title: A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme.
abstract: 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.
authors.affiliation: 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
authors.forename: Mauro
authors.initials: M
authors.lastname: Sola-Penna
authors.forename: Ana Cristina
authors.initials: AC
authors.lastname: dos Santos
authors.forename: Gutemberg G
authors.initials: GG
authors.lastname: Alves
authors.forename: Tatiana
authors.initials: T
authors.lastname: El-Bacha
authors.forename: Joana
authors.initials: J
authors.lastname: Faber-Barata
authors.forename: Monica F
authors.initials: MF
authors.lastname: Pereira
authors.forename: Fredson C
authors.initials: FC
authors.lastname: Serejo
authors.forename: Andrea T
authors.initials: AT
authors.lastname: Da Poian
authors.forename: Martha
authors.initials: M
authors.lastname: Sorenson
mesh_terms: 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
chemical_list: D005636:Fructosephosphates; D010761:Phosphorus Radioisotopes; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D025203:Phosphofructokinase-1, Muscle Type; D010732:Phosphofructokinase-1