Properties of plant nuclease 1 purified from Tradescantia leaves by binding to Concanavalin A-Sepharose

Abstract

A nuclease with 3′-nucleotidase activity was purified at least 2,500-fold from Tradescantia paludosa leaves by a process that included chromatography on Concanavalin A-Sepharose. The preparation contains several active isozymes without impurities as judged by gel electrophoresis, but immunological tests revealed one or two major and a few minor inactive components. Phosphatase, phosphodiesterase and 5′-nucleotidase were absent or minimal, as were the known glycoproteins, peroxidase and esterase. The nuclease was highly active on RNA and denatured DNA, and less active on native DNA; it hydrolyzed the substrates in endonuclease manner. The mononucleotides had 5′-phosphate ends. Nuclease activity was optimal at pH 5.5–6.5 in tris-acetate buffers, 0.05–0.1 M. DNase activity was insensitive to EDTA and not stimulated by Mg2+ or Ca2+. Poly A and poly U were good and poly C and poly G poor substrates for the nuclease, but (d)GMP was the predominant mononucleotide from digests of nucleic acids. 3′-AMP, GMP and UMP were about equally good substrates for the nucleotidase at pH 6 but 3′-CMP was degraded at 1/5th the rate with 3-AMP, and any activity on deoxynucleotides was very slight.