Affinity-chromatographic purification of S-adenosyl-L-homocysteine hydrolase. Some properties of the enzyme from rat liver.
S-Adenosyl-L-homocysteine hydrolase has been purified to apparent homogeneity from rat liver by means of affinity chromatography on 8-(3-aminopropylamino)adenosine linked to Sepharose. The purified enzyme was free from adenosine kinase and adenosine deaminase activities and was homogeneous on SDS/polyacrylamide-gel electrophoresis which gave a subunit mol.wt. of 47 000. The native enzyme showed some microheterogeneity on polyacrylamide-gel electrophoresis under increased-resolution conditions but was homogeneous on isoelectric focusing (pI 5.6). The molecular weight of the native enzyme was about 220 000 as judged by pore-gradient electrophoresis. The native enzyme bound adenosine tightly and showed Km values of 0.6 microM, 0.9 microM and 60 microM for adenosine, S-adenosyl-L-homocysteine and L-homocysteine respectively. The enzyme was rapidly inactivated when incubated in the presence of adenosine, S-adenosyl-L-homocysteine or several adenosine derivatives or analogues. Inactivation took place both at 0 and 37 degrees C. Freezing in the absence of glycerol resulted in the appearance of dissociation products of the oligomeric protein. Multimer formation was observed at low thiol concentrations.
D000241:Adenosine / Q000031:analogs & derivatives; D043383:Adenosylhomocysteinase; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006867:Hydrolases / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051381:Rats; D012685:Sepharose / Q000031:analogs & derivatives
C032701:8-(3-aminopropylamino)adenosine-sepharose; D012685:Sepharose; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine