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 activity in this type of cells. The isolated cDNA encodes a protein of 784 amino acids and 85.5 kDa, whose expression was constant along tumor growth and markedly decreased when cell proliferation stops. The enzyme was functionally expressed in a PFK-deficient strain of Saccharomyces cerevisiae and purified to homogeneity. Recombinant PFK-C exhibited the same subunit size as the tumor wild-type isozyme and its steady-state kinetic parameters were similar to those of the form present in normal cells. The regulatory properties 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 tumor preparations containing the various isozyme types. Nevertheless, PFK-C binds fructose-1,6-P(2) to an allosteric site as suggested by protection against thermal denaturation. Our results indicate that glucose metabolism in tumor cells is not regulated by a mutant form of PFK-C but by a high level expression of the normal C isozyme.
Sánchez-Martínez
C
C
Departamento de Bioquímica, Facultad de Medicina de la Universidad Autónoma, Madrid, 28029, Spain.
Estévez
A M
AM
Aragón
J J
JJ
D000494:Allosteric Regulation; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005635:Fructosediphosphates / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae
D004791:Enzyme Inhibitors; D005635:Fructosediphosphates; D007527:Isoenzymes; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D010728:Phosphoenolpyruvate; D010732:Phosphofructokinase-1; D005947:Glucose; C029063:fructose-1,6-diphosphate