Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei.
GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of a GMP residue from GTP to the 5' end of RNA to form a cap structure identified as G(5')pppN-, has been isolated from HeLa cell nuclei. The enzyme has been purified approximately 1000-fold and separated by column chromatography (using DEAE-cellulose, phosphocellulose, Cibacron blue-agarose, and GTP-agarose) from a variety of other activities, including RNA triphosphatase and mRNA (guanine-7)methyltransferase. The reaction product was identified by its resistance to Penicillium nuclease and alkaline phosphatase, sensitivity to venom phosphodiesterase, and electrophoretic and chromatographic mobilities relative to authentic standards. Optimal enzyme activity was obtained at pH 7.5 in the presence of Mn2+ or Mg2+, GTP, and an appropriate acceptor polyribonucleotide. The enzyme was inhibited by elevated concentrations of salt and by sulfhydryl-binding reagents but was unaffected by S-adenosylmethionine or S-adenosylhomocysteine. A molecular weight of 48,500 was estimated by sucrose gradient centrifugation of purified enzyme.
D002467:Cell Nucleus / Q000201:enzymology*; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009713:Nucleotidyltransferases; D012315:RNA Caps / Q000096:biosynthesis; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D000098556:mRNA Guanylyltransferases
D012315:RNA Caps; D008345:Manganese; D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D008274:Magnesium