Identification and purification of diphosphoinositol pentakisphosphate kinase, which synthesizes the inositol pyrophosphate bis(diphospho)inositol tetrakisphosphate

Diphosphoinositol pentakisphosphate (PP-IPs) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP₄) were recently identified as inositol phosphates which possess pyrophosphate bonds. The molecular mechanisms that regulate the cellular levels of these compounds are not yet characterized. To pursue this question, we have previously purified an inositol hexakisphosphate (IP₆) kinase from rat brain supernatants [Voglmaier, S. M., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4305-4310]. We now report the identification and purification of another novel kinase, diphosphoinositol pentakisphosphate (PP-IPs) kinase, which uses PP-IP₅ as a substrate to form bis(diphospho)inositol tetrakisphosphate (bisPP-IP₄) in soluble fractions of rat forebrain. The purified protein, a monomer of 56 kDa, displays high affinity (K(m) = 0.7 μM) and selectivity for PP-IP₅ as a substrate. The purified enzyme also can transfer a phosphate from bis-PP-IP₄ to ADP to form ATP. This ATP synthase activity is an indication of the high phosphoryl group transfer potential of bis-PP-IP₄ and may represent a physiological role for PP-IP₅ and bis-PP-IP₄.