Figure 4.

CPE is a metal-dependent enzyme. (A) Percent of cleavage of [³⁵S]methionine-labeled preRBCA in the presence of EDTA (▪), EGTA (○), and 1,10-phenanthroline (•). Aliquots of CPE-B extract were preincubated at 28°C for 30 min with each inhibitor and activity was assayed under standard conditions with 1 μl of in vitro synthesized preRBCA. Processing products were separated by SDS/PAGE and quantified using PhosphorImager (Molecular Dynamics). Extent of cleavage in absence of inhibitor was taken as 100%. (B) Reactivation of immobilized CPE-B by Zn²⁺ in chelating buffer. Untreated [³⁵S]methionine-labeled preRBCA (lane 1), preRBCA incubated with immobilized CPE-B without inhibitor preincubation (lane 2), and after preincubation with 1 mM 1,10-phenanthroline at 0 μM (lane 3), 5 μM (lane 4), and 50 μM (lane 5) ZnCl₂.