Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Abstract

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP(2)) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP(2) phospholipase C was dependent on Ca(2+) with maximum activity at 10 to 100 mum free Ca(2+) and half-maximal activation at 0.1 to 1 mum free Ca(2+). In the presence of 10 mum Ca(2+), 1 to 2 mm MgCl(2) or MgSO(4) further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba(2+), Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+)) inhibited the enzyme activity. The stimulatory effect by Mg(2+) was observed also when 35 mm NaCl was included. Thus, the PIP and PIP(2) phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP(2) as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca(2+), with maximum activity at 1 mm CaCl(2), and could not be further stimulated by Mg(2+).