Purification of mammalian ribonuclease using immobilized human ribonuclease inhibitor.
Affinity chromatography on immobilized ribonuclease (RNase) inhibitor was developed for purification of mammalian RNase. Human placental RNase inhibitor was conjugated to CNBr-activated Sepharose in the presence of dithiothreitol. About 80% of the immobilized RNase inhibitor was capable of binding bovine pancreatic RNase A. The bound RNase A was eluted with 3 M NaCl at pH 5.0. Two 25-kDa and 18-kDa RNases, which were obtained from human liver using a cellulose phosphate column, were bound to the immobilized RNase inhibitor and recovered in a pure and active form after treatment of the resin with p-hydroxymercuribenzoate. These enzymes were considered to be nonsecretory-type RNases with different sugar contents.
Nadano
D
D
Department of Legal Medicine, Fukui Medical School, Japan.
Yasuda
T
T
Takeshita
H
H
Sawazaki
K
K
Kishi
K
K
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002411:Cation Exchange Resins / Q000378:metabolism; D002417:Cattle; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010920:Placenta / Q000201:enzymology; D010926:Placental Hormones / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D002411:Cation Exchange Resins; D007527:Isoenzymes; D010926:Placental Hormones; C022052:placental ribonuclease inhibitor; D012313:RNA; D002482:Cellulose; C008881:phosphocellulose; D012260:Ribonucleases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase