.m.: 3.3+/-0.4, n=144), they were high in the case of colorectal cancer (56.1+/-15.3, n=60). At a cutoff value of 4 U ml(-1), the sensitivity was 73%. TNM and Dukes' classification of the tumours revealed a strong correlation between faecal Tumour M2-PK levels and staging. The determination of Tumour M2-PK in faeces provides a new promising screening tool for colorectal tumours.

Biomarkers, Tumor↗