Figure 9.

The effect of protein kinase inhibitors, CaM antagonist W7, and exogenous CaM on autophosphorylation and syntide-2 phosphorylation activities in the barley aleurone. Four protein kinase inhibitors, W7, or CaM (bv, bovine; sp, spinach [Sigma]) were added to autophosphorylation and syntide-2 phosphorylation assays at the indicated concentrations. Aleurone layers were extracted and assays were carried out in the presence of 50 μm CaCl₂ as described in Methods. A, Autophosphorylation of the 68- and 54-kD proteins was visualized by autoradiography; B, the effect of protein kinase inhibitors on syntide-2 phosphorylation expressed as a percentage of control (no inhibitor) is shown below the appropriate inhibitor lane on the autoradiograph. The inhibitors used were obtained from Calbiochem: H89, specific for cAMP-dependent protein kinase; KN93, specific for Ca²⁺/CaM-dependent protein kinase II; bisindolylmaleimide (bis), specific for protein kinase C; and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), a broad-range Ser/Thr kinase inhibitor. The experiments were repeated twice with two replicates each. The largest se for syntide-2 phosphorylation was 12%.