Purification, properties and specificity of an endonuclease from Agropyron elongatum seedlings

An endonuclease was isolated from 5 days old Agropyron elongatum 8× = Elytrigia turcica McGuire seedlings. The enzyme was purified by means of ammonium sulfate fractionation, DEAE-cellulose and Heparin Sepharose column. The final preparation, named nuclease A, gave a single band after silver staining had followed SDS-electrophoresis that was identified with nuclease activities. The enzyme also showed a single band after activity staining on gel polymerized in the presence of heat denatured DNA (ssDNA)/RNA. The Mr of native enzyme was 36 and the enzyme’s moiety consisted of one polypeptide chain. Nuclease A activity was stimulated in the presence of Zn2+ and was moderately reduced by NaCl yet strongly by spermine. The enzyme had pH optimum 5.5 and isoelectric point (pI) 4.7. It hydrolyzed the nucleic acids in the order ssDNA > dsDNA ≥ RNA; hence it was classified as a plant nuclease type I (EC 3.1.30.2). Synthetic homopolyribonucleotides were hydrolyzed in the order polyU > polyI ≥ polyA > polyG > polyC. Nuclease A nicked the supercoiled plasmid DNA while it was incapable of hydrolyzing dinucleoside monophosphates. With regard to nuclease A base linkage specificity towards a synthetic 5′-32P labeled deoxydecanucleotide [5′-32P]CCTGGCAGTT, the enzyme firstly exhibited a preference to Ap↓G bond and then to Gp↓T, Cp↓A and Gp↓G bonds while it was incapable of hydrolyzing the Cp↓C bond. The substrate’s products of nuclease A were oligonucleotides with the monoesterified phosphate at the 3′ position. Nuclease A may perform a crucial function in the metabolism of nucleic acids during seedling growth and could be used as a biochemical tool for analysis of nucleic acids structure.