CHARACTERIZATION OF M7G(5')PPPN-PYROPHOSPHATASE ACTIVITY FROM HELA-CELLS

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 252  (9) , 2815-2821
Abstract
The m7G(5'')pppN[7-methylguanosine 5''-triphosphoryl-5''-nucleoside]-pyrophosphatase activity previously detected in HeLa [human cervical carcinoma] cells was further characterized. Results from DEAE-cellulose column chromatography and polyacrylamide gel electrophoresis under nondenaturing conditions revealed only 1 enzyme activity in HeLa cell extracts which was capable of selectively hydrolyzing m7G(5'')pppN to yield m7pG [7-methylguanosine 5''-monophosphate] + ppN (where N = 2''-O-methylated or unmethylated ribonucleosides or oligonucleotides of up to 8 to 10 nucleosides in length). The majority (.apprx. 95%) of this activity was found in the cytoplasmic extract but appeared not to be associated with the lysosomal fraction. m7G(5'')pppG was hydrolyzed by the partially purified enzyme in the absence of divalent cations at a pH optimum of 7.5 and a temperature optimum of 45.degree. C, with a Km of 1.7 .mu.M. Sedimentation analysis and gel filtration showed the MW of the enzyme as approximately 81,000. Inhibition studies testing the effect of a number of prospective substrates on the rate of m7G(5'')pppG hydrolysis have confirmed the importance of the methyl moiety at the N7 position of guanosine for enzyme-substrate interaction. The trimethylated guanosine-containing 5''-terminal structure derived from U-2 RNA did not serve as substrate, and 7-methylinosine, unlike 7-methylguanosine, was not an effective inhibitor of m7G(5'')pppG hydrolysis. The 2-amino group of the 7-methylguanosine portion of m7G(5'')pppN is also important for substrate interaction with this specific pyrophosphatase.

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