Characterization of the m7G(5')pppN-pyrophosphatase activity from HeLa cells.
The m7(G(5')pppN-pyrophosphatase activity previously detected in HeLa cells has been further characterized. Results from DEAE-cellulose column chromatography and polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one enzyme activity in HeLa cell extracts which was capable of selectively hydrolyzing m7G(5')pppN to yield m7pG + ppN (where N = 2'-O-methylated or unmethylated ribonucleosides or oligonucleotides of up to 8 to 10 nucleosides in length). The majority (approximately 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 degrees, with a Michaelis constant (Km) of 1.7 micronM. Sedimentation analysis and gel filtration showed the molecular weight 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. Furthermore, the trimethylated guanosine-containing 5'-terminal structure derived from U-2 RNA was found not to serve as substrate, and 7-methylinosine, unlike 7-methylguanosine, was not an effective inhibitor of m7G(5')pppG hydrolysis. Thus, the 2-amino group of the 7-methylguanosine portion of m7G(5')pppN is also important for substrate interaction with this specific pyrophosphatase.
D003593:Cytoplasm / Q000201:enzymology; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology; D013696:Temperature
D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases