5′-Nucleotidase from Soybean ( Glycine max) Root Nodules. Partial Purification and Characterization. Regulation in Sterile Tissue Culture

Abstract

Summary Four isoforms of 5′-nucleotidase (EC 3.1.3.5) activity have been resolved and partly purified from soybean ( Glycine max ) root nodules. The purification procedure involved ammonium sulfate precipitation, anion- and cation exchange chromatography, Concanavalin A and Fractogel affinity chromatography and chromatofocusing. Isoelectric points were determined to be 8.5, 8.2, 8.0 and 7.5 for isoforms I, II, III and IV, respectively. A pH optimum around 5 was found and Mg ++ and Co ++ were most effective in stimulating activity of all isoforms. Forms I and II showed a preferential substrate specificity for AMP, IMP and GMP, while forms III and IV showed no such preference. All four isoforms of 5′-nucleotidase seem to be glycosylated and a native molecular mass of 70 kDa was estimated by gel filtration. In order to elucidate the regulation, response of 5′-nucleotidase activity to phosphate starvation was studied in sterile callus tissue and suspension culture. The phosphate starvation resulted in an increase in specific activity of 6-fold and 4-fold, respectively. An increase in mainly isoform III was demonstrated after isoelectric focusing of phosphate starved callus culture.