An enzymatically coupled assay for rat brain polyphosphoinositide phosphodiesterase in an optimized reaction mixture.
Bovine intestinal alkaline phosphatase was found to hydrolyze inositol phosphates many times faster than the monoester phosphate groups of the polyphosphoinositides. A convenient and sensitive in vitro assay for the Ca2+-dependent polyphosphoinositide phosphodiesterase was devised in which inositol trisphosphate released from exogenous phosphatidylinositol 4,5-bisphosphate was hydrolyzed by alkaline phosphatase. The resulting inorganic phosphate was measured by an automated method after solubilization of the reaction mixture with sodium dodecyl sulfate. The phosphodiesterase was maximally stimulated by combining the known positive effects of cetyltrimethylammonium bromide (at the optimum detergent-to-substrate ratio of 2.3), monovalent cations (0.1 M KCl), and Ca2+ (0.5 mM) with the additional enhancement by Triton X-100 (0.2% w/v). Activities obtained for rat brain homogenates and microsomal and cytosol fractions were 126 +/- 3.8 (17), 110 +/- 5.7 (10), and 252 +/- 15.5 (8) nmol X min-1 X mg protein-1 (mean +/- SE for n determinations), respectively.
D000469:Alkaline Phosphatase; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003902:Detergents; D004075:Diglycerides / Q000096:biosynthesis; D006868:Hydrolysis; D007295:Inositol Phosphates; D007422:Intestines / Q000201:enzymology; D010710:Phosphates / Q000032:analysis; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains
D003902:Detergents; D004075:Diglycerides; D007295:Inositol Phosphates; D010710:Phosphates; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C