Table 2. Kinetic parameters of PPIP5K2^KD:ATP synthesis.

Kinetic parameters were determined under pseudo first-order conditions [38] for dephosphorylation (ATP synthesis) of InsP₈ or 1-InsP₇ for both ADP and InsP substrates (see the Materials and methods section). When fixed, ADP was 10 mM and InsP 10 μM. Initial velocities were measured by conversion of an appropriate [³H]-radiolabelled inositol phosphate tracer (4000 DPM) as analysed via Partisphere 5 SAX HPLC (e.g., see, Figures 4A and 4C) for each data point. The Michaelis–Menten equation was fit to plots of initial velocity against substrate concentration using non-linear regression (GraphPad Prism v5.03). No substrate inhibition was observed (results not shown). Results are presented as the means±S.E.M. (n≥2 independent experiments).

		Km (μM)				kcat/Km (M−1·s−1)
Substrates		Nucleotide	InsP	Vmax (nmol·min−1·mg−1)	kcat (s−1)	Nucleotide	InsP
ADP	InsP8	900±280	0.022±0.011	270±30	0.19±0.02	2.1×102	8.5×106
ADP	1-InsP7	5.2±1.5	0.11±0.01	2.8±0.1	0.002±0.000	3.7×102	1.7×104