Heterogeneity of the calcium-dependent phosphatidylinositol phosphodiesterase in rat brain

1. The Ca(2+)-dependent phosphatidylinositol phosphodiesterase (phospholipase C-type) from the cytosolic supernatant of rat brain was active against exogenous [(32)P]-phosphatidylinositol from pH5.0 to pH8.5. However, the activity in the range pH7.0-8.5 could not be recovered after precipitation with (NH(4))(2)SO(4); most of the enzyme activity was recovered in the 30-50% fraction and showed a single sharp pH optimum at 5.5. 2. The cytosolic supernatant was analysed by isoelectric focusing on acrylamide gels, and assay at pH5.5. Four peaks of phosphodiesterase activity were found at pI ranges 7.4-7.2, 6.0-5.8, 4.8-4.4 and 4.2-3.8. 3. The cytosolic supernatant was also applied to a chromatofocusing column, and again assayed at pH5.5. Four peaks were eluted: minor, but consistent, activity at the beginning of the elution with a pI of near 7.2 or above; a second peak at pH6.0-5.85; a third broad peak with a wide range pH5.3-4.2; and a fourth peak, which was eluted by washing the column with 1m-NaCl, suggesting an isoenzyme with a pI below 4.0 (supported by the result of the isoelectric focusing). 4. If all the chromatofocusing fractions were assayed at pH7.0 or 8.0 (at 1mm-Ca(2+)), only a single sharp peak was detected, with a pI of 4.6-4.8. This peak disappeared on (NH(4))(2)SO(4) fractionation (30-50%) of the cytosolic supernatant, whereas the four peaks with activity at pH5.5 were virtually unaffected. 5. The four activities (assayed at pH5.5) separated by chromatofocusing produced inositol 1:2-cyclic monophosphate, inositol 1-monophosphate and diacylglycerol as enzymic products. 6. We conclude that the Ca(2+)-dependent phosphatidylinositol phosphodiesterase exhibits considerable heterogeneity, both with respect to pH optima of activity, and its isoelectric properties.