Three RNases in Senescent and Nonsenescent Wheat Leaves : Characterization by Activity Staining in Sodium Dodecyl Sulfate-Polyacrylamide Gels

Abstract

We have described three RNases in wheat leaves (Triticum aestivum L. cv Chinese Spring) and developed assays for measuring each RNase individually in crude leaf extracts. We initially used activity staining in sodium dodecyl sulfate-polyacrylamide gels to characterize RNases in extracts of primary and flag leaves. We thus identified acid RNase (EC 3.1.27.1, here designated RNase WL(A)), and two apparently novel enzymes, designated RNases WL(B) and WL(C). RNase WL(B) activity displays a distinctive isozyme pattern, a molecular mass of 26 kilodaltons (major species), a broad pH range with an optimum near neutrality, insensitivity to EDTA, and stimulation by moderate concentrations of KCl and by MgCl(2). RNase WL(C) activity exhibits a molecular mass of 27 kilodaltons, a neutral pH optimum, insensitivity to EDTA, and inhibition by KCl, MgCl(2), and tri-(hydroxymethyl)aminomethane. Based on distinctive catalytic properties established in gels, we designed conventional solution assays for selective quantitation of each RNase activity. We used the assays to monitor the individual RNases after gel filtration chromatography and native gel electrophoresis of extracts. In accompanying work, we used the assays to monitor RNases WL(A), WL(B), and WL(C), which are present in senescent and nonsenescent leaves, during the course of leaf senescence.