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PubMed1985

Metabolic zonation in liver of diabetic rats. Zonal distribution of phosphoenolpyruvate carboxykinase, pyruvate kinase, glucose-6-phosphatase and succinate dehydrogenase.

The activities and zonal distribution of key enzymes of carbohydrate metabolism were studied in livers of diabetic rats. 48 h after alloxan treatment the following alterations were observed, intermediate values being reached after 24 h: Blood glucose, acetoacetate and beta-hydroxybutyrate were increased to more than 500%; liver glycogen was reduced to about 10%. Portal vein insulin was reduced to below 10%, portal glucagon was increased to almost 200%. The glucogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were enhanced to 320% and 150%, respectively. The glycolytic enzymes glucokinase and pyruvate kinase L (differentiated from the M2 isoenzyme with a specific anti-L-antibody) were lowered to 50% and 75%, respectively. The citrate cycle enzyme succinate dehydrogenase remained unchanged. The normal periportal to perivenous gradient of phosphoenolpyruvate carboxykinase of about 3:1, as measured in microdissected tissue samples, was enhanced to about 4:1 with activities elevated to 230% and 190%, respectively, in the two zones. The normal periportal to perivenous gradient of pyruvate kinase L of about 1:1.7, as determined with the microdissection technique, was reduced to about 1:1.4 with levels lowered to 55% and 45%, respectively, in the two zones. The even zonal distribution of pyruvate kinase M2 remained unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Pyruvate kinase of sea bass liver: interrelationship in regulating properties.

Pyruvate kinase of sea bass liver show a joint modulation of both pH and temperature for PEP substrate. The effect of F-1,6-P2, alanine and ATP is not fundamentally affected by a variation in pH. The kinetic constants in the presence of ATP are not affected, but the intensity of its inhibitor effect varies with temperature. A study of different buffers: Tris-HC1, Tris-maleic and phosphate, on this enzymatic activity with or without effectors has been made.

Alanine↗
PubMed1968

The preparation and properties of pyruvate kinase attached to porous sheets, and the operation of a two-enzyme continuous-feed reactor.

1. The covalent attachment of pyruvate kinase to filter-paper disks and some kinetic properties of the resultant enzymically active porous sheets are described. 2. A two-enzyme reactor was constructed by using water-insoluble sheets of lactate dehydrogenease in conjunction with the above-mentioned sheets possessing pyruvate kinase activity.

Adenine Nucleotides↗
PubMedDate not supplied

Phylogenetic analysis of the putative phosphorylation domain in the pyruvate kinase of Bacillus stearothermophilus.

All sequenced phosphoenolpyruvate synthases (PPS), pyruvate:phosphate dikinases (PPDK) and enzymes I (EI) of the phosphoenolpyruvate:sugar phosphotransferase system comprise the PEP family. Linked to the C terminus of the sequenced pyruvate kinase from Bacillus stearothermophilus (PKBst) is a domain that is homologous to the putative phosphorylation domains of PEP family enzymes. We report sequence and phylogenetic analyses that lead to the following conclusions: (1) the phosphorylation domain of PKBst was derived from a PPS, late in the evolutionary process, after the divergence of PPSs from PPDKs and EIs; (2) this domain is probably functional in phosphoryl transfer; (3) the C-terminal phosphorylation domain in PKBst probably defines a compact domain in all PEP family proteins that is linked to other domains in these proteins via flexible linkers.

Amino Acid Sequence↗
PubMed1976

Simultaneous inheritance of mutant isoenzymes of erythrocyte pyruvate kinase associated with chronic haemolytic anaemia.

The heterogeneity of pyruvate kinase (PK) deficiency associated with hereditary haemolytic anaemia is emphasized by studies of a kindred harbouring two distinct mutant forms of this enzyme, both of which were kinetically defective with markedly decreased affinities for the substrate, phosphoenolypyruvate. The two isoenzymes, designated PK-Vancouver1 and PK-Vancouver2, were primarily distinguishable from one another by differences in maximum in vitro activities and by variations in response to fructose-I,6-diphosphate activation. When combined in proband erythrocytes to the exclusion of any normal PK, the isoenzymes were associated with a severe chronic haemolytic process with many of the features of PK deficiency of the more common quantitative type. Clinical laboratory screening tests for detecting PK deficiency may be falsely negative or equivocal in such cases.

Anemia, Hemolytic, Congenital↗
PubMed1986

Complete nucleotide and deduced amino acid sequences of rat L-type pyruvate kinase.

Four overlapping cDNA clones for L-type pyruvate kinase (PK-L) were isolated from carbohydrate-induced rat liver cDNA libraries. They contained all the coding sequence of the enzyme from the 7th codon and the entire 3'-untranslated extension up to the poly(A) tail. The sequence of the first 7 codons and that of the 5'-untranslated region were determined by primer extension. The analyzed PK-L mRNA has 19 5'-untranslated bases, 1629 coding bases and 1281 3'-untranslated bases without the poly(A) tail; it corresponds to the heavier, 3.2 kb species of the L-type mRNAs. The codons for the phosphorylatable site are located at the 5'-end of the messenger. The unusually long 3'-untranslated extension contains a repetitive element complementary to the 'brain-specific' identifier sequence described by Sutcliffe et al. [(1982) Proc. Natl. Acad. Sci. USA 79, 4942-4946].

Amino Acid Sequence↗
PubMed1989

The yeast pyruvate kinase gene does not contain a string of non-preferred codons: revised nucleotide sequence.

The sequence of the gene encoding pyruvate kinase from Saccharomyces cerevisiae was re-determined because of failures with oligonucleotide-directed mutagenesis experiments involving a region thought to contain a string of five contiguous non-preferred codons. This region was found to be difficult to sequence and was shown to have three extra bases when compared with the published sequence [(1983) J. Biol. Chem. 258, 2193-2201]. The revised sequence demonstrates that the yeast pyruvate kinase gene does not have a cluster of non-preferred codons, and that it therefore is not an example of the class of genes which possibly exhibit translational control by the presence of non-preferred codons.

Amino Acid Sequence↗
PubMed2003

Ineffective erythropoiesis in the spleen of a patient with pyruvate kinase deficiency.

In this study, possible adverse effects of pyruvate kinase (PK) deficiency on the maturation of erythroid progenitors were investigated. A 4-year-old Japanese girl with severe PK deficiency underwent splenectomy to reduce her need for blood transfusions. The spleen was examined a histologically, and the hematopoietic progenitors in the spleen were assayed to evaluate the extramedullary hematopoiesis of this PK-deficient subject. The number of hematopoietic progenitors including CFU-GM, BFU-E and CFU-GEMM in the spleen of the PK-deficient patient was much higher than those found in control spleens, indicating enhanced extramedullary hematopoiesis. TUNEL assay demonstrated apoptotic cells in the splenic red pulp of the PK-deficient patient. The expression of 7A6 antigen was detected in cells isolated from spleen and in cells cultured in vitro, but only in those cells that were positive for glycophorin A. These results provide evidence that the metabolic disturbances in PK deficiency affect not only the survival of red cells but also the maturation of erythroid progenitors, which results in premature cell death, i.e., apoptosis.

Apoptosis↗
PubMed1994

Transcriptional regulatory regions for expression of the rat pyruvate kinase M gene.

To study the regulatory mechanism of pyruvate kinase M gene transcription, we analyzed its chromatin structure and cis-acting DNA regions. Two DNase-I-hypersensitive sites were detected in dRLh-84 hepatoma cells, but not in hepatocytes, which coincides with expression of the M gene in the two types of cells. These sites, designated HS2 and HS1, were located around the major transcription start site and about 2.9 kb downstream from this site, respectively. A transient chloramphenicol acetyltransferase expression assay indicated that the region around HS1 did not show any activity, whereas the upstream region up to -457 had promoter activity in hepatoma cells. Most of this activity was lost by a 5'-deletion from -286 to -225. Further analysis identified a cluster of three cis-acting regions from -279 to -216, which are named boxes A, B and C. These regions did not have any independent effect, but the inclusion of all regions were synergistic. These regions were not active in hepatocytes, suggesting that they have cell-type specificity. A gel mobility shift assay indicated that unidentified, but distinct, nuclear proteins bound to the three boxes. These results suggest that transcriptional regulation of the M gene involves alteration of chromatin structure and binding of proteins to three cis-acting elements.

Animals↗
PubMed2000

A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency.

Mutations in the PKLR gene responsible for pyruvate kinase (PK)-deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A-->G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK-deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA-A element) in the R-type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R-PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the -72A-->G point mutation disables the binding of the erythroid transcription factor GATA-1 to the GATA-A element. Supporting these data, the two patients homozygous for the -72A-->G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C-->T had a mild condition.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1977

An investigation of the interactions of the allosteric modifiers of pyruvate kinase with the enzyme from Carcinus maenas hepatopancreas.

1. Pyruvate kinase purified from the hepatopancrease of Carcinus maenas exhibited sigmoidal saturation kinetics with respect to the substrate phosphoenolpyruvate in the absence of the allosteric activator fructose 1,6-bisphosphate, but normal hyperbolic saturation was seen in the presence of this activator. The activation appears to be the result of a decrease in the s0.5 (phosphoenolpyruvate) and not to a change in Vmax. 2. In the presence of ADP and ATP at a constant nucleotide-pool size the results indicate that phosphoenolpyruvate co-operativity is lost on increasing the [ATP]/[ADP] ratio. 3. Paralleling this change is the observation that the fructose 1,6-bisphosphate activation became less at the [ATP]/[ATP] ratio was increased. This was due to the enzyme exhibiting a near-maximal activity in the absence of activator. 4. L-Alanine inhibited the enzyme, but homotropic co-operative interactions were only seen with a cruder (1000000g supernatant) enzyme preparation. The inhibition by alanine could be overcome by increasing the concentration of either phosphoenolpyruvate or fructose 1,6-bisphosphate, although increasing the L-alanine concentration did not appear to be able to reverse the activation by fructose 1,6-bisphosphate. 5. In the presence of a low concentration of phosphoenolpyruvate, increasing the concentration of the product, ATP, caused an initial increase in enzyme activity, followed by an inhibitory phase. In the presence of either fructose 1,6-bisphosphate or L-alanine only inhibition was seen. 6. The inhibition by ATP could not be completely reversed by fructose 1,6-bisphosphate.

Adenosine Triphosphate↗
PubMed1991

Determination of erythrocyte pyruvate kinase deficiency in Basenjis with chronic hemolytic anemia.

Erythrocyte pyruvate kinase (PK) deficiency was first described in Basenjis 20 years ago. Although the approach to diagnosis had not been well established, a screening program to detect heterozygotes was thought to have eliminated PK deficiency from the Basenjis of the United States. Four not closely related Basenjis with severe chronic hemolytic anemia, from various parts of the United States, were studied. Their PCV ranged from 16 to 25% and their reticulocyte count was always above 15%. A progressive osteosclerosis was seen in all of the Basenjis and hepatic failure developed in 2 of them. The erythrocyte intermediary metabolite patterns indicated a glycolytic defect at the PK step. Erythrocyte PK activities were markedly increased in the anemic Basenjis, compared with those of a control group, but the enzyme in these Basenjis had abnormal kinetic properties and was thermolabile. An antibody against R-type PK, the regular erythrocyte PK form, did not neutralize the PK activity of affected Basenjis, and results of electrophoretic studies suggested the expression of M2-type PK, a leukocyte and fetal erythroid PK-type. Clinically healthy heterozygous Basenjis had half-normal R-type PK activity and did not express the M2-type in their erythrocytes. We conclude that severe chronic hemolytic anemia, caused by erythrocyte PK deficiency, and associated osteosclerosis still develop in Basenjis. A definitive diagnosis cannot be reached by simply measuring erythrocyte PK activity; rather, diagnosis requires measurement of glycolytic substrate accumulation and enzyme stability and immunologic or electrophoretic studies of erythrocyte PK.

Anemia, Hemolytic↗
PubMed1992

Elements responsible for hormonal control and tissue specificity of L-type pyruvate kinase gene expression in transgenic mice.

L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway specifically expressed in the liver and, to a lesser degree, in the small intestine and kidney. One important characteristic of L-PK gene expression is its strong activation by glucose and insulin and its complete inhibition by fasting or glucagon treatment. Having previously established that the entire rat L-PK gene plus 3.2 kbp of 5'-flanking region functions in mice in a tissue-specific and hormonally regulated manner, various deletions of these 3.2 kbp of 5'-flanking regions were tested in transgenic animals to map the cis-acting elements involved in transcriptional gene regulation. Our experiments indicate that the proximal region between -183 and +11 confers tissue specificity and contains all the information necessary for dietary and hormonal control of L-PK gene expression in vivo. We found, however, that the transcriptional activity generated by this proximal promoter fragment can be modulated by distal sequences in a tissue-specific manner. (i) Sequences between bp -183 and -392 seem to play a dual role in the liver and small intestine; they induce L-PK expression in the liver but repress it in the small intestine. (ii) Sequences from bp -392 up to -1170 do not seem to have any additional effect on promoter activity. (iii) Between bp -1170 and -2080, we found a putative extinguisher whose transcriptional inhibitory effect is much more marked in the small intestine than in the liver. (iv) Finally, between bp -2080 and -3200, we identified an activating sequence required for full expression of the gene in the liver.

Animals↗
PubMed2005

Nuclear factor 1 family members interact with hepatocyte nuclear factor 1alpha to synergistically activate L-type pyruvate kinase gene transcription.

Transcription of hepatic L-type pyruvate kinase (L-PK) gene is cell type-specific and is under the control of various nutritional conditions. The L-PK gene contains multiple cis-regulatory elements located within a 170-bp upstream region necessary for these regulations. These elements can synergistically stimulate L-PK gene transcription, although their mechanisms are largely unknown. Because nuclear factor (NF) 1 family members bind to specific cis-regulatory elements known as L-IIA and L-IIB and hepatocyte nuclear factor (HNF) 1alpha binds to the adjacent element L-I, we examined the functional and physical interactions between these two transcription factors. Reporter gene assay showed that these two factors synergistically activated the L-PK promoter containing the 5'-flanking region up to -189. Although two NF1-binding sites are required for the maximum synergistic effect of NF1 family members with HNF1alpha, significant functional interaction between the two factors was observed in the L-PK promoter containing two mutated NF1-binding sites and also in the promoter containing only the HNF1alpha-binding site, raising the possibility that NF1 proteins function as HNF1alpha co-activators. Chromatin immunoprecipitation assay revealed that both NF1 proteins and HNF1alpha bound to the promoter region of the L-PK gene in vivo. In vitro binding assay confirmed that NF1 proteins directly interacted mainly with the homeodomain of HNF1alpha via their DNA-binding domains. This interaction enhanced HNF1alpha binding to the L-I element and was also observed in rat liver by co-immunoprecipitation assay. Thus, we conclude that cooperative interaction between NF1 family members and HNF1alpha plays an important role in hepatic L-PK transcription.

Animals↗
PubMed1975

The immunological properties of pyruvate kinase. II. The relationship of the human erythrocyte isozyme to the human liver isozymes.

We have examined the hypothesis that the human erythrocyte isozyme of pyruvate kinase (EC 2.7.1.40) is a hybrid of the two isozymes present in liver. Rabbit antiserum against purified human erythrocyte pyruvate kinase inactivates the erythrocyte isozyme and the major liver isozyme from human tissue but does not inactivate the minor liver isozyme. The electrophoretic mobilities of the erythrocyte and major liver isozymes are altered by anti-erythrocyte enzyme antibody while the mobility of the minor liver isozyme is unaffected. Gel diffusion analysis indicates cross-reactivity between the erythrocyte and major liver isozyme but no cross-reactivity with the minor liver isozyme. The hybrid hypothesis would predict cross-reactivity including changes in activity and mobility of all isozymes and we conclude, therefore that the hypothesis is incorrect.

Animals↗
PubMed1984

Insulin and glucagon's reciprocal mediation of a dual regulation mechanism for pyruvate kinase.

The addition of glucagon to hepatocytes in primary culture produced a rapid and sustained increase in the Km (1.27 mM phosphoenol pyruvate) of pyruvate kinase. The low Km (0.4 mM) form of the enzyme was seen when cells were retreated with insulin, demonstrating a short-term regulation mechanism. Injections of insulin, glucagon or glucagon followed by insulin demonstrated that a similar mechanism occurs in vivo. Results from longer times after injection indicated that another mechanism occurs when altered activity was the result of changes in V max and not Km. Thus, a dual mechanism for regulation of pyruvate kinase occurs. A rapid responding system functions by modification of the enzyme, while a long-term system functions by altering the rate of synthesis, thus changing the amount of enzyme present.

Animals↗