Polynucleotide‐acrylamide gel electrophoresis of soluble nucleases from tobacco leaves

To detect zones of hydrolytic activity after electrophoresis in polyacrylamide gels, advantage can be taken of the inclusion of high molecular weight substrate in the gel during polymerization [ 11. This technique obviates the need for diffusion of either enzyme into a substrate-containing medium, or low molecular weight substrate and, if necessary, coupling factors into the gel, thus allowing longer incubation times and producing sharper bands. After electrophoresis under conditions in which the enzyme is not active, gels are incubated in a suitable medium for hydrolysis to take place. Subsequently, staining of residual substrate in the gel leaves bands of activity as colourless zones. Methods based upon this principle have been described for both deoxyribonuclease [2] and ribonuclease [3], but found only limited application, probably due to the complicated procedures involved (cf. [4] ) and because the nucleic acids used are not necessarily uniformly degraded, leaving blurred zones on the gels instead of sharp, colourless bands. In the soluble protein fraction of tobacco leaves, at least four types of nuclease activity have been characterized on the basis of their substrate specificity [5,6]. Although cultured tobacco cells upon electrophoresis yielded four bands of RNase activity as detected by diffusion of low molecular weight RNA into the gel [4] , in our hands similar methods led to a poorly defined, remarkably complex pattern when applied to soluble proteins from tobacco leaves. Therefore, a variant of the substrate-gel method was developed which, by making use of simple high molecular weight polynucleotides, offered increased resolu-