Influence of Ionic Strength, pH, and Chelation of Divalent Metals on Isolation of Polyribosomes from Tobacco Leaves

Abstract

A procedure was developed for extracting polysomes from tobacco (Nicotiana sp) leaves. Unexpanded leaves ground in a medium consisting of 200 mm tris-HCl, pH 9, 400 mm KCl, 200 mm sucrose, and 35 mm MgCl(2) yielded larger amounts of polysomes with less degradation than polysomes from leaves extracted with buffers of lower ionic strength or pH. Extraction of polysomes from expanded leaves required the inclusion of ethyleneglycol-bis(2-aminoethyl ether)tetraacetic acid (EGTA, a divalent cation chelator with a high affinity for Ca(2+), Cu(2+), and Zn(2+)). EGTA also improved isolation of polysomes from unexpanded leaves. Addition of 25 mm Ca(2+), Cu(2+), or Zn(2+) to extracts from young leaves precipitated polysomes, and density gradient profiles of polysome preparations from the cation treatments mimicked profiles from expanded leaves which were extracted without EGTA. Polysome precipitation by Ca(2+) was prevented by EGTA. Endogenous Ca(2+) was present in unexpanded leaves in sufficient concentrations (25 mm) to cause some precipitation of polysomes during extraction, and this cation increased by 60% in expanded leaves. Cu(2+) and Zn(2+) were not present in amounts sufficient to cause polysome precipitation. The results show that recovery of polyribosomes may be reduced by divalent cations in leaf tissue, and this can be overcome by chelation of these ions with EGTA.