FIG. 1.

Phosphorylation of PVA VPg by plant protein kinase activity is stimulated by Mn²⁺. (A) Increasing concentrations of manganese were introduced into assays containing bacterially expressed PVA VPg, total protein kinase activity from leaves of N. tabacum, and [γ-³³P]ATP. Phosphoproteins were separated by SDS-PAGE and transferred to membranes, and their positions were identified by staining with Ponceau S. Radioactivity associated with phosphoproteins was compared by PhosphorImager densitometry and plotted against Mn²⁺ concentration. In a control experiment, manganese was removed from phosphorylation reaction in a 1:1 molar complex with EDTA. (B) Effect of Mn²⁺, Mg²⁺, or Ca²⁺ on phosphorylation of PVA VPg. Proteins were assayed for phosphorylation in a reconstituted system containing plant enzymes, [γ-³³P]ATP, and 10 mM Mn²⁺, Mg²⁺, or Ca²⁺. Proteins were subjected to SDS-PAGE and transferred to membranes. Autoradiograms of phosphorylated proteins are shown together with stained membranes.