Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line.
Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein.
D000241:Adenosine / Q000378:metabolism; D000243:Adenosine Deaminase / Q000302:isolation & purification / Q000378:metabolism*; D058892:Adenosine Deaminase Inhibitors; D000818:Animals; D002460:Cell Line; D003070:Coformycin / Q000031:analogs & derivatives / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D006207:Half-Life; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D009700:Nucleoside Deaminases / Q000378:metabolism*; D015649:Pentostatin
D058892:Adenosine Deaminase Inhibitors; D003070:Coformycin; D015649:Pentostatin; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase; D000241:Adenosine