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PubMed1985

Transcriptional and post-transcriptional regulation of L-type pyruvate kinase in diabetic rat liver by insulin and dietary fructose.

Regulation of the expression of the hepatic L-type pyruvate kinase gene by insulin and dietary fructose was studied in diabetic rats. Insulin increased the levels of putative nuclear RNA precursor species of this enzyme in parallel with that of total cellular pyruvate kinase L mRNA. These changes occurred more slowly than those induced by dietary fructose. Insulin caused a 3-fold increase in transcription of the pyruvate kinase L gene after 6 h and a 6-fold increase after 16 h. The increase caused by insulin was inhibited by glucagon, but not by adrenalectomy. Cycloheximide inhibited the induction caused by insulin, suggesting that insulin may stimulate transcription of the pyruvate kinase L gene by stimulating synthesis of some unknown protein. On the other hand, feeding fructose had no effect on transcription of the pyruvate kinase L gene. We previously showed that increases in the levels of putative nuclear RNA precursor species of the pyruvate kinase L after fructose feeding preceded changes in the levels of cytosolic pyruvate kinase L mRNA (Inoue, H., Noguchi, T., and Tanaka, T. (1984) J. Biochem. (Tokyo) 96, 1457-1462). Thus, dietary fructose may increase the levels of pyruvate kinase L mRNA by stabilizing nuclear RNA species. Glucagon inhibited the increase in pyruvate kinase L mRNA caused by dietary fructose. However, plasma levels of glucagon and thyroid hormones were not decreased in diabetic rats after fructose feeding. In addition, treatment with triiodo-L-thyronine caused no change in the pyruvate kinase L mRNA level. Furthermore, adrenalectomy did not impair enzyme induction by fructose in diabetic rats. Thus, the effect of fructose on pyruvate kinase L seems to be directly on the liver.

Adrenalectomy↗
PubMed1998

Severe pyruvate kinase deficiency anemia. A case report.

BACKGROUND: Pyruvate kinase deficiency is a rare cause of hemolytic anemia and, in its most severe form, requires splenectomy in childhood. During pregnancy, severe cases have been traditionally managed with prophylactic blood transfusions to keep the hemoglobin concentration above arbitrary thresholds of 7-8 g/dL. CASE: A case of severe pyruvate kinase deficiency anemia was managed conservatively without blood transfusions even though the hemoglobin concentration reached a nadir of 6.8 g/dL. The perinatal outcome was good. CONCLUSION: In cases of severe pyruvate kinase deficiency anemia, pregnancy per se might not be an indication for prophylactic blood transfusions.

Adult↗
PubMed1974

The reaction of diethyl pyrocarbonate with pyruvate kinase.

Diethyl pyrocarbonate inactivates muscle pyruvate kinase with the substitution of 3-4 histidine residues per subunit. Phosphoenolpyruvate, ATP and ADP to a lesser extent, and Mg(2+) and pyruvate to a small extent, protect against inactivation.

Adenosine Diphosphate↗
PubMed1983

An approach to the elucidation of the quaternary structure role in the activity of pyruvate kinase studies on the immobilized enzyme.

1. Studies on pyruvate kinase immobilized via -S-S- linkages showed that single subunits of this tetrameric enzyme are inactive. 2. Pyruvate kinase was coupled with different preparations of CNBr-activated Sepharose via one and more than one bond, and after removal of non-covalently bound subunits by denaturant treatment the subunit derivatives were obtained composed of different pyruvate kinase species. 3. Heat inactivation studies of immobilized enzyme derivatives suggested that not only tetramers of pyruvate kinase are active. 4. This idea is further supported by experiments with 2 M urea dissociation of immobilized tetrameric enzyme carried out in the presence of Mg2+ ions.

Animals↗
PubMed1980

Comparative studies of kinetic and optical properties of rabbit muscle, sturgeon muscle, and yeast pyruvate kinase.

The kinetic and optical properties of pyruvate kinase isolated from rabbit muscle, sturgeon muscle, and yeast were compared using various activating divalent metal ions as probes for functional features and using ultraviolet circular dichroism (cd) measurements for conformational features, respectively. All three preparations of pyruvate kinase were similar in many aspects, such as activating efficiencies of the four activating metal ions, Mg(II), Co(II), Mn(II), and Ni(II) and pH-rate profiles, suggesting the presence of a similar metal binding locus of these enzymes as well as a common underlying mechanism of action. L-Phe inhibited the rabbit muscle enzyme and turned the hyperbolic kinetics into a sigmoidal kinetic with respect to phosphoenolpyruvate at alkaline pH, while fructose-1,6-biphosphate activated the sturgeon muscle and yeast enzymes and turned the sigmoidal kinetics into hyperbolic kinetics with respect to phosphoenolpyruvate. The ultraviolet cd spectral changes qualitatively correlated well with kinetic observations of all three native enzymes in the presence and absence of allosteric effectors. Our results suggested that there are at least two conformational states of pyruvate kinase which are inducible by the binding of substrate and (or) allosteric effectors. The conformational changes from one form to another in these enzymes are very similar, especially between the rabbit and sturgeon muscle enzymes.

Animals↗
PubMed1977

[Deficiency of pyruvate kinase in erythrocytes: biochemical studies. Preliminary communication].

Pyruvate kinase deficiency was found to be the causative factor in a family with nonspherocytic hemolytic anemia. Biochemical findings were the following: decrease in adenosine-5'-triphosphate (ATP) concentration (59 micromoles per 100 ml of erytrocytes; normal: 150 +/- 59), increase in 2,3-diphosphoglycerate (2,3-DPG) concentration (13611 micromoles per 100 ml of erythrocytes; normal: 480 +/- 80) and low level of pyruvate kinase activity (955 U) mmol Hb; normal 2700 +/- 498. The deficiency of enzyme activity in the hexose-monophosphate shunt was excluded on bases of normal or elevated glucose-6-phosphate and 6-phosphogluconate dehydrogenase activities, normal reduced glutathione concentration and normal glutathione stability in vitro. The Michaelis constant for phosphoenolpyruvate of the patient's pyruvate kinase was found to be normal; thus, the mutation affected only the Vmax of the enzyme.

Adult↗
PubMed1992

Isolation and characterization of the Aspergillus niger pyruvate kinase gene.

The Aspergillus niger gene encoding pyruvate kinase was cloned by heterologous hybridization using a fragment from the corresponding yeast gene as a probe. The primary structure of the gene, including 5' and 3' flanking sequences, was determined. The structural part of the A. niger pkiA gene is 2054 bp long and is interrupted by seven putative introns. Splicing of the intron sequences results in an open reading frame of 1578 bp, encoding a protein of 526 amino-acid residues and a molecular weight of 58,130 Da. Extensive homology is found with pyruvate kinase from A. nidulans; only 33 amino acids are different between both proteins. Transformation experiments using the pyrA gene as a selection marker and the subcloned pkiA gene as a co-transforming marker led to increased levels of pyruvate kinase. Analysis of the transformants showed that in none of the transformants integration had occurred at the pkiA locus. Predominantly co-integration of the pyrA- and the pkiA-containing plasmids was found in the cases examined.

Amino Acid Sequence↗
PubMed1975

The reversibility of skeletal muscle pyruvate kinase and an assessment of its capacity to support glyconeogenesis.

The kinetics of pyruvate phosphorylation by rabbit skeletal muscle pyruvate kinase (EC 2.7.1.40) has been studied with a coupled assay using P-enolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). The reaction sequence is (See journal for formula). Although the equilibrium of the pyruvate kinase reaction by itself strongly favors pyruvate production, the over-all equilibrium of this coupled system favors the depletion of pyruvate, thus greatly reducing the problem of back reaction during the assay. In addition, the oxidation of NADH by malate dehydrogenase makes it possible to monitor the system with a spectrophotometer. The Michaelis constant of pyruvate kinase was found to be 0.9 mM for ATP and 7 mM for pyruvate, values that agree reasonably well with earlier studies using direct assays. However, the maximum velocity is about 6 mumol of pyruvate phosphorylated/min/mg of enzyme, which is very much faster than that indicated by earlier studies. These results suggest that the metabolic significance of the reverse reaction of muscle pyruvate kinase may have been underestimated. In particular, the data given here suggest that its rate in vivo is probably comparable to the observed rate of glycogen synthesis from lactate, making possible glyconeogenesis in muscle by pyruvate kinase reversal without the need for an enzymatic bypass of the kind employed by liver and kidney.

Adenosine Triphosphate↗
PubMedDate not supplied

[Effect of Ca2+ ions on the pyruvate kinase isoenzymes from rabbit kidney cortex].

Ca2 ions showed the various effect on isoenzymes of pyruvate kinase from rabbit kidney cortex. Ca2 activated the "L" type of pyruvate kinase at low concentrations of PEP and inhibited -- at high concentrations of the latter. "M2" type of pyruvate kinase was inhibited by Ca2 under all the conditions studied. In presence of Ca2+ the activating effect of PDP on "L" and "M2" types of pyruvate kinase was absent; the inhibitory action of ATP on the "M" type of pyruvate kinase was increased at all the concentrations above 1.3 mM. The effect of Ca2+ on the pyruvate kinase isoenzymes depended on content of Mg2+ in the medium.

Adenosine Triphosphate↗
PubMed1982

Extreme deficiency of L-type pyruvate kinase with moderate clinical expression.

In the erythrocytes and liver of a patient with hereditary non-spherocytic hemolytic anemia and increased serum aminotransferases, almost complete deficiency of L-type pyruvate kinase was detected. The parents of the patient are second cousins and the pyruvate kinase activity in their erythrocytes was decreased to about half normal values. Pyruvate kinase from the patient is characterized by extreme lability. Pyruvate kinase from the parents' red cells showed no molecular abnormalities. No cross-reactive material could be precipitated with a monospecific antibody raised against L-type pyruvate kinase in the patient's erythrocytes. In the red cells of the parents a decreased amount of cross-reactive material against pyruvate kinase antibodies was found, indicating that the lowered pyruvate kinase activity in the erythrocytes of the parents is caused by a decreased level of the pyruvate kinase protein. In the liver of the patient no L-type pyruvate kinase activity and no immunologically recognizable L-type pyruvate kinase could be detected. The increased lability of the enzyme protein may explain the low residual activity. However, this decreased activity is shown to be sufficient to perform a normal glycolytic flux resulting only in moderate clinical expression.

Adult↗
PubMed1983

Evidence for two distinct pyruvate kinase genes in Escherichia coli K-12.

A strain of Escherichia coli K-12 defective in pyruvate kinase F has been produced. The existence of this mutant, in conjunction with earlier results, strongly suggests that the two pyruvate kinases in this bacterium are distinct forms and not interconvertible. Either form of pyruvate kinase appeared to be equally effective in the glycolytic conversion of phosphoenolpyruvate to pyruvate. Genes specifying pyruvate kinase A and pyruvate kinase F were present on the small F-prime F506 and the locus for pyruvate kinase F was found to be at minute 36.5 on the E. coli genetic map.

Chromosome Mapping↗
PubMed1984

Plasma pyruvate kinase and creatine kinase activity in Becker muscular dystrophy.

Plasma creatine kinase (CK) and pyruvate kinase (PK) were measured in 31 obligate carriers of Becker muscular dystrophy (BMD), 36 BMD patients and appropriate controls. Mean plasma CK was 108 U/l in obligate carriers and 62 U/l in 43 age- and sex-matched controls (P less than 0.001 carriers vs controls). Control CK reference range was 31-125 U/l (mean +/- 2 SD of log transformed values). Mean plasma PK was 40 U/l in obligate carriers and 34 U/l in 56 controls (P less than 0.02 carriers vs controls). Control PK reference range was 18-61 U/l. Values of CK above the reference range upper limit were found in 13 of 31 BMD obligate carriers but only 2 showed elevated PK values. The sensitivity of CK in determining BMD carrier status, although only 42%, was markedly better than PK at 6.5%. Mean plasma CK in BMD patients was 2366 U/l, a 19-fold increase over the control value of 127 U/l (P less than 0.001 patients vs controls). Control CK reference range was 40-316 U/l. In contrast, mean plasma PK in BMD patients was 353 U/l, only 7-fold higher than the mean control value of 57 U/l (P less than 0.001 patients vs controls). Control PK reference range was 22-126 U/l. Clearly, the estimation of plasma PK as a means of determining BMD carrier status is markedly inferior to CK. Previous reports of increased sensitivity of PK compared with CK may have been due to artefactually elevated PK levels produced during sample preparation.

Adolescent↗
PubMed1994

Dietary polyunsaturated fatty acids interfere with the insulin/glucose activation of L-type pyruvate kinase gene transcription.

L-type pyruvate kinase (L-PK) is a key glycolytic enzyme regulating the flux of metabolites through the pyruvate-phosphoenolpyruvate cycle (1). The regulation of L-PK is complex involving both hormones and nutrients. We have found that feeding rats diets containing polyunsaturated fatty acids (PUFA) significantly inhibits hepatic pyruvate kinase enzyme activity (> 60%) and suppresses mRNAPK abundance (> 70%). Studies with primary hepatocytes indicate that PUFA act directly on hepatocytes. Specifically, arachidonic (20:4, omega 6) and eicosapentaenoic (20:5, omega 3) acid suppressed both mRNAPK llevels and the activity of a transfected PKCAT (-4300/+12) fusion gene by > 70%. This is due to an inhibition of the insulin/glucose-mediated transactivation of L-PKCAT. Deletion analysis localized PUFA-regulated cis-acting elements to a region within the L-PK proximal promoter, i.e. between -197 and -96 base pairs. This region binds two transcription factors involved in the hormone/nutrient regulation of L-PK gene transcription, i.e. a major late transcription factor-like factor and HNF-4. Linker scanning mutation analysis localized the PUFA-regulated cis-acting elements to the vicinity of the HNF-4 binding site. Thus, PUFA-regulated factors abrogate the insulin/glucose activation of L-PK gene transcription by targeting the HNF-4 elements. These studies suggest that PUFA may have significant effects on hepatic carbohydrate metabolism by inhibiting the L-PK side of the pyruvate-phosphoenolpyruvate cycle.

Animals↗
PubMed1984

Hepatic L-type pyruvate kinase: separation of unphosphorylated, phosphorylated and proteolytically modified in vivo forms.

After partial chromatographic purification of rat liver cell sap on DEAE-cellulose, including the removal of type M2 pyruvate kinase, different forms of type L pyruvate kinase were separated by chromatofocusing. Three fractions of pyruvate kinase activity were found, eluting at pH 5.0, 5.2, and 5.3, respectively. The first one was identified as phosphorylated and the second one as unphosphorylated pyruvate kinase. There were strong indications that the third fraction represented a proteolytically modified form of the enzyme, since it co-migrated with a form modified in vitro and had a similarly increased apparent Km for phosphoenolpyruvate. To rule out the possibility of this being a phosphorylated form of pyruvate kinase, the enzyme was incubated with a phosphoprotein phosphatase and then phosphorylated with cAMP-dependent protein kinase. The enzyme was not phosphorylated, like pyruvate kinase modified with subtilisin or calcium-activated protease. There is some evidence that a proteolytically modified pyruvate kinase exists in vivo. This enzyme form has not previously been demonstrated in cell sap, prior to exposure to proteolytic enzymes. The relative amounts of the three forms were determined in livers from starved rats and rats fed on a normal or a carbohydrate-rich diet.

Animals↗
PubMed1982

Glucocorticoid hormones have a permissive role in the phosphorylation of L-type pyruvate kinase by glucagon.

The incorporation of [32P]phosphate into L-type pyruvate kinase in response to glucagon was studied in hepatocytes isolated from control and adrenalectomized rats. In control cells, pyruvate kinase phosphorylation was maximally stimulated by 210% by glucagon. Adrenalectomy reduced both the basal extent of phosphorylation and the response to glucagon to 25% of those measured in control hepatocytes. This impaired pyruvate kinase phosphorylation was associated with altered kinetic properties of L-type pyruvate kinase. Glucagon injected in vivo (1 mg/kg) into adrenalectomized rats did not increase the substrate-co-operativity of L-type pyruvate kinase. These results suggest a molecular basis for the well-recognised glucocorticoid hormone-dependence for the stimulation by glucagon of hepatic gluconeogenesis. Further effects of this defective enzyme regulation on hepatic metabolism are suggested by the observation that adrenalectomy abolished the ability of glucagon to inhibit hepatic fatty acid biosynthesis both in vivo and in isolated hepatocytes.

Adrenalectomy↗
PubMed1985

The purification and kinetic characterization of eel white muscle pyruvate kinase.

A stable, homogeneous preparation of pyruvate kinase from white muscle of the American eel, Anguilla rostrata with a specific activity of 350 units/mg has been obtained. The enzyme has a pH optimum in the range 6.3-6.5 and requires Mg2+ and K+ for maximum activity. Eel muscle pyruvate kinase exhibits slight co-operativity in the binding of the substrate phosphoenol-pyruvate. It is activated by fructose-1,6-bisphosphate in a pH dependent manner and is inhibited by both alanine and phenylalanine. These properties are very similar to the properties of the mammalian M2 isozyme.

Anguilla↗
PubMed2002

Cloning and functional analysis of pyruvate kinase promoter region from Drosophila melanogaster.

Pyruvate kinase (PK; EC 2.7.1.40) is a key glycolytic enzyme of Drosophila melanogaster. It catalyzes the conversion of phosphoenolpyruvate into pyruvate with the transfer of a phosphate group to ADP to form ATP. The ATP provides energy for cell growth and metabolism, and pyruvate participates in many metabolic reactions. Therefore, PK plays an important role in cell metabolism. Southern blot analysis, PCR, and sequencing were used to determine the content of a Drosophila pyruvate kinase (Pyk) genomic clone, lambdaPK61. The results indicated that the insert of lambdaPK61 comprised 8330 bp upstream of and 7186 bp downstream of the transcription start point of the Pyk gene. The size of the insert was 15,516 bp in total, which contained six genes including Pyk. Deletion mapping was applied to identify the promoter region and cis-acting elements 5' of PyK. Ten serial deletions produced by PCR were inserted upstream of the reporter gene (LacZ) to form recombinant plasmids, which were then transfected into Drosophila S2 cells. The results revealed that the regions -1475 approximately -1033 and -1033 approximately -534 of the 5' end of PyK possessed positive regulatory function for Pyk expression; i.e., increased gene expression. There were redundant putative cis-acting elements, including ecdysone response element (EcRE), E74A, and broad complex zinc finger (BRCZ) binding sites. Both E74A and BRCZ belong to the early genes regulated by ecdysone. This result suggested that Pyk might be regulated by ecdysone, directly or indirectly. However, the results of the developmental profile of Pyk expression by Northern blot analysis suggested that the effects of ecdysone on Pyk were repressive, not inductive. In addition, it was found that in these regions, there were many cis-acting elements related to egg and embryo development. Both -258 approximately -254 and -167 approximately -163 contained a CAAT box, and deletion of these regions decreased reporter gene expression. Therefore, it is suggested that both CAAT boxes are functional and that the promoter of Pyk might be located in the region of -258 approximately +109. No TATA box or downstream promoter element were identified around the transcription start site of Pyk. Additionally, PyK might share a regulatory region with an unknown neighboring gene. It was concluded that Pyk has the characteristics of a housekeeping gene.

Animals↗
PubMed1993

Divergent binding sites in pyruvate kinases I and II from Escherichia coli.

Pyridoxal 5'-phosphate incorporation into pyruvate kinase II from E. coli was decreased by the substrate phosphoenolpyruvate and increased by the allosteric activator ribose 5-phosphate, the total incorporation being linearly related to inactivation. Four lysyl residues were substantially modified, whatever the incubation conditions were while two additional residues became reactive only in the presence of the allosteric activator. Six tryptic peptides containing modified lysines were purified and sequenced. They defined five regions of pyruvate kinase II, since one of them contained two labelled lysines and included a peptide which also appeared independently. Sequence comparison with E. coli type I, yeast and cat muscle pyruvate kinases shows that the binding regions of pyruvate kinase II are clearly divergent from those of pyruvate kinase I and of the eukaryotic enzymes.

Allosteric Regulation↗