Pyruvate kinase expression

Pyruvate kinase (PK) is one of the three postulated rate-controlling enzymes of glycolysis. The high-energy phosphate of phosphoenolpyruvate is transferred to ADP by this enzyme, which requires for its activity both monovalent and divalent cations. Enolpyruvate formed in this reaction is converted spontaneously to the keto form of pyruvate with the synthesis of one ATP molecule. PK has four isozymes in mammals M, M2, L, and R. The M2 type, which is considered to be the prototype, is the only form detected in early fetal tissues and is expressed in many adult tissues. This form is progressively replaced by the M( type in the skeletal muscle, heart, and brain by the L type in the liver and by the R type in red blood cells during development or differentiation (M26). The M, and M2 isozymes display Michaelis-Menten kinetics with respect to phosphoenolpyruvate. The Mj isozyme is not affected by fructose-1,6-diphosphate (F-1,6-DP) and the M2 is al-losterically activated by this compound. Type L and R exhibit cooperatively in... 

Fig. 4. Schematic representation of expression of the rat pyruvate kinase (PK) gene. Exons specific to each isozyme are indicated by marked boxes. The exons common to Mj- and M2-type PK and common to L- and R-type PK are shown by open boxes. CAAT, CAT box TATA, TATA box AATAAA, polyadenylation signal.

T10. Tani, K Fujii, H.,Nagata, S and Miwa, S Human liver type pyruvate kinase Complete amino acid sequence and the expression in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 85, 1792-1795 (1988). 

Til. Tani, K., Yoshida, M. C., Satoh, H., Mitamura, K Noguchi, T Tanaka, T., Fujii, H., and Miwa, S., Human M2-type pyruvate kinase cDNA cloning, chromosomal assignment and expression in hepatoma. Gene 73,509-516 (1988). 

Most cases are due to decreased expression of pymvate kinase activity, usually to 5-25% of normal levels complete loss of pyruvate kinase activity can cause embryonic death. 

Yamada, K. Noguchi, T. (1999) Nutrient and hormonal regulation of pyruvate kinase gene expression. Biochem J. 337, 1—11. Detailed review of recent work on the genes and proteins of this system and their regulation. 

In view of these problems with Ni2+, Mna+ has been used as a probe for Mg2+ with some success. However, it should be noted that there is a difference in radius which may be manifested in different biochemical behaviour (Mn2+, 0.80 A Mg2+, 0.65 A). Thus Mn2+ has been used to probe the Mg2+ site in pyruvate kinase.95 While the Mg2+-activated enzyme is inhibited by Ca2+ and Li+, the Mn2+-activated enzyme is inhibited by Ca2+ and not by Li+. There are also differences in the catalytic and regulatory properties of the NAD+-specific malic enzyme of E. coli,104 depending upon whether the divalent activator is Mn2+ or Mg2+. It is necessary, therefore, to express a cautionary note. These two cations may act in slightly different ways to bring about a similar final result. In the second example it appears that the metal cofactors stabilize two different conformational states of the enzyme. 

The multienzymic nature of pyruvate kinase has been investigated in detail in M. expansa (98), H. diminuta (117) and S. erinacei (240). These worms possess FBP-sensitive and FBP-insensitive pyruvate kinase isoenzymes. In H. diminuta, as many as five pyruvate kinase isoenzymes (for definition and usefulness, see Chapter 6) occur during development (Fig. 5.3) and it seems likely that differential expression of these different forms of the enzyme may help to control the specific composition of excreted end-products by the various life cycle stages. The nature and regulation of the end-products secreted in H. diminuta are discussed further below. 

Finally, in the third mammlian grouping (dog, horse, rabbit, seal, swine), the fetal Hb is not expressed. In these species, even without HbF, the 02 affinity of fetal blood is kept higher than that of the adult. This difference is achieved by means of low concentrations of RBC DPG in the fetus and by higher-than-usual concentrations of DPG in the RBCs of adults. In most of these species, this fetal-adult difference in RBC DPG concentration is due to an enhanced catalytic capacity of the terminal part of the glycolytic path (indexed by pyruvate kinase activity, for example), which increases DPG flux towards pyruvate and lactate (see Poyart et ah, 1992). 

Hnang B, Wn P, Bowker-Kinley MM, and Harris RA (2002) Regnlation of pyruvate dehydrogenase kinase expression hy peroxisome proliferator-activated receptor-alpha ligands, glucocorticoids, and insulin. Diabetes 51,276-83. 

HK deficiency (OMIM 235700) is a rare, recessively inherited disease with chronic nonspherocytic hemolytic anemia as the predominant clinical feature. The phenotypic expression of the disease is heterogeneous, as with most glycolytic red cell enzyme deficiencies. The spectrum ranges between severe neonatal hemolysis and death to a fully compensated chronic hemolytic anemia. Patients benefit in general from a splenectomy. Red cell morphology is normal. Since HK activity is dependent on red cell age, reticulocytosis, always present in HK-deficient patients, may obscure enzyme deficiency. Other age-dependent red cell enzymes (e.g., pyruvate kinase and/or G6PD) should be measured simultaneously as an internal control to assess the influence of reticulocyte enzyme activity. 

Second, we selected or made several yeast glycolytical promoters including pyruvate kinase (Pyk) promoter, phosphoglucose isomerase (PCI) promoter, and phosphoglycerol kinase (PGK) promoter that can actively express genes in both E. coli and Saccharomyces yeasts and also use these promoters to express the E. coli genes both in yeast and E. coli. 

A number of other enzymopathic substances (e.g., pyruvate kinase. Chapter 13 and pyrimidine-5 -nucleotidase. Chapter 27), abnormal hemoglobins (Chapter 28), and abnormalities of the erythrocyte cytoskeleton (Chapter 10) may cause hemolytic anemia. Because many enzymes in the red cell are identical to those in other tissues, defects in these enzymes may have pleiotropic effects. Thus, in addition to hemolytic anemia, triose phosphate isomerase deficiency causes severe neuromuscular disease, and phospho-fructokinase deficiency causes a muscle glycogen storage disease (Chapter 13). Mutations that result in decreased enzyme stability are usually most strongly expressed in erythrocytes because of their inability to synthesize proteins. 

However, is it possible that malaria parasites also have a functional Krebs cycle Gene expression profiles of blood-stage parasites have indicated an apparent coordinated expression of genes encoding TCA cycle enzymes however, since the expression is so low, it is understandable why biochemical assays failed to detect such activities. (Unexplained is how Speck, Moulder and Evans (1946) were able to detect TCA cycle enzymes.) Since plasmodial mitochondria lack pyruvate kinase the... 

Miquerol, L., Lopez, S., Cartier, N., Tulliez, M., Raymondjean, M., and Kahn, A. (1994) Expression of the L-type pyruvate kinase gene and the hepatocyte nuclear factor 4 transcription factor in exocrine and endocrine pancreas. J. Biol. Chem. 269, 8944-8951. 

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