Open AccessPartial Purification and Characterization of mRNA (Guanine‐7‐) Methyltransferase from the Yeast Saccharomyces cerevisiae
Author(s) -
LOCHT Camille,
BEAUDART JeanLuc,
DELCOUR Jean
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07539.x
Subject(s) - lysis , guanosine , biochemistry , yeast , sephadex , chemistry , saccharomyces cerevisiae , guanine , size exclusion chromatography , methyltransferase , uridine , enzyme , chromatography , microbiology and biotechnology , divalent , methylation , nucleotide , dna , biology , rna , organic chemistry , gene
As a tool for the study of the capping‐methylation process of yeast mRNA, we developed a procedure for the purification of the mRNA (guanine‐7‐)methyltransferase using the commercial cap analog guanosine(5′)triphospho(5′)guanosine as a substrate and radioactive S ‐adenosylmethionine (AdoMet) as the methyl group donor. The osmotic‐sensitive yeast strain VY 1160 was used as the enzyme source. Little methyltransferase activity was detectable in a crude lysate obtained after osmotic shock. We showed that this was due to the presence of a low‐molecular‐weight inhibitor which could easily be eliminated by Sephadex G‐25 gel filtration. The 10000 × g supernatant from the crude lysate was submitted to DEAE‐cellulose and DNA‐agarose chromatography. The resulting preparation was enriched about 450‐fold in specific activity. Under standard assay conditions, the incorporation rate remained constant for at least 6 h at 30°C. Transmethylation was not stimulated by KCl nor NaCl. Divalent cations were strong inhibitors. The partially purified enzyme was able to methylate undermethylated poly(A)‐rich mRNA isolated from an AdoMet auxotrophic yeast strain briefly exposed to AdoMet‐free medium.