Purification of bovine liver S-adenosylhomocysteine hydrolase by affinity chromatography on blue dextran-agarose.
S-Adenosylhomocysteine (AdoHcy) hydrolase (adenosylhomocysteinase, EC 3.3.1.1) was purified from bovine liver by conventional protein purification procedures (differential centrifugation, ammonium sulfate fractionation and DEAE-cellulose chromatography) followed by affinity chromatography on blue dextran coupled to agarose. The enzyme was eluted from the blue dextran-agarose column with adenosine and the adenosine was removed by chromatography on Sephadex G-75. The affinity chromatography step resulted in a substantial increase in total AdoHcy hydrolase activity (about 600%) suggesting either removal of some inhibitory substance or a change in the structure of the protein producing a more catalytically efficient enzyme. The isolation procedure afforded over 3400-fold purification of the enzyme, which was shown to be homogeneous by polyacrylamide gel electrophoresis. Using high pressure liquid chromatography, the nucleotide content of the freshly purified enzyme was determined to be 2 mol of nicotinamide adenine nucleotide per mol of enzyme tetramer. The ratio of the reduced to the oxidized form of the nucleotide was correlated to the activity of the enzyme preparation.
Narayanan
S R
SR
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66045.
Borchardt
R T
RT
D043383:Adenosylhomocysteinase; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D006867:Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D009243:NAD / Q000032:analysis / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives
D004396:Coloring Agents; D046911:Macromolecular Substances; D009243:NAD; C024908:blue dextran-sepharose; D012685:Sepharose; D006867:Hydrolases; D043383:Adenosylhomocysteinase