Phosphofructokinase C isozyme from ascites tumor cells: Cloning, expression, and properties

The phosphofructokinase C isozyme (PFK-C) from ascites tumor cells has been cloned and characterized to investigate the particular properties of PFK a ctivity in this type of cells. The isolated cDNA encodes a protein of 784 a mino acids and 85.5 kDa, whose expression was constant along tumor growth a nd markedly decreased when cell proliferation stops. The enzyme was functio nally expressed in a PFK-deficient strain of Saccharomyces cerevisiae and p urified to homogeneity, Recombinant PFK-C exhibited the same subunit size a s the tumor wild-type isozyme and its steady-state kinetic parameters were similar to those of the form present in normal cells. The regulatory proper ties of the C isozyme accounted for the lack of fructose-1,6-P-2 activation and the P-enolpyruvate inhibition of PFK activity observed in ascites tumo r preparations containing the various isozyme types. Nevertheless, PFK-C bi nds fructose-1,6-P-2 to an allosteric site as suggested by protection again st thermal denaturation. Our results indicate that glucose metabolism in tu mor cells is not regulated by a mutant form of PFK-C but by a high level ex pression of the normal C isozyme, (C) 2000 Academic Press.