Regulation of 6-phosphofructo-1-kinase activity in ras-transformed rat-1 fibroblasts.
Fructose 2,6-bisphosphate (F-2,6-P2) stimulated glycolysis in cell-free extracts of both normal and ras-transfected rat-1 fibroblasts. The extract of the transformed cell glycolyzed more rapidly in both the absence and the presence of F-2,6-P2 than the extract of the parent fibroblast. Addition of mitochondrial ATPase (F1) or inorganic phosphate (Pi) further stimulated lactate production in both cell lines. F-2,6-P2 stimulated the 6-phosphofructo-1-kinase (PFK-1) activity in extracts of normal and transfected cells. The activity in extracts of transformed cells tested with a fructose 6-phosphate regenerating system was considerably higher than in the extract of normal cells. Stimulation of PFK-1 activity by cAMP of both cell lines was not as pronounced as that by F-2,6-P2. In the absence of F-2,6-P2 the PFK-1 activity was strongly inhibited in the transformed cell by ATP concentrations higher than 1 mM, whereas in the normal cell only a marginal inhibition was noted even at 2 or 3 mM ATP. F-2,6-P2 reversed the inhibition of PFK-1 by ATP. Nicotinamide adenine dinucleotide (NAD) at 100 microM (in the presence of 2 mM ATP and 1 microM F-2,6-P2) stimulated PFK-1 activity only in the transformed cell, whereas nicotinamide adenine dinucleotide phosphate (NADP) inhibited PFK-1 activity (in the presence or absence of 1 microM F-2,6-P2) in extracts of both cell lines. No previous observations of stimulation or inhibition by NAD or NADP on PFK-1 activity appear to have been reported. A threefold increase in the intracellular concentration of F-2,6-P2 was observed after transfection of rat-1 fibroblast by the ras oncogene. We conclude from these data that the PFK-1 activity of ras-transfected rat-1 fibroblasts shows a greater response to certain stimulating and inhibitory regulating factors than that of the parent cell.
Kole
H K
HK
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Resnick
R J
RJ
Van Doren
M
M
Racker
E
E
D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002474:Cell-Free System; D005347:Fibroblasts / Q000201:enzymology; D005632:Fructose / Q000494:pharmacology; D005635:Fructosediphosphates / Q000378:metabolism / Q000494:pharmacology; D011905:Genes, ras; D006019:Glycolysis / Q000187:drug effects; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D051381:Rats
D005635:Fructosediphosphates; D007773:Lactates; D010710:Phosphates; D005632:Fructose; C027652:fructose 2,6-diphosphate; D010732:Phosphofructokinase-1