The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes.
AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.
Spychala
J
J
Human Purine Center, Internal Medicine and Biological Chemistry Clinical Research Center, University of Michigan, Ann Arbor 49109-0108.
Fox
I H
IH
D015720:5'-Nucleotidase / Q000302:isolation & purification*; D000659:AMP Deaminase / Q000302:isolation & purification*; D000243:Adenosine Deaminase / Q000302:isolation & purification*; D000248:Adenosine Kinase / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D009700:Nucleoside Deaminases / Q000302:isolation & purification*; D009709:Nucleotide Deaminases / Q000302:isolation & purification*; D010770:Phosphotransferases / Q000302:isolation & purification*; D010924:Placental Extracts / Q000032:analysis*
D010924:Placental Extracts; D010770:Phosphotransferases; D000248:Adenosine Kinase; D015720:5'-Nucleotidase; D009700:Nucleoside Deaminases; D009709:Nucleotide Deaminases; D000243:Adenosine Deaminase; D000659:AMP Deaminase