Plant Physiology 100:1296-1303 (1992)
© 1992 American Society of Plant Biologists
Metabolism and Enzymology
Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) 1
Orientation of Active Site and Activation by Ca2+ and Mg2+
Christophe Pical,
Anna Stina Sandelius,
Per-Martin Melin and
Marianne Sommarin
Department of Plant Biochemistry, University of Lund, P.O. Box 7007, S-220 07 Lund, Sweden,
Department of Plant Physiology, Botanical Institute, University of Göteborg, Carl Skottsbergs Gata 22, S-413 19 Göteborg, Sweden,
Department of Biochemistry, Chemical Centre, University of Lund, P.O. Box 124, S-220 01 Lund, Sweden
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 (PIP2) 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 PIP2 phospholipase C was dependent on Ca2+ with maximum activity at 10 to 100 µM free Ca2+ and half-maximal activation at 0.1 to 1 µM free Ca2+. In the presence of 10 µM Ca2+, 1 to 2 mM MgCl2 or MgSO4 further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mM Ba2+, Co2+, Cu2+, Mn2+, Ni2+, and Zn2+) inhibited the enzyme activity. The stimulatory effect by Mg2+ was observed also when 35 mM NaCl was included. Thus, the PIP and PIP2 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 PIP2 as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca2+, with maximum activity at 1 mM CaCl2, and could not be further stimulated by Mg2+.
1 This work was supported by grants from the Swedish Council for Forestry and Agricultural Research (M.S.), the Swedish Natural Science Research Council (A.S.S.), and the Carl Tesdorpf Foundation (M.S.).
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