Purification and characterization of double-stranded RNA adenosine deaminase from bovine nuclear extracts.

U Kim,T.L Garner,T Sanford,D Speicher,J.M Murray,K Nishikura

doi:10.1016/s0021-9258(17)36857-6

Abstract

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.

Highlights

From the SWistar Institute of Anatomy and Siology and the ~ epartme notf Cell and Developmental Biology, Uniuersity of Pennsy~vaniaS, chool of Medicine, P h i ~ a d e ~ p hP~ean,nsylvania 19104
The precise biological function(s) of this newly dis-. These values indicate that DRADA has a much higher covered enzyme are currently not known, several hypotheses affinity forits substrate than other deaminases sucahs have beenproposed in recent yea(rKs im and Nishikura1, 993a, adenosine deaminase anAdMP deaminase.DRADA may 1993b)
We found that 5 mM vanadyl ribonucleoside complex (VRC) significantly decreased the DRADA to dsRNA involves different amino acid residues, as

Summary

EXPERIMENTAL PROCEDURES

P, nuclease, micrococcal nuclease, poly(I).poly(pCo)l,y(U), poly(I).poly(C)-agarose(type 61, DEAE-SepharoseCL-GB,and Superose 12 HR 10/30 were purchased from Pharmacia LKB Biotech Inc. 1,lOPhenanthroline was purchased from Sigma. After centrifugation a t 30,000 rpm in a type Ti-45 Beckman rotor for 30min, the nuclear pellet was suspended in a hypertonic buffer containing 20 m~ Hepes (pH 7.9),0.42 M NaCl, 1.5 m~ MgCl,, 0.2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol (DTT),and 0.5 m~ phenylmethylsulfony~fluoride. 5 g of crude nuclear extract (after adjusting the salt concentration to 0.35 M KCI) was passed through 85 ml(2.6 x 16 cm) of a ssDNA-agarose columnequilibrated with buffer A containing 0.35 M KC1 at a flow rate of 20 mlh. The reactions were incubated for 5 min at 37 "C, quenched by the addition of 10 volumes of the DRADA assay buffer (ice-cold), and immediately filtered through a nitrocellulose membrane (25 mm and 0.45 pm; Millipore, BedfordM , A) on a manifold filtration apparatus (Schleicher & Schuell) at a slowflow rate. The column was washed and eluted as for the first poly(I).poly(C) (Biirk et al, 1983)

M Kc1 Eluate Pllrieed DRADA

RESULTS

Findings

DISCUSSION