Conditions affecting depolymerization of actin in plant homogenates

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Abstract
The successful isolation of actin depends on the ability to control its polymerization state. Conditions that affect the depolymerization of actin in plant homogenates were determined using a qualitative assay for both polymeric and monomeric forms. In this assay, phalloidin was added to homogenates to stabilize actin polymers, which were separated from monomers by gel electrophoresis and detected by immunoblotting. By this criterion, actin in pea and rice root homogenates was initially in fila-mentous form, but depolymerized rapidly at pH 8 or above. If the pH was neutral to slightly acidic, some actin remained in filamentous form. The presence of Mg2+ or EGTA had little effect on the stabilization of filaments compared to the effect of pH. A slightly acidic pH was not by itself able to induce the polymerization of concentrated, purified pea actin, while Mg2+ was sufficient to polymerize the actin at both neutral and alkaline pH. F-actin binding proteins did not appear to prevent the depolymerization of the filaments in alkaline homogenates. Our data suggest that most actin in root cells is filamentous.