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

Abstract

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 Zn 2+ and was moderately reduced by NaCl yet strongly by spermine. The enzyme had pH optimum 5.5 and isoelectric point (p I) 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.