Partial purification and characterization of ribonucleases from roots, stem and leaves of cowpea

Abstract

Partial purification and characterization of ribonucleases (RNase; EC 3.1.27.1) present in roots, stem and leaves of 5 day-old Pitiúba cowpea [Vigna unguiculata (L.) Walp.] seedlings are described. Crude extracts from the different tissues were precipitated with ammonium sulfate followed by ionic exchange chromatography (CM-Cellulose) resulting in purification factors of 48-fold for roots, 21 for stem and 42 for leaves. No deoxyribonuclease activity was practically observed. The molecular masses of the RNases did not significantly differ, averaging 16.3 kDa. Leaf RNase was stable up to 50ºC while the others were inactivated at this temperature. The maximal inactivation for both stem and roots RNases was reached at 70ºC while for leaf it occurred at 80ºC. The addition of KCl to the assay medium caused a shift of optimal pH from 6.0 toward the range of 5.2 - 5.6 for the enzymes extracted from the different tissues. RNase activities were strongly inhibited by Hg2+, Zn2+ and Cu2+, partially inhibited by Co2+ and Fe2+ and were not affected by EDTA, Ca2+ or Mg2+. In contrast to the leaf RNase, roots and stem enzymes were inactivated by urea and 2-mercaptoethanol (2-ME). Although there is a great similarity among the enzymes studied, leaf RNase appears to be more stable to heat and to chemical denaturation than root and stem RNases. The results also suggest that the enzymes extracted from different tissues of Pitiúba cowpea seedlings are ribonucleases and not nucleases.