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. 1992 Nov;100(3):1296–1303. doi: 10.1104/pp.100.3.1296

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) 1

Orientation of Active Site and Activation by Ca2+ and Mg2+

Christophe Pical 1,2,3, Anna Stina Sandelius 1,2,3, Per-Martin Melin 1,2,3, Marianne Sommarin 1,2,3
PMCID: PMC1075781  PMID: 16653120

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 (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+.

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Selected References

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