Nuclease I from suspension-cultured Nicotiana tabacum: Purification and properties of the extracellular enzyme

摘要: Abstract Nuclease I, which hydrolyzes polynucleotides and 3′-mononucleotides, was found to be released from suspension-cultured tobacco cells throughout the active phase of growth. The enzyme purified homogeneity filtrates late-log cultures by a procedure utilizing affinity interactions with NADP-Sepharose concanavalin A-Sepharose followed gel chromatography. extracellular nuclease glycoprotein carbohydrate content 9%. A single band corresponding polypeptide chain molecular weight 35,000 observed on sodium dodecyl sulfategel electrophoresis. Two forms enzyme, isoelectric points 5.2 5.6, could resolved disc electrophoresis or electrofocusing. No significant differences in catalytic properties two were observed. Base specificity for 3′-nucleotides, expressed as V K m , order > G ⪢ U C. Greater when 3′-mononucleotide substrate contained an additional phosphate at 5′-position. study rates hydrolysis dinucleoside monophosphates revealed strong preference purine nucleosides 5′-residue slight uridine 3′-residue. With both Np NpN substrates, ribonucleotides occurred >30 times rate deoxyribonucleotides. Action ribohomopolymers determined mainly secondary structural features substrate. degree inhibition I EDTA function concentration, time, temperature preincubation.