期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1982
卷号：79
期号：6
页码：1693-1697
DOI：10.1073/pnas.79.6.1693
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Rat liver RNA guanylyltransferase catalyzes a GTP-PPi exchange reaction in the absence of acceptor RNA [Mizumoto, K. & Lipmann, F. (1979) Proc. Natl. Acad. Sci. USA 76, 4961-4965] suggesting that the reaction proceeds through the formation of a covalent guanylylated intermediate. We now present more direct evidence for the existence of the enzyme-GMP intermediate: (i) the enzyme-[32P]GMP intermediate was formed on incubation of rat liver guanylyltransferase with [alpha-32P]GTP and migrated as a single radioactive band with Mr 69,000 on NaDodSO4/polyacrylamide gel electrophoresis, and (ii) the intermediate isolated on gel filtration can transfer its GMP moiety to ppGpCpC-poly(A2,U2,G) to form the capped RNA molecule or it can react with PPi to regenerate GTP. The formation of the intermediate was dependent on Mg2+ and was strongly inhibited by PPi. The addition of pyrophosphatase markedly increased the amount of the intermediate complex. On blue dextran-Sepharose affinity column chromatography, the activity of guanylyltransferase to form an enzyme-[32P]GMP intermediate comigrated with activities of cap formation and GTP-PPi exchange. A phosphoamide type linkage between GMP and enzyme is suggested by its acidlabile and alkali-stable nature and also by the susceptibility to acidic hydroxylamine. These results indicate that the reaction catalyzed by rat liver guanylyltransferase occurs through the following two partial steps: (i) E + GTP in equilibrium E-pG + PPi; and (ii) E-pG + ppN .....leads to GpppN .....+ E.
