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PubMed1991

Isoenzymes of pyruvate kinase, lactate dehydrogenase and alkaline phosphatase in epithelial cell lines of rat liver.

In cultured epithelial cells of rat liver the isoenzyme patterns of pyruvate kinase, lactate dehydrogenase and alkaline phosphatase were studied and compared with those of freshly isolated parenchymal and non-parenchymal liver cells. In all epithelial cell lines pyruvate kinase was not activated by fructose 1,6-bisphosphate, suggesting the absence of the L-isoenzyme. Cell lines derived from livers of newborn rats expressed LDH-4 and -5, whereas cell lines developed from fetal rat livers contained all 5 lactate dehydrogenase isoenzymes. In the latter case the pattern was found to depend on the state of confluence. All cell lines exhibited only a single alkaline phosphatase form, however, differences were found with respect to electrophoretic mobility.

Alkaline Phosphatase↗
PubMed1998

Gene expression of mouse M1 and M2 pyruvate kinase isoenzymes correlates with differential poly[A] tract extension of their mRNAs during the development of spermatogenesis.

In eukaryotes, different isoenzymes for pyruvate kinase have been characterized. M2-type Pk cDNA from a mouse fetal ovary library was isolated and differential expression for M1 and M2-types during testis development was observed. While the presence of M2 mRNAs decreases throughout the development of spermatogenesis, we deduced that M1 type expression increases in adult testis coinciding with the presence of elongating spermatids in the seminiferous epithelium. Polyadenylation tests showed a concurrent increase in the length of the polyadenylation tail of transcribed M1-type pyruvate kinase mRNAs in prepuberal to adult seminiferous tubules. A similar relationship between poly[A] tail extension and differential increase of gene expression was detected for M1-type mRNA in adult brain and muscle. Length of poly[A] tail of M2-type transcripts is shown to decrease during the development of mouse testis. These results suggest that changes in the length of the poly[A] tail of transcripts are associated with differential expression of both regulated isoenzymes during testicular development.

Animals↗
PubMed1986

The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing.

The complete nucleotide sequences of rat M1- and M2-type pyruvate kinase mRNAs were determined by sequencing the cDNAs and by analyses of S1 nuclease mapping and primer extension. The sequences have an identical molecular size of about 2220 nucleotides excluding a poly(A) tail and include 1593-nucleotide coding region. Their nucleotide sequences are identical except for 160-nucleotide sequences within the coding regions. The amino acid sequences of the M1- and M2-type subunits deduced from the cDNA sequences differ by only 45 residues within domain C, which constitutes the main region responsible for intersubunit contact. The sequence of this region of the M2-type shows higher homology than that of the M1-type with the corresponding sequence of the L-type. Since the M2- and L-types are allosteric enzymes, unlike to the M1-type, the residues common to the M2- and L-types, but not the M1-type may be important for mediating the allosteric properties. Genomic clones encoding both M1- and M2-type isozyme mRNAs were isolated. By partial sequence analysis of a clone lambda MPK37 four exons were identified, of which two adjacent exons coded the M1- and M2-specific sequences, respectively. The two remaining exons present downstream coded amino acids common to the two isozymes. Thus, we conclude that the M1- and M2-type isozymes of pyruvate kinase are produced from the same gene probably by alternative RNA splicing.

Amino Acid Sequence↗
PubMed1992

Regulation of M2-type pyruvate kinase from human meningioma by allosteric effectors fructose 1,6 diphosphate and L-alanine.

In the present study the mechanism of action of M2-type pyruvate kinase from human meningioma in the simultaneous presence of fructose 1,6 diphosphate and L-alanine was investigated. Purified pyruvate kinase from human meningioma was allosterically inhibited by L-alanine with respect to substrates phosphoenolpyruvate and ADP. The inhibitory effects of L-alanine was partially removed by fructose 1,6 diphosphate. The purified enzyme was slightly susceptible to ATP inhibition.

Adenosine Diphosphate↗
PubMed1982

Isozyme distribution of hexokinase, phosphofructokinase and pyruvate kinase in lymphocytes from patients with chronic lymphocytic leukemia.

The enzyme activities and isozyme distribution of the three glycolytic regulator enzymes hexokinase, phosphofructokinase and pyruvate kinase were studied in lymphocytes of patients with chronic lymphocytic leukemia. Isozyme distribution patterns were determined by kinetic measurements, electrophoresis and immunoprecipitation. The CLL lymphocytes were different from normal non-T lymphocytes with respect to hexokinase residual activity in the presence of glucose-1,6-P2, pyruvate kinase residual activity in the presence of alanine, and phosphofructokinase activity after stimulation by glucose-1,6-P2. No differences could be discerned in enzyme activities between the CLL and the normal T and non-T lymphocytes.

Hexokinase↗
PubMed2006

Plasma levels of tumor M2-pyruvate kinase should not be used as a tumor marker for hematological malignancies and solid tumors.

It has been reported that the dimeric isoform of the enzyme pyruvate kinase M2 was overexpressed in various solid tumor cells. Hence, it was suggested that circulating levels of the so-called tumor M2-pyruvate kinase (Tu M2-PK) could be used as a tumor marker for monitoring systemic therapies of various solid tumors. We analyzed its validity as a tumor marker by comparing plasma levels of Tu M2-PK in patients with different non-malignant diseases to levels in healthy individuals and in patients with hematological diseases. Plasma levels of Tu M2-PK were measured using an ELISA assay in a total of 284 patients. The mean Tu M2-PK concentration of 32 U/mL was significantly higher in the group of patients with hematological malignancies (n = 121) (p < 0.001). However, 37% of healthy individuals (n = 63) and 44% of patients with non-malignant diseases (n = 100), especially patients with an acute inflammatory reaction (67%), were found to have elevated levels of Tu M2-PK using a cutoff level of 15 U/mL. The specificity was 59% and the sensitivity was 51%. There was no significant correlation between the prevalence of a hematological malignancy and positive Tu M2-PK result. Thus, our data imply that Tu M2-PK is not a useful tumor marker for hematological malignancies and solid tumors, as a significant number of false positive results were detected in healthy individuals and patients with non-malignant diseases.

Biomarkers, Tumor↗
PubMed1987

Pyruvate kinase activity and response to allosteric effectors in rat erythrocytes and reticulocytes fractionated by multiple partitioning in aqueous two-phase systems.

Specific activity of pyruvate kinase decreases as the age of rat erythrocytes increases in fractions obtained by counter-current distribution in dextran-polyethylene glycol biphasic systems; the enzyme is inhibited by ATP and activated by fructose-1,6-bisphosphate at low phosphoenol pyruvate concentrations. Specific activity does not change in fractions from greater than 95 per cent-rich reticulocytes (anaemic rats); the enzyme is inhibited by ATP but not activated by fructose-1,6-bisphosphate. These results can be explained on the basis of different pyruvate kinase isozymes and suggest that decrease in activity is not affecting regulatory properties during erythrocytes aging.

Adenosine Triphosphate↗
PubMed1976

[Pyruvate kinase deficiency. I. Clinical and hematological observations (author's transl)].

Clinical and hematological studies were performed on ten homozygous and seven heterozytous individuals with pyruvate kinase deficiency, aged between 2 and 71 years. Five of the homozygotes were splenectomized. With the exception of a decreased enzyme activity between 41 and 55 per cent and minor changes in their red cell metabolism the heterozygotes showed no abnormal results. In the homozygotes the following results could be demonstrated: 1. Pyruvate kinase activity was decreased to 11 to 35 per cent of normal enzyme activity. 2. There is no relation between the severity of hemolysis and the degree of the enzyme defect. 3. The reticulocyte counts correlated inversely with the hemoglobin concentrations. 4. There is a close correlation between the activities of hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and glutamate oxalacetate transaminase on the one side and reticulocyte counts on the other. 5. Adenosine triphosphate or adenosine reduced the increased autohemolysis in all cases. 6. Following splenectomy, anemia was less pronounced than before. Splenectomized patients did not need further transfusions, though hemolysis persisted.

Adenosine↗
PubMed1976

Pyruvate kinase activity and gluconeogenesis in rat liver after glycogen depletion with nicotinic acid.

Nicotinic acid administration, which depletes liver glycogen, leads to an increase of both pyruvate kinase L and phosphoenolypyruvate carboxykinase in liver by a factor of nearly two. The former is not prevented by either cycloheximide or actinomycin D. L-Cysteine, an allosteric inhibitor of pyruvate kinase L, favors gluconeogenesis from lactate in both nicotinic acid treated and starved animals.

Animals↗
PubMedDate not supplied

[Activity of pyruvate kinase and glucose-6-phosphate dehydrogenase in polymorphonuclear granulocytes of patients with atopic dermatitis in the active stage and in remission].

The activity of granulocyte pyruvate kinase and glucose-6-phospho dehydrogenase in 12 patients with severe atopic dermatitis in active stage of disease and in remission was investigated. It was found that in active stage the activity of both enzymes was not significantly different than in healthy individuals. In remission pyruvate kinase activity was significantly decreased and glucose-6-phospho dehydrogenase activity was found to be non significantly decreased.

Adolescent↗
PubMed1977

Yeast pyruvate kinase: a mutant from catalytically insensitive to fructose 1,6-bisphosphate.

The paper describes some of the characteristic properties of an altered form of pyruvate kinase from a mutant of Saccharomyces cerevisiae. The partially purified enzyme does not require fructose 1,6-bisphosphate for activity but is stabilised in its presence both at low and at high temperatures. The enzyme displays in the absence of fructose 1,6-bisphosphate hyperbolic kinetics with phosphoenolpyruvate (Km, 0.11 mM), ADP (Km, 0.12 mM) and K+ (Km, 11 mM). Sedimentation velocity experiments indicate that the mutated enzyme and the wild type enzyme have S20,w values of 8.9 and 8.6 S respectively. The mutant with the pyruvate insensitive to fructose 1.6-bisphosphate is capable of growing on synthetic media with alcohol or malate as the sole carbon source. The steady-state intracellular levels of phosphoenolpyruvate in the mutant suggest mechanisms that prevent depletion of this metabolite despite an active pyruvate kinase. Spontaneous reversion of this mutant yields clones with normal enzyme activated by fructose 1,6-bisphosphate.

Adenosine Diphosphate↗
PubMed2003

The proton transfer step catalyzed by yeast pyruvate kinase.

The nature of the proton donor to the C-3 of the enolate of pyruvate, the intermediate in the reaction catalyzed by yeast pyruvate kinase, was investigated by site-directed mutagenesis and physical and kinetic analyses. Thr-298 is correctly located to function as the proton donor. T298S and T298A were constructed and purified. Both mutants are catalytically active with a decrease in k(cat) and k(cat)/K(m)(,PEP). Mn(2+)-activated T298S and T298A do not exhibit homotropic kinetic cooperativity with phosphoenolpyruvate (PEP) in the absence of fructose 1,6-bisphosphate, although PEP binding to enzyme-Mn(2+) is cooperative. The pH dependence of k(cat) for T298A indicates the loss of pK(a)(,2) = 6.4-6.9. Thr-298 affects the ionization (pK(a) approximately 6.5) responsible for modulation of k(cat). Fluorescence studies show altered dissociation constants of ligands to each enzyme complex upon Thr-298 mutations. The rates of the phosphoryl transfer and proton transfer steps in the pyruvate kinase-catalyzed reaction are altered; pyruvate enolization is affected to a greater extent. Proton inventory studies demonstrate solvent isotope effects on k(cat) and k(cat)/K(m)(,PEP). Fractionation factors are metal-dependent and significantly <1. The data suggest that a water molecule in a water channel is the direct proton donor to enolpyruvate and that Thr-298 affects a late step in catalysis.

Amino Acid Substitution↗
PubMed1980

Studies of the structure--function relationships of Neurospora crassa pyruvate kinase: interaction with blue dextran--sepharose and Cibacron blue 3G-A.

Blue dextran--Sepharose and Cibacron blue 3G-A interact with pyruvate kinase of Neurospora crassa. The enzyme is readily released from the substituted Sepharose column by elution with 0.17 M potassium phosphate buffer (pH 7.9), or 2 mM fructose 1,6-diphosphate (FDP), but not with either of the substrates, ADP and phosphoenolpyruvate (PEP), at 2 mM. Cibacron blue 3G A is a noncompetitive inhibitor of pyruvate kinase with respect to both substrates. It appears to compete with the allosteric effector, FDP, for binding to the enzyme surface. A lack of elution of the enzyme from the immobilized blue dextran matrix by adenine nucleotides and the absence of a difference spectrum in the 650- to 700-nm range suggest that a "dinucleotide-fold" substructure is not implicated in the dye binding sites on pyruvate kiase. The interaction of Cibacron blue 3G-A and this enzyme can be followed fluorometrically; incremental additon of the dye to the enzyme solution results in a progressive decrease in the fluorescence of surface tryptophanyl residues. The quenching of fluorescence of exposed aromatic groups is subject to reversal following addition of FDP to the pyruvte kinase--Cibacron blue complex.

Adenosine Diphosphate↗
PubMedDate not supplied

The aldolase and pyruvate kinase isoenzyme patterns in a malignant testicular tumor from a 27 year old male.

On the basis of performed tests, we observed the change of pattern of pyruvate kinase isoenzyme in a malignant testicular tumor (only isoenzyme K) and the same isoenzyme in the tissue of other testis not being tumorously changed. In control group pyruvate kinase isoenzyme K and M was observed. Similarly, another pattern of aldolase isoenzymes in tumorous tissue was stated (AC) comparing with control group (arrangement AC, BC). The changes referred also to the activity of both examined enzymes in tumorous tissue and in the tissue of testis not being tumorously changed. The authors suggest the existence of a factor with depressor characteristics, which is produced by neoplasm and changes the function of genes that are responsible for synthesis examined isoenzymes in the tissue not being tumorously changed.

Adult↗
PubMed1982

Influence of morphinization on the development of isoenzymes of pyruvate kinase in rat liver.

1. Acute administration or morphine to foetuses and neonates either directly or via the mother leads to a decrease in the total pyruvate kinase activity. 2. The L-type isoenzyme is selectively suppressed while the M-type isoenzyme is not altered to any significant extent with a consequent lowering of the L/M ratio. 3. The decrease in the total pyruvate kinase activity and the L/M ratio is more prominent when the route of administration of morphine is via the mother. 4. The possible explanation for this difference in the route of administration is that morphine can be converted into a more active metabolite with greater biological potency in adult animals.

Animals↗
PubMed1977

Mechanism of "L"-type pyruvate kinase from rabbit liver. Evidence against phosphoenzyme formation.

The "L"-type pyruvate kinase from rabbit liver does not catalyse exchange between phosphoenol[1-14C]pyruvate and pyruvate at either pH 8.5 or 6.2. Spectrophotometric experiments at pH 8.5 and 6;2 and gel-filtration experiments with [32P]phosphoenolpyruvate at pH 8,5 also fail to demonstrate phosphoenzyme formation. It is concluded that it is very unlikely that the enzyme has a phosphoenzyme mechanism.

Animals↗
PubMed1993

Determination of pyrimidine nucleoside diphosphates by use of combined reactions of pyruvate kinase and lactate dehydrogenase.

ADP is known to be easily determined spectrophotometrically after it is utilized to produce the corresponding amount of NAD by combined reactions of pyruvate kinase and lactate dehydrogenase. We studied whether CDP and UDP can be also determined in a similar manner if they were incubated for a longer period with an increased amount of pyruvate kinase. It was shown that CDP and UDP could be utilized to produce the corresponding amount of NADH oxidized after an incubation of at least 25 min and that 0 to 300 nmols of these nucleotides were able to be determined spectrophotometrically.

Animals↗