Title

The phosphoinositide-specific phospholipase C in oat roots: Association with actin cytoskeleton

Date of Completion

January 1997

Keywords

Biology, Cell|Biology, Plant Physiology

Degree

Ph.D.

Abstract

Phosphoinositide-specific phospholipase C (PI-PLC) is an important signal transduction enzyme in animal cells, generating the second messengers inositol trisphosphate and diacylglycerol. Studies in this dissertation show that there are at least four variants of phosphoinositide-specific phospholipase C in the roots of oat seedlings, two cytosolic and two plasma membrane-associated. The two cytosolic and two plasma membrane variants can be separated on the basis of their affinity for binding to heparin. Both the cytosolic and the plasma membrane heparin-binding forms have apparent molecular weight of about 50-70 kDa by size exclusion chromatography.^ The partially purified heparin binding plasma membrane and cytosolic phospholipase C have essentially identical substrate specificities, hydrolyzing PtdIns(4)P and PtdIns(4,5)P$\sb2$ with nearly the same affinity, but showing little or no activity against PtdIns. These two enzymes are both activated at Ca$\sp{2+}$ concentration above 10 $\mu$M, with maximal hydrolysis of both PtdIns(4)P and PtdIns(4,5)P$\sb2$ at about 1 mM Ca$\sp{2+}.$ Above 1-2 mM Ca$\sp{2+},$ activity was reduced.^ In this dissertation, I also demonstrate that PI-PLC associated with actin microfilaments. First, 41% of the PI-PLC activity remained associated with the cytoskeleton fraction obtained by sedimentation of a purified plasma membrane fraction treated with 1% Triton X-100 Further 80% of the PI-PLC was solubilized when actin filaments were depolymerized by addition of either 1 M Tris buffer or 20 mM latrunculin B to this pellet. Incubation of the resulting supernatant under conditions favoring repolymerization of actin resulted in return of PI-PLC activity to a pelletable form. Finally, immunoblotting with anti-actin, anti-tropomyosin and anti-profilin antibodies demonstrates that actin and some actin-binding co-purified with PI-PLC through several chromatography steps. ^