Purification, properties and specificity of an endonuclease from Agropyron elongatum seedlings.
An endonuclease was isolated from 5 days old Agropyron elongatum 8x = 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 (pI) 4.7. It hydrolyzed the nucleic acids in the order ssDNA > dsDNA > or = 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 > or = 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'-(32)P labeled deoxydecanucleotide [5'-(32)P]CCTGGCAGTT, the enzyme firstly exhibited a preference to Ap downward arrow G bond and then to Gp downward arrow T, Cp downward arrow A and Gp downward arrow G bonds while it was incapable of hydrolyzing the Cp downward arrow 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.
Yupsanis
Traianos
T
Laboratory of Biochemistry, Department of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece. yupsanis@chem.auth.gr
Symeonidis
Lazaros
L
Kalemi
Theodora
T
Moustaka
Helen
H
Yupsani
Anastasia
A
D031702:Agropyron / Q000201:enzymology*; D002384:Catalysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D013379:Substrate Specificity
D004720:Endonucleases