Limited tryptic digestion of messenger RNA capping enzyme from Artemia salina. Isolation of domains for guanylyltransferase and RNA 5'-triphosphatase.
The partially purified preparation of messenger RNA guanylyltransferase from Artemia salina contains, as in the case of the rat liver enzyme (Yagi, Y., Mizumoto, K., and Kaziro, Y. (1983) EMBO J. 2, 611-615), the RNA 5'-triphosphatase activity which specifically removes the gamma-phosphoryl group from the 5'-triphosphoryl end of the newly synthesized mRNA molecule. The enzyme consists of a single polypeptide chain of Mr = 73,000 and forma a covalent enzyme-GMP complex as an intermediate for the guanylyltransferase reaction. Upon limited hydrolysis with trypsin, the enzyme-[32P]GMP complex is converted to a smaller 32P-containing fragment of Mr = 44,000. When the free enzyme, not complexed with GMP, is digested with trypsin under the same condition as above, the digests retain almost full activities of both guanylyltransferase and RNA 5'-triphosphatase and can form an enzyme-[32P]GMP complex of the size of Mr = 44,000 on incubation with [alpha-32P]GTP. Functional domains harboring the activities of guanylyltransferase and RNA 5'-triphosphatase are separated by gel filtration on a Sephacryl S-200 column at positions corresponding to Mr = 44,000 and 20,000, respectively. They can be separated completely from each other by CM-Sephadex column chromatography. While the native, undigested enzyme can transfer the GMP moiety to mRNA molecules with either triphosphoryl (pppN-) or diphosphoryl (ppN-)5'terminal, the purified Mr = 44,000 domain with the guanylyltransferase activity can utilize only the latter as an acceptor.
D017766:Acid Anhydride Hydrolases; D000818:Animals; D001156:Artemia / Q000201:enzymology*; D002467:Cell Nucleus / Q000201:enzymology; D004625:Embryo, Nonmammalian; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014357:Trypsin
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; C041026:capping enzyme, Artemia salina; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase