Purification and properties of double-stranded RNA-specific adenosine deaminase from calf thymus.
A double-stranded RNA-specific adenosine deaminase, which converts adenosine to inosine, has been purified to homogeneity from calf thymus. The enzyme was purified approximately 340,000-fold by a series of column chromatography steps. The enzyme consists of a single polypeptide with a molecular mass of 116 kDa as determined by electrophoresis on a SDS/polyacrylamide gel. The native protein sediments at 4.2 s in glycerol gradients and has a Stokes radius of 42 A upon gel-filtration chromatography. This leads to an estimate of approximately 74,100 for the native molecular weight, suggesting that the enzyme exists as a monomer in solution. Enzyme activity is optimal at 0.1 M KCl and 37 degrees C. Divalent metal ions or ATP is not required for activity. The Km for double-stranded RNA substrate is approximately 7 x 10(-11) M. The Vmax is approximately 10(-9) mol of inosine produced per min per mg and the Kcat is 0.13 min-1.
O'Connell
M A
MA
Department of Cell Biology, University of Basel, Switzerland.
Keller
W
W
D000243:Adenosine Deaminase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*; D014981:Xenopus
D002413:Cations, Divalent; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D017886:Durapatite; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase