6-PHOSPHOFRUCTO-1-KINASE FROM THE LIPID-ACCUMULATING, NON-FERMENTATIVE, RED YEAST RHODOTORULA-GLUTINIS

6-Phosphofructo-1-kinase (PFK, EC 2.7.1.11) activity was detected in e xtracts of Rhodotorula glutinis grown on different nitrogen and carbon sources. The activity of PFK varied depending on the composition of t he culture medium and on the state of growth. Using a carbon-limited m edium containing glucose and a mixed organic/inorganic nitrogen source , maximal yield, activity and stability of PFK were found under aerobi c conditions at the end of the exponential growth phase. Native Rhodot orula-PFK could be separated by polyacrylamide gel electrophoresis. Ap plying this method for molecular mass estimation a value of 450 +/- 90 kDa was calculated. Taking into account a molecular mass of 130 +/- 5 kDa for the subunit, as determined by sodium dodecyl sulfate-polyacry lamide gel electrophoresis following immunoblotting, a tetrameric stru cture of the native PFK is likely. Polyclonal antibodies, raised again st PFK from Saccharomyces cerevisiae, were able to cross-react with Rh odotorula-PFK. Therefore, there are similarities in the primary struct ure of both enzymes. Fructose 2,6-bisphosphate was identified as a sig nificant activator of Rhodotorula-PFK leading to a 10-fold activation of the enzyme: maximal activation was achieved with 5 mu M fructose 2, 6-bisphosphate.