Identification and purification of diphosphoinositol pentakisphosphate kinase, which synthesizes the inositol pyrophosphate bis(diphospho)inositol tetrakisphosphate.
Diphosphoinositol pentakisphosphate (PP-IP5) 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-IP5) kinase, which uses PP-IP5 as a substrate to form bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) in soluble fractions of rat forebrain. The purified protein, a monomer of 56 kDa, displays high affinity (Km = 0.7 microM) 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.
Department of Neuroscience, Pharmacology & Molecular Sciences, and Psychiatry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
C F
CF
Huang
S M
SM
Voglmaier
M E
ME
Bembenek
A
A
Saiardi
S H
SH
Snyder
D000818:Animals; D001921:Brain / Q000201:enzymology; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000096:biosynthesis*; D007700:Kinetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011817:Rabbits; D051381:Rats
D007295:Inositol Phosphates; C079848:bis(1,4)-diphosphoinositol tetrakisphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C064990:inositol pentakisphosphate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); C407349:diphosphoinositol pentakisphosphate kinase; C099438:inositol hexakisphosphate kinase; D006180:Proton-Translocating ATPases