Isolation of mutant adenosine deaminase by coformycin affinity chromatography.
Adenosine deaminase is a purine salvage enzyme that catalyzes the deamination of adenosine and deoxyadenosine. Deficiency of the enzyme activity is associated with T-cell and B-cell dysfunction. Mutant adenosine deaminase has been isolated from heterozygous and homozygous deficient lymphoblast cell lines with the aid of an affinity matrix consisting of coformycin (a potent inhibitor of the enzyme) as the affinity ligand, bound to 3,3'-iminobispropylamine-derivatized Sepharose. Routinely, 80-90% of adenosine deaminase in crude cell homogenates could be bound to the material. Adenosine deaminase was specifically eluted by enzyme inhibitors or less efficiently by high substrate concentrations. Protein preparations isolated from several different deficient cell lines were highly purified and exhibited molecular weights identical to wild-type adenosine deaminase. This method produces a protein that is suitable for structural studies.
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000243:Adenosine Deaminase / Q000235:genetics / Q000302:isolation & purification*; D058892:Adenosine Deaminase Inhibitors; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D003070:Coformycin / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology; D009154:Mutation; D009700:Nucleoside Deaminases / Q000302:isolation & purification*; D015649:Pentostatin
D058892:Adenosine Deaminase Inhibitors; D003070:Coformycin; D015649:Pentostatin; C010192:9-(2-hydroxy-3-nonyl)adenine; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase; D000225:Adenine