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PubMed1978

Erythrocyte populations in pyruvate kinase deficiency anaemia following splenectomy. I. Cell morphology.

Light and scanning electron microscopic examination of fresh erythrocytes from the blood of four patients splenectomized for severe pyruvate kinase (PK) deficiency haemolytic anaemia has revealed three morphologically different cell populations. Most striking in these patients was the presence of crenated red cells (30-50% of total) which fit current criteria for definition as echinocytes, but also with unusual surface retraction compatible with the existence of intracellular shrinkage, a phenomenon known to occur after incubation of PK deficient red cells in vitro. Additional cell types include immature reticulocytes (7-8% of total) and thin macrocytic discocytes (45-65% of total) none of which are considered specific for the disease. These morphological findings are in sharp contrast with the paucity of red cell shape abnormalities usually described in PK deficient patients before splenectomy and may provide insight into the mechanisms leading to accelerated erythrocyte destruction in this disease.

Adolescent↗
PubMed1983

Developmental changes of mouse red cell pyruvate kinase.

The increase in red cell 2,3-diphosphoglycerate (2,3-DPG) concentration during mouse postnatal development is highly correlated with the decrease in red cell pyruvate kinase (PK) activity. Immunoneutralization curves for newborn and adult red cell PK are parallel, which suggests that the higher PK activity in newborn mouse red cells is due primarily to a greater quantity of PK protein. Mouse newborn and adult red cell PK also differ in mobility on cellulose acetate electrophoresis (pH 7.5). This is true for two strains of mice (C57BL/6 and IS/Cam) whose adult red cell PK electrophoretic patterns differ (single vs. double bands, respectively). Trypsinization does not alter the electrophoretic mobilities of C57BL/6 newborn and adult red cell PK, but it does convert the IS/Cam adult PK to a single band of greater mobility than the single band of C57BL/6 adult PK. These data are consistent with the hypothesis that mice have structurally different forms of PK for newborn and adult red cells, analogous to the fetal gamma- and adult beta-globins of humans.

Aging↗
PubMed1988

Pyruvate kinase Greensboro. A four-generation study of a high K0.5s (phosphoenolpyruvate) variant.

The proband with lifelong hemolytic anemia has a high K0.5s phosphoenolypyruvate (PEP) erythrocyte pyruvate kinase (PK) variant substantially but incompletely normalized by the allosteric modifier fructose-1,6-diphosphate (F-1,6-P2) with conversion of sigmoidal to hyperbolic kinetics. Heterozygotes in four generations express qualitatively identical but less severely abnormal kinetics and lack overt hemolysis. Kinetic abnormalities are closely mimicked by sulfhydryl modification of normal PK. Three distinct clinical and metabolic phenotypes characterize the proband and two sisters: variant PK and hemolytic anemia, variant PK without clinical manifestations or hemolysis, and complete normality. Their mother, whose red cell PK is entirely normal except for a questionably slightly low Vmax, is postulated to express the gene products of nonidentical alleles, one encoding a product with mildly less favorable catalytic characteristics. At low PEP concentrations, the proband and heterozygotes for the PK mutant express only a very small fraction of normal PK activity despite apparent inheritance of one normal allele in the latter. Evidence suggests that disproportionately lowered PK activity may be a property of a heterotetrameric PK. Illusory abnormalities in nucleotide specificity are artifacts of diminished substrate affinity characterizing the mutant PK.

Adenine Nucleotides↗
PubMedDate not supplied

Iron status and HFE genotype in erythrocyte pyruvate kinase deficiency: study of Italian cases.

We evaluated the iron status and searched for mutations C282Y and H63D in the hereditary hemochromatosis gene (HFE) in 34 pyruvate kinase (PK)-deficient patients from 29 unrelated families. Nine had received multiple transfusions. Thirteen of the 25 nontransfused patients displayed increased serum ferritin concentration, in the absence of conditions known to raise this parameter. HFE genotype was abnormal in 9 of 34 patients. The allele frequency was 1.8% for mutation 845G--> (C282Y) and 16.1% for mutation 187C-->G (H63D). Nontransfused subjects with abnormal genotype had serum ferritin and transferrin saturation values significantly higher than those with wild-type genotype. Of the 12 adult nontransfused patients with increased iron status parameters, 1 was C282Y homozygous, 1 compound heterozygous for C282Y and H63D, 3 H63D heterozygous, and 7 had a normal HFE genotype. Serum ferritin and transferrin saturation were not related to hemoglobin, reticulocytes, and bilirubin concentration. At multivariate analysis serum ferritin was independently associated with age and gender, but not with splenectomy and HFE genotypes. The retrospective evaluation of the iron status profile of 10 patients (3 with abnormal and 7 with wild-type HFE genotype) with at least 10 years follow-up showed that overt iron accumulation requiring iron chelation had occurred only in the 3 patients (2 of whom were splenectomized) with the mutated HFE gene.

Adolescent↗
PubMed1997

Metal-ion-mediated allosteric triggering of yeast pyruvate kinase. 2. A multidimensional thermodynamic linked-function analysis.

A role has been proposed for the free divalent metal in triggering the allosteric responses of yeast pyruvate kinase based upon a kinetic linked-function analysis [Mesecar, A. D., & Nowak, T. (1997a) (preceding paper in this series)]. The major conclusion from the analysis is that the allosteric activator, fructose 1,6-diphosphate (FDP), does not directly communicate with the substrate, phosphoenolpyruvate (PEP), at the active site of the enzyme: it is Mn2+ that mediates the allosteric communication between the PEP and FDP sites in an allosteric relay mechanism. Assumptions were necessary to treat kinetic parameters as thermodynamic parameters, and the presence of the substrate ADP was necessary for the kinetic analysis. In this study, the influence of FDP on the interactions of PEP and Mn2+ and the influence of PEP and Mn2+ on the interaction of FDP with YPK were measured, where possible, by direct binding methods in the absence of ADP. Direct binding data were then subjected to a thermodynamic linked-function analysis for a heterotropic, three ligand coupled system in order to ascertain the two and three ligand coupling free energies. The two ligand coupling free energies deltaG(Mn-PEP), deltaG(Mn-FDP), and deltaG(PEP-FDP) are -3.88, -1.09, and -0.22 kcal/mol, respectively. These values indicate that positive, heterotropic interactions exist between each of these ligand pairs. The three ligand coupling free energy term, deltaG(Mn-PEP-FDP), indicates that simultaneous binding of Mn2+, PEP, and FDP is considerably favored over the sum of their independent binding free energies by -6.6 kcal/mol. These results demonstrate the key role of the metal in the modulation of ligand binding and are consistent with the values and the relationships of the kinetic parameters obtained from the kinetic linked-function analysis.

Adenosine Diphosphate↗
PubMed1991

Erythrocyte pyruvate kinase deficiency in newborns of eastern Saudi Arabia.

Cord blood specimens from 513 normal Saudi neonates from the Eastern Province were screened for the deficiency of erythrocyte pyruvate kinase enzyme using the fluorescent spot test. The enzyme activity of the deficient specimens and 108 randomly selected samples was assayed spectrophotometrically. The results indicate that the incidence of this enzyme deficiency is 3.12% which is higher than that reported previously. The findings are compared with those reported by others and the possible factor responsible for the high incidence of this deficiency is discussed.

Erythrocytes↗
PubMed1968

Pyruvate Kinase Activity of Wheat Plants Grown under Potassium Deficient Conditions.

The activity of pyruvate kinase was determined in the first leaves of wheat plants grown under K(+)-deficient conditions. An enhancement of the enzyme activity compared with the normal plants was found to start from eighth day of growth, and about 4-fold increase in the enzyme activity was observed in 14-day wheat leaves. The addition of K(2)SO(4) to the nutrient solution given to the K(+)-deficient plants at tenth day resulted in the restoration of the enzyme activity to the normal level after 3 days. The levels of K(+) as well as carbohydrates and chlorophyll were found to return normal over the same period. These findings are discussed in relation to the metabolic pattern of plants at the early stages of K(+)-deficiency.

Journal Article↗
PubMed1984

Change in kinetic property of M1-type pyruvate kinase from hyperbolic type to sigmoidal type by limited proteolysis with an acid fraction from rabbit liver.

An acid extract of rabbit liver contained M1-type pyruvate kinase inactivating activity, and was separated to three fractions. The optimal inactivation of the enzyme with Fraction II (Mr 42,000) was observed at pH 5.5, and this inactivation was completely prevented by leupeptin and antipain, but not by pepstatin. With Fraction III (Mr 22,000), on the other hand, optimal inactivation of the enzyme was observed at pH 8-9, and was not prevented by these inhibitors. The kinetic properties, with phosphoenolpyruvate, of the enzyme were changed from hyperbolic type to sigmoidal type by the limited proteolysis with Fractions II and III. The subunit molecular weight of the enzyme (57,300) was decreased to 55,800 via 56,400 in the former case and to 56,400 in the latter case.

Animals↗
PubMed1993

Molecular basis of impaired pyruvate kinase isozyme conversion in erythroid cells: a single amino acid substitution near the active site and decreased mRNA content of the R-type PK.

Conversion of pyruvate kinase (PK) isozymes from M2- to R-PK has been observed during erythroid cell maturation. To understand this mechanism, we analyzed the PK gene of a R-PK deficient patient, in whose erythrocytes the M2-PK was persistently expressed. A point mutation, 1102 GTC-->TTC was identified in the R-PK cDNA, and it caused a single amino acid substitution from 368Val-->Phe. The residue is very close to the 372nd Gln, the putative binding site of the monovalent cation (K+). The impaired K+ binding would cause the decreased affinity for phosphoenolpyruvate, consequently the variant PK may be extremely unstable. Although the proband's other PK allele did not have any structural change, the R-PK mRNA level in reticulocytes was decreased. These findings suggested that both the structural mutation near the active site and the decreased mRNA level of the R-PK were responsible for the disorder.

Amino Acid Sequence↗
PubMed1989

[Immunohistochemical demonstration of L- and M2-pyruvate kinase in primary renal cell carcinomas and their metastases].

Seventeen renal cell carcinomas (RCC) were classified histologically and investigated for their expression of L- and M2-pyruvate kinase (PK) immunohistochemically. Using monoclonal antibodies we were able to demonstrate L-PK within 15 RCC and two metastases (in thyroid gland and bone) after fixation with aceton and paraffin embedding. In normal renal tissue the enzyme was localized within proximal tubules selectively. No enzyme reaction of L-PK could be demonstrated in renal oncocytoma, thyroid carcinoma, and in carcinomas of renal pelvis and lung. In contrast to this the M2-PK was detectable in all tumors and metastases investigated in this study. The results presented in this paper are able to show that (1) RCC derive from the proximal renal tubules, but not the renal oncocytoma, (2) the detection of L-PK may be helpful for identification of metastases of RCC even if they are undifferentiated and (3) there is an enzyme shift from L-PK to M2-PK within tumor cells of RCC resulting in alteration of glucose utilization from energy supply to syntheses of substrates essential for tumor cells.

Carcinoma, Renal Cell↗
PubMed1976

Chromium(III)-adenosine triphosphate as a paramagnetic probe to determine intersubstrate distances on pyruvate kinase. Detection of an active enzyme-metal-ATP-metal complex.

The interaction of CrATP, a stable, substitution-inert, paramagnetic tridentate complex of ATP, with muscle pyruvate kinase has been studied by measuring the effects of CrATP on the kinetics of pyruvate enolization and on the longitudinal nuclear magnetic relaxation rate (1/T1) of the protons of water and the protons and carbon atoms of pyruvate to investigate the existence and activity of bimetallic enzyme-M(II)-CrATP complexes and to determine intersubstrate distances on a kinase. The paramagnetic effect of CrATP on 1/T1 of water protons is enhanced upon complexation with the enzyme. Titrations of the enzyme with CrATP yielded characteristic enhancements of 1/T1 for the binary enzyme-CrATP, ternary enzyme-Mg(II)-crATP, and quaternary enzyme-Mg(II)-crATP-pyruvate complexes of 3.5, 1.7, and 1.2 and dissociation constants of CrATP of 400, 200, and 200 muM, respectively. From the frequency dependence of 1/T1, the number of fast exchanging water protons in the coordination spheres of Cr(III) is approximately 6 in CrATP and in both the ternary enzyme-Mg(II)-CrATP complex and the quaternary enzyme-Mg(II)-CrATP-pyruvate complex. The paramagnetic effect of enzyme-bound Mn(II) on 1/T1 of water protons decreases upon the addition of CrATP. Titration of the binary enzyme-Mn(II) complex with CrATP decreases the characteristic enhancement due to Mn(II) from 24 +/- 3 to 6 +/- 1. Titration of the ternary eznyme-Mn(II)-pyruvate complex with CrATP decreases the enhancement from 6 +/- 1 to 0.5 +/- 0.1. The affinity of the enzyme for Mn(II) is increased 2-fold upon binding of CrATP as indicated by decreases in the amplitude of the EPR spectrum of free Mn(II). The dissociation constants of CrATP from the enzyme-Mn(II)-CrATP complex, the enzyme-CrATP-pyruvate complex, and the enzyme-Mn(II)-CrATP-pyruvate complex are all 200 muM. The observed titration behavior, the characteristic enhancement values, the tightening by Mg(II) of the binding of CrATP to the enzyme, and the tightening of the binding of Mn(II) to the enzyme by CrATP establish the existence of enzyme-M(II)-CrATP and enzyme-M(II)-CrATP-pyruvate complexes containing two cations, Mg(II) or Mn(II) and Cr(III), at the active site.

Adenosine Triphosphate↗
PubMed1987

The effects of alterations within the 3' untranslated region of the pyruvate kinase messenger RNA upon its stability and translation in Saccharomyces cerevisiae.

A 53 basepair deletion was constructed within the 3' untranslated region (3' UTR) of the yeast pyruvate kinase (PYK) gene borne upon a centromeric plasmid. Various modular assemblies of the pUC13 polylinker DNA (single unit = 44 bp) were used to replace the deleted region, and the effects of these modifications upon both transcript stability and translation ascertained in yeast. The use of a differential probing stratagem, based on the hybridisation of specific oligonucleotides to either pUC13 polylinker or unaltered PYK 3' UTR sequences, allowed for discrimination between mutant (plasmid borne) and wild-type (chromosomal) PYK transcripts. In no construct was there any significant alteration in mRNA stability, but translation of the PYK mRNA was severely curtailed by truncation of the 3' UTR or the presence of a strong hairpin-loop structure in the 3' UTR. A specific mutation in the N-terminal coding sequences, which created a premature termination codon in both a 3' 'tagged' PYK plasmid and a PYK/LacZ fusion gene, aborted the translation of a majority of their transcripts but left their chemical half-lives unaltered. This observation is at variance with some previously published data (Losson & Lacroute (1979) Proc Natl Acad Sci USA 76, 5134; Pelsey & Lacroute (1984) Curr Genet 8, 277), but is consistent with our own earlier observation that there is no obvious link between ribosome loading and mRNA stability in yeast (Santiago et al. (1986) Nucleic Acids Res 14, 8347). Possible reasons for this disparity are discussed.

Fungal Proteins↗
PubMed1977

Alanine inhibition of pyruvate kinase in gliomas and meningiomas. A diagnostic tool in surgery for gliomas?

In gliomas a shift was found in the composition of enzymes, manifested by abnormal inhibition of pyruvate kinase by alanine. This study demonstrates the appearance of the MII-type isoenzyme in various gliomas. This MII type is also found in meningiomas but not in normal brain tissue. The method of enzyme examination described may be valuable as a diagnostic aid in the surgery of gliomas.

Alanine↗
PubMed1992

25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase.

25Mg NMR spectroscopy was used to study the interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase (PK). Titration of Mg2+ with enolase allows for the calculation of 1/T2 for Mg2+ bound at site I of 1510 s-1 and a quadrupolar coupling constant chi = 0.30 MHz. Titration of Mg2+ with enolase in the presence of 2-phosphoglycerate (PGA) and Zn2+, where Zn2+ binds specifically at site I, gives a 1/T2 for Mg2+ bound at site II of 4000 s-1 (chi = 0.49 MHz). The Mg2+ at site II appears to be more anisotropic than Mg2+ at site I. The titration of site I of the enolase-Mg-PGA-Mg complex with Zn2+ or Mn2+ shows a simple displacement of the Mg2+. No paramagnetic effects by Mn2+ on 25Mg relaxation were observed. Temperature studies of the 25Mg resonance show that fast exchange of the Mg2+ occurs under these conditions. From the lack of a paramagnetic effect, the distance between the cations at sites I and II must be more than 6-9 A. This distance limits the location, hence the function, of the cation at site II for catalytic activity. Titration of Mg2+ with PK gives a 1/T2 for bound Mg2+ of 2200 s-1 (chi = 0.24 MHz). A titration of Mg2+ with PK in the presence of the inhibitor oxalate gives a 1/T2 of 400 s-1. The temperature dependence of 25Mg relaxation in the PK-Mg-oxalate complex is consistent with slow exchange (Ea = 6.1 +/- 1.6 kcal/mol). The enzyme-bound cation is more tightly sequestered by the addition of a ligand that binds directly to the cation. An investigation of the 25Mg relaxation in the PK-Mn-oxalate-Mg-ATP complex, where the Mg2+ is bound to the nucleotide and the Mn2+ was enzyme bound, was not successful due to precipitation of PK under experimental conditions and the short T2 relaxation for 25Mg in this complex. The applications of 25Mg NMR have been useful in partially describing the properties of the bound Mg2+ in these two metal-requiring enzymes.

Adenosine Triphosphate↗
PubMed1977

Assignment of the structural genes for the alpha subunit of hexosaminidase A, mannosephosphate isomerase, and pyruvate kinase to the region q22-qter of human chromosome 15.

Concordant segregation of the expression of the alpha subunit of human hexosaminidase A, human mannosephosphate isomerase, and pyruvate kinase was observed in somatic cell hybrids between either thymidine kinase-deficient mouse cells or thymidine kinase-deficient Chinese hamster cells and human white blood cells carrying a translocation of the distal half (q 22-qter) of the long arm of chromosome 15 to chromosome 17. A positive correlation was established between the expression of these human phenotypes and the presence of the distal half of the long arm of human chromosome 15.

Carbohydrate Epimerases↗
PubMed1988

The pyruvate kinase isoenzyme shift in human gliomas: a potential marker in the treatment of gliomas.

In primary human brain tumours a shift occurs in the synthesis of isoenzymes of pyruvate kinase from the M towards the K-type. In astrocytomas, oligodendrogliomas and glioblastomas, which were localised in the cerebral hemispheres of adult patients over 20 years of age, the shift correlated well with histological grading and growth rate as observed in postoperative survival. Gliomas of adults, localised in midline structures, as well as childrens gliomas were characterised too by a strong shift from M towards the K type. However, in these tumours, less correlation with histological grading and growth rate was found. The isoenzyme shift can be rapidly demonstrated with an alanine inhibition test. The application of this assay may have a diagnostic value during operation for gliomas in grading of malignancy in adults as well as demarcation of the resection of gliomas in all age groups. The test can be performed within 10-15 min and can thus fit easily into a surgical procedure. A case report is presented for illustration.

Adolescent↗
PubMedDate not supplied

Fecal pyruvate kinase concentrations (ELISA based on a combination of clone 1 and clone 3 antibodies) for gastric cancer screening.

BACKGROUND: Recently we were able to demonstrate that Tumor M2 Pyruvate Kinase (Tumor M2-PK) is detectable in the feces of patients with gastrointestinal (GI) cancer. In this study an ELISA based on a different combination of antibodies was used to investigate stool samples of GI cancer patients. PATIENTS AND METHODS: The ELISA (ScheBoBiotech AG, Giessen, Germany) was based on one antibody specific for Tumor M2-PK and a second antibody reacting with Tumor M2-PK, M1-PK and the tetrameric M2-PK. Stool samples of healthy controls, patients with inflammatory bowel disease (IBD) and different kinds of GI cancer were tested. RESULTS: Compared to controls, samples of IBD or different tumors did not show significant differences, except those patients with gastric cancer (elevated fecal PK in 80% (p = 0.005)). CONCLUSION: If the present results can be confirmed in larger patient samples, this new test might provide an excellent screening tool for gastric cancer.

Adult↗