Purification and characterization of a polyphosphoinositide phosphatase from rat brain.
A novel membrane-bound polyphosphoinositide phosphatase has been purified 7700-fold from rat brain. A combination of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated that the enzyme is a monomer with a molecular weight of 85,000-90,000. Biochemical analysis of the polyphosphoinositide phosphatase demonstrated that the enzyme utilizes phosphatidylinositol(4)phosphate (PtdIns(4)P), phosphatidylinositol(3)phosphate (PtdIns(3)P), and phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P2) as substrates. In the case of PtdIns(4,5)P2, the substrate is doubly dephosphorylated to yield PtdIns. The apparent Km values for PtdIns(4)P and PtdIns(4,5)P2 are 45 and 5 microM, respectively. Inositol(1,4)bisphosphate and inositol(1,4,5)trisphosphate neither serve as direct substrates for the polyphosphoinositide phosphatase nor inhibit its activity even at concentrations as high as 100 microM. Thus, the substrate specificity of the polyphosphoinositide phosphatase is distinct from that of previously identified phosphatases that utilize both inositol phospholipids and soluble inositol phosphates as substrates. The ability of the polyphosphoinositide phosphatase to hydrolyze phosphate from the 3-, 4-, or 5-position of the inositol ring suggests that this enzyme may play a key role in maintaining homeostasis among all forms of polyphosphoinositides.
Hope
H M
HM
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Pike
L J
LJ
D000818:Animals; D001921:Brain / Q000201:enzymology*; D006358:Hot Temperature; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D010716:Phosphatidylinositols; C089504:polyphosphoinositide phosphatase; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium