PFK1 activity assays

PFK1 activity was determined using an auxiliary enzyme assay (Brüser et al., 2012) with kinetic analysis using 200-µl reactions containing 50 mM Tris-HCl, pH 7.4, 100 mM KCl, 10 mM MgCl₂, 0.15 mM NADH, 0.675 U/ml aldolase, 5 U/ml triosephosphate isomerase, and 2 U/ml glycerol phosphate dehydrogenase. ATP and F6P were used as indicated. Auxiliary enzymes were desalted using an Amicon Ultracel-10K Centrifugal Filter Unit (EMD Millipore) before use. The temperature was equilibrated to 25°C for 10 min before initiating the reaction with the addition of magnesium chloride. The absorbance at 340 nm was measured using a SpectraMax M5 microplate reader (Molecular Devices). Kinetic parameters were generated by linear regression analysis of the Michalas-Menton or Hill equations using Prism (GraphPad Software) and are the mean of a minimum of three measurements from two independent preparations of protein. One unit of activity was defined as the amount of enzyme that catalyzed the formation of 1 mmol of fructose 1,6-bisphosphate per minute at 25°C.