Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line.

摘要: Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of immune system. For purpose studying function structure this enzyme, we have isolated selection a mouse cell line, B-1/50, in which levels were increased 4,300-fold over parent line. The enzyme was purified from these cells large quantity high yield simple two-step purification scheme. derived B-1/50 indistinguishable that parental as judged several criteria. Km (30 microM) Ki (4 nM) values using substrate 2'-deoxycoformycin inhibitor, respectively, identical well gene amplification mutants. both types exhibited multiple isoelectric focusing forms co-purified our protocol. Electrophoretic analysis sodium dodecyl sulfate-polyacrylamide gels showed migrated with an apparent molecular weight 41,000 or 36,000 depending on whether reduced oxidized, respectively. This shift reversible, indicating proteolysis not responsible faster migrating form. Monospecific antibodies raised against cross-reacted precipitated 37% total soluble protein cells. Continued resulted isolation overproduced 11,400-fold accounted 75% protein.