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)

S. Shuman, M. Surks, H. Furneaux, J. Hurwitz

Research output: Contribution to journalArticle

72 Scopus citations

Abstract

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzymes requires MgCl2 for activity, has an alkaline pH optimum, an dspecifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PP(i) exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase)through successive chromatography steps on DEAE-celulose, DNA-cellulose, and phosphocellulose. GTP-PP(i) exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PP(i) exchange, capping and methylating activities co-sediment with an RNA triphosphate activity and a nucleoside triphosphate phophohydrolase 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-PP(i) exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PP(i) exchange reaction to the mechanism of transguanylylation is considered.

Original languageEnglish (US)
Pages (from-to)11588-11598
Number of pages11
JournalJournal of Biological Chemistry
Volume255
Issue number23
StatePublished - Dec 1 1980

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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