DNase A, a poly(dA) and poly(dT)-specific deoxyribonuclease from Achatina fulica. Purification and characterization.
DNases A1 and A2 have been purified to homogeneity from the hepatopancreas of Achatina fulica by a series of steps: acetate buffer extraction, ammonium sulfate precipitation and column chromatography on hydroxylapatite, phosphocellulose, Blue-Sepharose, and poly(A)-Sepharose. The purified enzymes are free of acidic phosphomonoesterase, phosphodiesterase, and RNase activities. They are slightly acidic glycoproteins with identical isoelectric point (6.90). On 0.1% SDS gel electrophoresis, DNase A2 had a molecular weight of 30,000 when dissolved in 1% SDS, but it had molecular weights of 17,500, 8,000, and 4,800 when dissolved in 1% SDS and 1% 2-mercaptoethanol. This was evidence that the enzyme consists of three different subunits joined by interchain disulfide bonds. DNases A1 and A2 are endonucleases working at acidic pH (3.5--6.0) and do not require divalent cations for their activities. The enzymes degrade poly(dA) 5 times faster and poly(dT) 3 times faster than heat-denatured DNA under optimal conditions but do not appreciably digest poly(dG) and poly(dC). We developed an analytical procedure for oligodeoxynucleotides by high-performance liquid chromatography. The phosphomonoester end group and the mode of degradation were examined by the method. The termini produced by the enzymes have 3'-phosphoryl and 5'-hydroxy end groups. The products of exhaustive hydrolysis contain di-, tri-, tetra-, and pentanucleotides and mononucleotide was barely detected. The hydrolyzing activities of DNases A1 and A2 are stimulated by polyamines such as spermine, spermidine, and putrescine, but are inhibited by synthetic polynucleotides and various drugs. Adenosine deaminase highly active on oligoadenylic acids was found in a crude DNase A fraction. The enzyme preparation has higher activity on 3'-adenylic acid than on 5'-adenylic acid. The first adenosine residue of oligoadenylic acids was deaminated considerably more rapidly than the second or succeeding ones.
D000818:Animals; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003851:Deoxyribonucleases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D011061:Poly A / Q000378:metabolism; D011071:Poly T / Q000378:metabolism; D012908:Snails / Q000201:enzymology*; D013379:Substrate Specificity
D003838:Deoxyadenine Nucleotides; D011061:Poly A; D011071:Poly T; C015465:poly(dA); D003851:Deoxyribonucleases; C019328:deoxyribonuclease A