Purification and properties of a decapping enzyme from rat liver cytosol.
A decapping enzyme has been purified about 2400-fold from rat liver cytosol. The decapping enzyme was shown to be fairly homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme had an apparent molecular weight of 110,000 and consisted of two equal subunits. The enzyme hydrolyzed m7Guo5'PPP5'Ado to m7GMP and ADP. Analysis of the products produced from radioactively capped oligonucleotides and intact mRNA having 3H-cap suggests that the enzyme can hydrolyze capped mono- to pentanucleotides (m7Guo5'PPP5'N (where N = 1-5 nucleotides)) but not intact mRNA. The existence of methyl group at the N7 position of guanosine moiety of cap structure was necessary for the action of the decapping enzyme. This was confirmed by the comparison of the rates of hydrolysis of m7Guo5'PPP5'Ado by the enzyme in the presence of various nucleotides. The activity of enzyme was slightly stimulated by Na+, K+, NH4+, Ca2+ and polyamines. Mg2+ and Mn2+ were without effect on the enzyme activity.
Kumagai
H
H
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Kon
R
R
Hoshino
T
T
Aramaki
T
T
Nishikawa
M
M
Hirose
S
S
Igarashi
K
K
D000818:Animals; D002458:Cell Fractionation; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D004722:Endoribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009843:Oligoribonucleotides / Q000138:chemical synthesis; D051381:Rats; D013379:Substrate Specificity
D009843:Oligoribonucleotides; C030052:mRNA decapping enzymes; D004722:Endoribonucleases