Characterization of an ATPase/dATPase activity associated with the Drosophila nuclear matrix-pore complex-lamina fraction. Identification of the putative enzyme polypeptide by direct ultraviolet photoaffinity labeling.
An ATPase/dATPase activity found associated with the nuclear matrix-pore complex-lamina fraction isolated from embryos of Drosophila melanogaster has been characterized. In the presence of either Ca2+ or Mg2+, this activity hydrolyzed either ATP or dATP to ADP or dADP, respectively, and Pi. Hydrolysis was optimal from pH 6.5-7.2, did not require either Na+ or K+, and was not significantly inhibited by NaF, ouabain, quercetin, Na3VO4, CTP, or GTP. In contrast, hydrolysis was inhibited by N-ethylmaleimide, EDTA, and cordycepin 5'-triphosphate. In all respects tested, hydrolysis of ATP was indistinguishable from that of dATP and when incubated in the same reaction mixture, each was linearly competitive with the other. Based upon these properties, a series of direct UV photoaffinity labeling experiments was performed. Using alpha-[32P]dATP, alpha-[32P]ATP, or gamma-[32P]ATP, only a single polypeptide (Mr approximately 174,000) was photolabeled in a manner completely consistent with the enzymology of ATP and dATP hydrolysis; cell fractionation studies revealed a predominantly or exclusively nuclear localization. A polypeptide with virtually the identical mobility on sodium dodecyl sulfate-polyacrylamide gels was similarly identified as the major photolabeled species in nuclear envelope fractions obtained from chickens, opossums, rats, and guinea pigs. Thus, it seems probable that this 174-kilodalton polypeptide constitutes at least the active site-containing subunit of the major insoluble ATPase/dATPase found in structural protein subfractions prepared from higher invertebrate as well as vertebrate nuclei.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology* / Q000528:radiation effects; D004331:Drosophila melanogaster / Q000201:enzymology*; D004625:Embryo, Nonmammalian / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism; D014466:Ultraviolet Rays
D011494:Protein Kinases; C037694:ATPase-dATPase; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases; D005033:Ethylmaleimide