The triphosphoinositide phosphomonoesterase of brain tissue.

Preparation of triphosphoinositide phosphomonoesterase from brain extract8. To 40 ml. of the extract of acetone-dried brain powder (Thompson & Dawson, 1964a) was added 20 ml. of 0 132M-tris-HCl buffer, pH 7-2, and the mixture was cooled to 00. Solid (NH4)2SO4 (10-64 g.) was slowly added with stirring, and the mixture was left for 30 min. at 00. After centrifuging at 0° for 10 min. at 10000gay. the supernatant was separated and an additional 6 g. of (NH4)2SO4 was added. The mixture was stirred until the salt was completely dissolved, kept at 0° for 30 min. and the precipitate recovered by centrifuging at l0000gav.. The precipitate was taken up in 3 ml. of 0 132M-tris-HCl buffer, pH 7-2, and the solution dialysed overnight at 40 against 1 1. of 5 mM-dimethylglutaric acid-NaOH buffer, pH 6-4. A 21-3-fold concentration of activity and 60% yield were obtained at this point. This 25-40% saturated (NH4)2SO4 fraction was then fractionated by densitygradient electrophoresis (Svensbon, 1960) as modified by Bangham & Dawson (1962) and Dawson (1963). The light buffer was 2-5 mM-dimethylglutaric acid-NaOH and the heavy component was the same buffer in 45% (v/v) glycerol. The enzyme fraction mixed with the appropriate amount of glycerol and buffer was inserted after about 2-3 cm. of the gradient had been formed. The electrophoresis was carried out for 16-18 hr. at 850 v (5 mA) at the temperature of running tap water. The fractions recovered from the column were assayed for enzymic activity and extinction at 280 mi,. The fractions constituting the main monophosphoesterase peak were combined and stored at -15°. Conditions of incubation and assay of activity. The incubation medium usually contained: triphosphoinositide (sodium or ammonium salt), 0 43 umole; 0-132M-tris-HCl buffer, pH 7-2, 0 3 ml.; 6 mM-MgCl2, 0-15 ml.; 0-2Mreduced glutathione, 0-1 ml.; enzyme preparation (0-050-2 ml.); and water to give a final volume of 1-2 ml. Incubation was carried out for 30 min. at 37°. The subsequent procedure was the same as that described for the