Phosphatidylinositol synthetase activities in neuronal nuclei and microsomal fractions isolated from immature rabbit cerebral cortex

Abstract

The synthesis of phosphatidylinositol was studied using a nuclear fraction N 1, a microsomal fraction P 3, rough (R) and smooth (S) microsomal fractions and a microsomal fraction P derived from isolated nerve cell bodies. Each fraction was prepared using cerebral cortices of 15-day-old rabbits. In assays using CDP-diacylglycerol (prepared from egg phosphatidylcholine) and myo[ 3H]inositol at pH 7.4, fraction N 1 had the highest maximal specific rates of phosphatidylinositol synthetase (EC 2.7.8.11) (expressed per μmol phospholipid in the fraction). However the three microsomal fractions achieved maximal specific activities at liponucleotide concentrations close to 50 μM, while fraction N 1 required 200 μM concentrations. In certain cases (25–120 μM CDP-diacylglycerol, and at higher pH values) fraction R had specific activities which equalled or surpassed those of N 1. However, with respect to inositol, fraction N 1 had a distinctly lower K m than was shown for fractions R or P 3. Each of the microsomal fractions and N 1 required Mg 2+ for the reaction, but for N 1, maximal rates could be sustained at 0.1 mM, while for the microsomal fractions the optimal Mg 2+ concentration was 1 mM. For each fraction Mn 2+ could not replace Mg 2+ in the reaction and Mn 2+ was inhibitory. The optimal pH for the reaction was between 8.0 and 9.0. Phosphatidylinositol synthetase could also be shown using fraction N 1 enriched in endogenous CDP-diacylglycerol. The relatively high specific activities of fraction N 1, and the differences found between N 1 and the microsomal fractions, for optimal CDP-diacylglycerol and Mg 2+ concentrations and for K m values for inositol, support the existence of a neuronal nuclear phosphatidylinositol synthetase.