An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. II. Purification and characterization of multi-forms of RNase T2.
RNase T2 bound to an affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, specifically at pH 4.5. The colorless enzyme was eluted only by the simultaneous addition of 2'(3')-AMP (1 mM) and NaCl (greater than 1 M) at pH 4.5. By applying this affinity chromatography to the purification of RNase T2, pure enzyme with a specific activity of 60 was obtained in only four steps and the yield was about 10 times higher than that of the previous purification method. This enzyme preparation was found to be heterogeneous in molecular weight and was separated into two fractions on Sephadex G-75 gel filtration. As the smaller enzyme with a molecular weight of 36,000 was identical with RNase T2 in every property examined, we tentatively designated the larger one with an apparent molecular weight of 80,000 as high molecular weight RNase T2 (RNase T2-L). RNase T2-L was still heterogeneous and was separated into five fractions, RNases T2-L 1-5, by repeated Sephadex G-150 gel filtration. The amino acid and carbohydrate analyses revealed that each of these fractions has a protein moiety in common with RNase T2 and the heterogeneities were due to the carbohydrate content, mainly galactose content.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives*; D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000302:isolation & purification* / Q000378:metabolism; D004722:Endoribonucleases; D007526:Isoelectric Point; D008970:Molecular Weight; D012260:Ribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives*
D000596:Amino Acids; D002241:Carbohydrates; C014325:N(6)-(6-aminohexyl)adenosine 5'-monophosphate-sepharose; D000249:Adenosine Monophosphate; D012685:Sepharose; D004720:Endonucleases; D004722:Endoribonucleases; D012260:Ribonucleases; C022543:ribonuclease T(2)