Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts.
Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.
Niemeyer
H
H
Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago.
Rabajille
E
E
D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D005635:Fructosediphosphates / Q000502:physiology*; D005954:Glucosephosphate Dehydrogenase / Q000502:physiology; D006593:Hexokinase / Q000037:antagonists & inhibitors*; D006598:Hexosediphosphates / Q000502:physiology*; D008099:Liver / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000502:physiology*; D011869:Radioligand Assay; D051381:Rats; D013053:Spectrophotometry
D005635:Fructosediphosphates; D006598:Hexosediphosphates; C027652:fructose 2,6-diphosphate; D005954:Glucosephosphate Dehydrogenase; D006593:Hexokinase; D010732:Phosphofructokinase-1