Messenger RNA guanylyltransferase from Saccharomyces cerevisiae. Large scale purification, subunit functions, and subcellular localization.
Messenger RNA capping enzyme (GTP:mRNA guanylyltransferase) purified from yeast Saccharomyces cerevisiae consisted of two polypeptides (45 and 39 kDa) and possessed two enzymatic activities, i.e. mRNA guanylyltransferase and RNA 5'-triphosphatase (Itoh, N., Mizumoto, K., and Kaziro, Y. (1984) J. Biol. Chem. 259, 13923-13929). In this paper, we describe an improved procedure suitable for the large scale purification of the enzyme. The steps include glass beads disruption of the cells and several ion-exchange and affinity column chromatographies. The enzyme was purified from kilogram quantities of yeast cells to apparent homogeneity. The purified enzyme had an approximate Mr of 180,000 and consisted of two heterosubunits of 80 and 52 kDa and had the same two enzymatic activities as above. We consider that this is the more intact form of the enzyme. Using the in situ assays on sodium dodecyl sulfate-polyacrylamide gels, RNA 5'-triphosphatase, and mRNA guanylyltransferase activities were located on the 80- and 52-kDa chains, respectively. In agreement with this, the 52-kDa enzyme-[32P]GMP complex was formed on incubation of the enzyme with [alpha-32P]GTP. Guinea pig antisera against purified yeast capping enzyme recognized both 80- and 52-kDa chains in Western blot analysis. The antibody did not cross-react with the enzymes from rat liver. Artemia salina, or vaccinia virus. Nuclear localization of the enzyme was demonstrated by immunofluorescence microscopy.
D017766:Acid Anhydride Hydrolases; D000818:Animals; D001156:Artemia / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology; D014616:Vaccinia virus / Q000201:enzymology; D000098556:mRNA Guanylyltransferases
D046911:Macromolecular Substances; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D010744:Phosphoric Monoester Hydrolases; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase