Novel Isolation and Filamentation of Drosophila melanogaster Phosphofructokinase-1Cellular and Molecular Biology and Biochemistry
- Kevin Nieves Pichardo
The rate-limiting step of glycolysis, the process by which glucose is broken down to produce energy, is catalyzed by the enzyme phosphofructokinase-1 (PFK). The liver isoform of human PFK has recently been found to organize into chains of tetramers, forming filament structures. Drosophila melanogaster, the fruit fly, is commonly used as a model system to study human development and disease. This study aimed to determine if Drosophila melanogaster PFK (DmPFK) forms filaments, and if so, under what conditions. We hypothesized that filamentation occurs in the presence of factors that inhibit PFK, such as ATP. To determine the conditions under which PFK forms filaments, the protein was purified from Escherichia coli cells by capturing it with nickel resin and additionally using anion exchange fast protein liquid chromatography. Successful purification was detected via western blot and Coomassie stained SDS-PAGE gel with bands at 89 kDa. Out of the five buffers tested, DmPFK was concentrated to 0.1 mg/ml and had the highest enzymatic activity rate in: 100 mM sodium phosphate, 50 mM NaCl, 1 mM EDTA, 1 mM BME, 5% glycerol, adjusted to pH 7.5 at room temperature. ATP and Mg2+ in the presence of F6P and (NH₄)₂SO₄ were required for filamentation, supporting the hypothesis. Future research will investigate the role filamentation may play in regulation of DmPFK, a critical metabolic enzyme, affecting cancer cell proliferation, autoimmune diseases, and neurodegenerative diseases.