Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme).
A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.
D002413:Cations, Divalent; D011756:Diphosphates / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases; D014616:Vaccinia virus / Q000378:metabolism*; D014764:Viral Proteins; D014771:Virion / Q000378:metabolism*
D002413:Cations, Divalent; D011756:Diphosphates; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D014764:Viral Proteins; D006160:Guanosine Triphosphate; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus