Heterogeneity of the calcium-dependent phosphatidylinositol phosphodiesterase in rat brain

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Abstract
The Ca2+-dependent phosphatidylinositol phosphodiesterase (phospholipase C-type) from the cytosolic supernatant of rat brain was active against exogenous [32P]-phosphatidylinositol from pH 5.0-8.5. The activity in the pH 7.0-8.5 range could not be recovered after precipitation with (NH4)2SO4; most of the enzyme activity was recovered in the 30-50% fraction and showed a single sharp pH optimum at 5.5. The cytosolic supernatant was analyzed by isoelectric focusing on acrylamide gels, and assay at pH 5.5. Four peaks of phosphodiesterase activity were found at pI [isoelectric point] ranges 7.4-7.2, 6.0-5.8, 4.8-4.4 and 4.2-3.8. The cytosolic supernatant was also applied to a chromatofocusing column and again assayed at pH 5.5. Four peaks were eluted: minor, but consistent, activity at the beginning of the elution with a pI of near 7.2 or above; a 2nd peak at pH 6.0-5.85; a 3rd broad peak with a wide range pH 5.3-4.2; and a 4th peak, which was eluted by washing the column with 1 M-NaCl, suggesting an isoenzyme with a pK < 4.0 (supported by the result of the isoelectric focusing). If all the chromatofocusing fractions were assayed at pH 7.0 or 8.0 (at 1 mM-Ca2+), only a single sharp peak was detected, with a pI of 4.6-4.8. This peak disappeared on (NH4)2SO4 fractionation (30-50%) of the cytosolic supernatant, whereas the 4 peaks with activity at pH 5.5 were virtually unaffected. The 4 activities (assayed at pH 5.5) separated by chromatofocusing produced inositol 1:2-cyclic monophosphate, inositol 1-monophosphate and diacylglycerol as enzymic products. The Ca2+-dependent phosphatidylinositol phosphodiesterase exhibits considerable heterogeneity, both with respect to pH optima of activity and its isoelectric properties.