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 channel gene transcripts in mammalian brains has been proposed as one of the biological functions for this recently identified cellular enzyme. We purified a mixture of three forms, 93, 88, and 83 kDa, of bovine DRADA 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 adenosine deaminase and AMP deaminase, suggesting that the catalytic mechanism of DRADA might be different from that of other deaminases. DRADA binds 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 modification of relatively rare substrate RNAs in the cell nucleus.
Kim
U
U
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Garner
T L
TL
Sanford
T
T
Speicher
D
D
Murray
J M
JM
Nishikura
K
K
D000243:Adenosine Deaminase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D058892:Adenosine Deaminase Inhibitors; D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology*; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D016601:RNA-Binding Proteins; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D058892:Adenosine Deaminase Inhibitors; D002614:Chelating Agents; D016601:RNA-Binding Proteins; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase; D015032:Zinc