Purification and Characterization of Phosphatidylinositol-specific Phospholipase C in Suspension-cultured Cells of Rice (Oryza sativa L.)

Abstract

Phosphatidylinositol-specific phospholipase C was purified from the soluble fraction of suspension-cultured rice cells. The apparent molecular weight of rice enzyme was estimated to be 50, 000 by both Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is composed of a single polypeptide. The enzyme had an isoelectric point of 6.3. The soluble phospholipase C had a high degree of specificity toward phosphatidylinositol and a weak activity toward phosphatidyl-inositol monophosphate, while the enzyme did not hydrolyze the other phospholipids or p-nitrophenylphosphorylcholine. V_max and K_m values were 5.0 μmol/min/mg protein and 0.3 mM, respectively. The pH dependency of the enzyme activity was sharp with an optimum of 5.2. In addition, the phospholipase C was a Ca²⁺-dependent enzyme. The marked activation of enzyme was observed in the presence of 10 to 250μM Ca²⁺ and higher Ca²⁺ concentrations than 1 mM had a strong inhibitory effect. A possible regulation of the phospholipase C activity by pH and Ca²⁺ concentrations in the rice cells is discussed.