Isolation and characterization of multiple forms of ovine pancreatic deoxyribonuclease. Chromatograhpic behavior of the enzyme on concanavalin A-agarose and carboxymethylcellulose columns.
A new procedure has been devised for the purification of ovine DNase, including (NH/4)2SO4 fractionation, two steps of CM-cellulose chromatography, concanavalin A-agarose chromatography, and gel filtration on Sephadex G--100. The enzyme, like bovine DNase, exhibits multiplicity due to changes in the primary structure and the sugar structure of the carbohydrate moiety. Unlike bovine DNase, ovine DNase does not have sialic acid in any of its multiple forms. Concanavalin A-agarose is useful in the purification of not only ovine but also bovine DNase. For ovine DNase, it is a necessary and key step of purification; for bovine DNase, it can be used to purify commercial preparations of DNase free from proteases in a single step as judged by its stability in Ca2+-free media at pH 8.0. The purified enzyme has a specific activity equal to that of a highly purified DNase and presumably contains predominantly DNases A and C. Two of the four forms of ovine DNase have been purified to apparent homogeneity and subjected to chemical analysis. The present results show that bovine and ovine DNases have indistinguishable molecular weights and identical end groups, suggesting that they may have the same number of amino acid residues. The amino acid composition indicates that two enzymes may have six residues of amino acids subject to substitution which can be explained by single base changes in their genetic code words. Amino acid analyses also indicate that the most likely difference between two forms of ovine DNase is the substitution of Leu for Arg.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D003208:Concanavalin A; D003851:Deoxyribonucleases / Q000302:isolation & purification*; D007527:Isoenzymes / Q000302:isolation & purification*; D010179:Pancreas / Q000201:enzymology*; D012756:Sheep; D012794:Sialic Acids / Q000032:analysis
D000596:Amino Acids; D007527:Isoenzymes; D012794:Sialic Acids; D003208:Concanavalin A; D003851:Deoxyribonucleases