Partial purification of inositol polyphosphate 1-phosphomonoesterase with characterization of its substrates and products by nuclear magnetic resonance spectroscopy.
A study of the enzyme activities that degrade Ins(1,3,4)P3 in rat brain showed that it was dephosphorylated primarily by a Mg2+-dependent inositol polyphosphate 1-phosphomonoesterase to Ins(3,4)P2 and then to Ins(3)P by a 4-phosphomonoesterase. A less active enzyme activity with the properties of a 4-phosphomonoesterase that converted Ins(1,3,4)P3 to Ins(1,3)P2 was also detected. The inositol polyphosphate 1-phosphomonoesterase was separated from the 4-phosphomonoesterase and the inositol monophosphate phosphomonoesterase by chromatography on phosphocellulose, DE-52 anion exchange and hydroxylapatite columns. Kinetic characterization of the partially purified inositol polyphosphate 1-phosphomonoesterase indicated that both Ins(1,3,4)P3 and Ins(1,4)P2 were substrates with apparent Km values of 0.9 microM and 0.7 microM, respectively. Either substrate was a competitive inhibitor of the other substrate and dephosphorylation of both substrates was directly inhibited by Li+ in an uncompetitive manner. These data strongly suggest that a single enzyme dephosphorylates both Ins(1,3,4)P3 and Ins(1,4)P2. The 4-phosphomonoesterase that dephosphorylated Ins(3,4)P2 to Ins(3)P was insensitive to Mg2+ and Li+ and was probably the same enzyme that degraded Ins(1,3,4)P3 to Ins(1,3)P2. The isomeric configurations of the major inositol polyphosphates formed from the degradation of Ins(1,3,4,5)P4 were determined using 1H- and 31P-NMR spectroscopy, and confirmation of the structures assigned to Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(3,4)P2 was obtained.
Hansen
C A
CA
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104.
Inubushi
T
T
Williamson
M T
MT
Williamson
J R
JR
D000818:Animals; D001921:Brain / Q000201:enzymology; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism; D000072181:Inositol Polyphosphate 5-Phosphatases; D008094:Lithium / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats
D007295:Inositol Phosphates; C051854:inositol 3,4-bisphosphate; D015544:Inositol 1,4,5-Trisphosphate; D008094:Lithium; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases; C053087:inositol-1,4-bisphosphate 1-phosphatase; D008274:Magnesium