MESSENGER RNA(NUCLEOSIDE-2'-)-METHYLTRANSFERASE FROM VACCINIA VIRUS - PURIFICATION AND PHYSICAL-PROPERTIES

  • 1 January 1978
    • journal article
    • research article
    • Vol. 253  (21) , 7692-7697
Abstract
An S-adenosyl-L-methionine:mRNA(nucleoside-2''-)-methyltransferase, one of at least 3 activities required for the 5''-terminal modification of mRNA, was purified from vaccinia virus particles. Employing brome mosaic virus RNA ending in m7G(5'')pppG- as substrate, a simple DEAE-cellulose filter assay measuring the incorporation of methyl groups from S-adenosyl[methyl-3H]methionine to position 2'' of the penultimate nucleoside was devised. Starting from disrupted vaccinia virus cores, a 350-fold enzyme purification was achieved by successive chromatography on columns of DEAE-cellulose, CM-Sephadex and ADP-agarose. Analysis of the isolated enzyme by sodium dodecyl sulfate-polyacrylamide discontinuous gel electrophoresis revealed a single polypeptide with a MW of 38,000. Similar MW were obtained by sucrose gradient centrifugation and gel filtration of the native methyltransferase. The isoelectric point of the purified enzyme occurs at pH 8.4.