Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues.
Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5' cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity.
Tuck
M T
MT
Department of Chemistry, Ohio University, Athens 45701.
D000225:Adenine / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D006367:HeLa Cells / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008297:Male; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D012333:RNA, Messenger; C065638:6-methyladenine mRNA methyltransferase; D008780:Methyltransferases; D000225:Adenine