Zinc inhibition of pyruvate kinase of M-type isozyme

Inhibitory effect of Zn on the pyruvate kinase of M (muscle)-type isozyme was analyzed for the purpose of elucidating the cytotoxicity of Zn. Zn inhibited pyruvate kinase uncompetitively with respect to the substrate PEP, and competitively with respect to ADP. Quotient velocity plot calculated from the Zn-inhibition curves showed that Zn²⁺ as a ZnADP complex acted as competitive and uncompetitive inhibitors of the enzyme with respect to the substrate ADP and PEP, respectively: Zn²⁺ forms a ZnADP complex, which may bind to the ADP-binding site of the free enzyme with the K_i value of 1.4 μM causing competitive inhibition, or to the ADP-site of the enzyme-PEP complex with 2.6 μM resulting in uncompetitive inhibition. The inhibition of pyruvate kinase by Zn²⁺ may be responsible for the cytotoxicity of this metal by decreasing glycolytic flux.     

Phylogeny congruence analysis and isozyme classification: the pyruvate kinase system

As the isozymes of pyruvate kinase (PK) are best known in rats, the characteristics of the rat isozymes are generally used to classify the PK isozymes in other species. Given the discrepancies generated by this classification by analogy, we evaluated a classification using a phylogeny congruence analysis of the compositional relatedness of vertebrate PK's. While our phylogenetic analysis confirmed the well established separation of the L and R isozymes from the K and M isozymes, its power became most evident in the identification of non-orthologous (or variant) forms of PK. Our analysis emphasized the uniqueness of chicken liver PK which cannot be classified either as a K or an L isozyme, confirmed that tumors express a variety of forms of PK, and indicated that lungs systematically express PK's which are not orthologous with PK's from other tissues. The determination of orthology by the phylogeny congruence analysis assumes that the structural data from different sources are subject to similar methodological error. However, we cannot reject the possibility that an apparent lack of orthology be due to artifacts during purification and analysis.     

A new pyruvate kinase mutation with hyperactivity in the mouse

A mouse mutant with pyruvate kinase (PK) hyperactivity has been found in offspring of 1-ethyl-1-nitrosourea (ENU)-treated male mice. The activity alteration was detected in the blood and could also be found in the liver but not in the muscle, kidney, heart, spleen, lung, or brain. Heterozygous mice have erythrocyte PK activity enhanced up to about 160% and homozygotes up to about 240%, compared to homozygous wild types. The mutation is codominantly expressed. The heterozygous and homozygous mutants are viable and fully fertile and do not show symptoms of erythrocytosis. The mutation does not affect the heat stability, the electrophoretic mobility, or the Km (for phosphoenolpyruvate) of the PK molecule. It is suggested that the regulatory locus of PK-1 is affected by this mutation. The observations support also the theory of one structural locus for the erythrocyte and liver isozymes.     

A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion

We previously reported a cytosolic pyruvate kinase (EC 2.7.1.40) from Toxoplasma gondii (TgPyKI) that differs from most eukaryotic pyruvate kinases in being regulated by glucose 6-phosphate rather than fructose 1,6-diphosphate. Another putative pyruvate kinase (TgPyKII) was identified from parasite genome, which exhibits 32% amino acid sequence identity to TgPyKI and retains pyruvate kinase signature motifs and amino acids essential for substrate binding and catalysis. Whereas TgPyKI is most closely related to plant/algal enzymes, phylogenetic analysis suggests a proteobacterial origin for TgPyKII. Enzymatic characterization of recombinant TgPyKII shows a high pH optimum at 8.5, and a preference for GDP as a phosphate recipient. Catalytic activity is independent of K+, and no allosteric or regulatory effects were observed in the presence of fructose 1,6-diphosphate, fructose 2,6-diphosphate, glucose 6-phosphate, ribose 5-phosphate, AMP, or ATP. Unlike TgPyKI, native TgPyKII activity was exclusively associated with the membranous fraction of a T. gondii tachyzoite lysate. TgPyKII possesses a long N-terminal extension containing five putative start codons before the conserved region and localizes to both apicoplast and mitochondrion by immunofluorescence assay using native antibody and fluorescent protein fusion to the N-terminal extension. Further deletional and site-directed mutagenesis suggests that a translation product from 1st Met is responsible for the localization to the apicoplast, whereas one from 3rd Met is for the mitochondrion. This is the first study of a potential mitochondrial pyruvate kinase in any system.     

Rat hyperplastic hepatic nodules and primary hepatomas retaining abnormally high pyruvate kinase L type activity.

As reported in our previous paper (Sato et al., Cancer Res., 38: 3086-3093, 1978), most of the hyperplastic hepatic nodules and primary hepatomas induced by N-2-fluorenylacetamide (2-FAA), diethylnitrosamine (DENA), and 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) showed that pyruvate kinase liver (L) type decreases and the prototypic M2 type increases concomitantly with dedifferentiation of tissues. However, at least 14 samples among 120, mostly hyperplastic nodules and highly differentiated hepatomas induced by 2-FAA or DENA, retained exceptionally high activities of the L type, while other enzymes of carbohydrate metabolism in these tissues deviated toward a common pattern similar to that in the fetal liver. Individual patterns of 9 enzymes including pyruvate kinase of carbohydrate metabolism in these samples are arranged and discussed as examples of unbalanced enzyme deviation in hepatocarcinogenesis.     

Lack of evidence for a pyruvate kinase isozyme shift in hepatocytes of the regenerating rat liver

1. Pyruvate kinase isozyme shift in a regenerating rat liver was studied. Rats were subjected to a 70% hepatectomy and the liver homogenate or hepatocyte preparations were obtained from the regenerating liver. 2. Using thin layer polyacrylamide gel electrophoresis, liver homogenates from an intact normal rat appeared to contain the L-type isozyme in the greatest number and M2-type to a lesser extent. 3. The ratio of the M2- to L-type increased in the preparations obtained from the regenerating liver. 4. In the hepatocyte preparations prepared from a regenerating rat liver by the conventional method, a small amount of M2-type isozyme was detected. 5. However, the M2-type isozyme was hardly detected in the highly purified hepatocyte preparations prepared using Percoll. 6. Similar results were obtained by separation of the enzyme by DEAE cellulose column chromatography. 7. These results suggest that there is no pyruvate kinase isozyme shift from L- to M2-type in hepatocytes in the course of regeneration. 8. The increased M2-type isozyme in the regenerating rat liver is considered to originate from nonparenchymal cells.     

Protein kinase activity in Morris hepatomas

Rat liver protein kinase has been fractionated into five peaks of activity on isoelectrofocusing columns. The major liver peak, which was activated by cAMP, was decreased in two fast growing Morris hepatomas. The second major liver peak, independent of cAMP, was increased in the tumors.     

A hexokinase-coupled radioassay for pyruvate kinase

A procedure for the assay of low activities of pyruvate kinase (0.01 to 4 mIU) is described. The method consists of coupling the formation of ATP by the pyruvate kinase reaction to hexokinase in the presence of uniformly labeled [¹⁴C]glucose. The labeled glucose 6-phosphate thus formed is easily separated from the unreacted glucose using small columns of Dowex 1-X8 formate and detected by liquid scintillation spectrometry. Chromatographic patterns of pyruvate kinases from 25 mg of rat liver, 3.5 mg of frog oocyte, and 0.5 mg of the whole body of the fruit fly Drosophila melanogaster are presented as illustrations of the sensitivity of the radioassay.     

A method of determining pyruvate kinase activity

A method is suggested for determining the activity of pyruvate kinase (ATP: pyruvate-phosphotransferase). The method is based on evaluation of the amount of ATP formed in the course of phosphoenolpyruvic-to-pyruvic acid transformation in the presence of ADP and magnesium ions. The ATP amount in the reaction medium is determined by paper electrophoresis with subsequent spectrophotometry.     

A direct radioassay for pyruvate kinase activity

Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. A direct radioassay for this enzyme using [14C]PEP as substrate has been developed. The product, [14C]pyruvate, can be separated from the substrate rapidly and easily by applying the mixture to a hydroxyapatite column, and eluting the [14C]pyruvate directly into a scintillation vial. The [14C]PEP is bound to the column which can be regenerated and used indefinitely. The assay is sensitive, rapid, and particularly well suited for the simultaneous assay of large numbers of samples.     

Kinetic characterization of yeast pyruvate carboxylase isozyme pyc1

Yeast (Saccharomyces cerevisiae) is unusual in being the only organism thus far identified as having two genes for pyruvate carboxylase. The expression of the two isozymes Pyc1 and Pyc2 appears to be differentially regulated, and since both are expressed cytoplasmically, this suggests that they have different properties. To the present, little has been done to characterize these isozymes, and almost all of the published kinetic information on yeast pyruvate carboxylase comes from measurements of enzyme prepared from bakers' yeast which is likely to be a mixture of both isozymes. Here we have measured basic kinetic parameters for Pyc1 and found that the K(a) of this isozyme for acetyl CoA is in the order of 8-10-fold higher than previously recorded, suggesting that Pyc1 and Pyc2 may be differentially regulated by this effector. Pyc1 is highly dependent on the presence of acetyl CoA for activity and in this respect is similar to chicken liver pyruvate carboxylase. However, unlike the chicken liver enzyme, the quaternary structure of the enzyme is quite stable in the absence of acetyl CoA, and the major locus of action of this effector appears to lie outside of the stimulation of the biotin carboxylation reaction.     

A kinetic study of rabbit muscle pyruvate kinase

The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg(2+), pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg(2+), form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.