A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme.
Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and alpha-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32P in the gamma position ([gamma-32P]ATP), and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32P ([1-32P]fructose-1,6-bisphosphate). Activated charcoal is used to adsorb unreacted [gamma-32P]ATP, and the radioactive product in the supernatant, [1-32P]fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme.
Sola-Penna
Mauro
M
Laboratório de Enzimologia e Controle do Metabolismo (LabECoM), Departamento de Fármacos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, RJ 21944-910, Brazil. maurosp@ufrj.br
dos Santos
Ana Cristina
AC
Alves
Gutemberg G
GG
El-Bacha
Tatiana
T
Faber-Barata
Joana
J
Pereira
Monica F
MF
Serejo
Fredson C
FC
Da Poian
Andrea T
AT
Sorenson
Martha
M
D000255:Adenosine Triphosphate; D000818:Animals; D002522:Chlorocebus aethiops; D004912:Erythrocytes / Q000201:enzymology; D005636:Fructosephosphates; D006801:Humans; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D010732:Phosphofructokinase-1 / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification; D025203:Phosphofructokinase-1, Muscle Type / Q000032:analysis / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D011817:Rabbits; D011874:Radiometry / Q000379:methods; D012588:Scintillation Counting; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D014709:Vero Cells
D005636:Fructosephosphates; D010761:Phosphorus Radioisotopes; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D025203:Phosphofructokinase-1, Muscle Type; D010732:Phosphofructokinase-1