PURIFICATION AND CHARACTERIZATION OF DOUBLE-STRANDED-RNA ADENOSINE-DEAMINASE FROM BOVINE NUCLEAR EXTRACTS

The double-stranded RNA (dsRNA) adenosine deaminase (DRADA) deaminates adenosine residues to inosines and creates I-U mismatched base pairs in dsRNAs. Its involvement in RNA editing of glutamate-gated ion chann el gene transcripts in mammalian brains has been proposed as one of th e biological functions for this recently identified cellular enzyme. W e purified a mixture of three forms, 93, 88, and 83 kDa, of bovine DRA DA proteins, all likely to be active enzymes. We determined that DRADA has a native molecular mass of approximately 100 kDa, suggesting that the enzyme exists as a monomer. The purified enzyme was not inhibited by 2'-deoxycoformycin, a transition state analog inhibitor of adenosi ne deaminase and AMP deaminase, suggesting that the catalytic mechanis m of DRADA might be different from that of other deaminases. DRADA bin ds specifically to dsRNA with a dissociation constant of 0.23 nM for a synthetic dsRNA, and the Michaelis constant is 0.85 nM. These values indicate that DRADA has a much higher affinity for its substrate than other deaminases such as adenosine deaminase and AMP deaminase. DRADA may need this extremely high affinity to catalyze efficiently the modi fication of relatively rare substrate RNAs in the cell nucleus.