Solubilization and reconstitution of a nuclear envelope-associated ATPase. Synergistic activation by RNA and polyphosphoinositides.
Treatment of isolated rat liver nuclear envelopes with 1% Triton X-100 solubilized 20-30% of the nuclear envelope protein and 85% of the ATPase activity. Chromatography on DEAE-Sepharose in 1% Triton X-100 at pH 7.5 removed all of the chemically measurable phospholipid from the bound ATPase activity; however, endogenously synthesized phosphatidylinositol [4-32P]phosphate (PIP) co-chromatographed with the ATPase activity on this column. Further purification by chromatography on heparin-agarose removed all of the [32P]PIP and RNA from the ATPase; however, the recovery of ATPase activity from this column was very low. RNA, polyadenylic acid, and polyguanylic acid stimulated the delipidated ATPase activity 4-6-fold. This activity was not further stimulated by the addition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid, but was stimulated 2-fold by the addition of phosphatidylinositol. Addition of 40 microM PIP and 50 micrograms of RNA/ml resulted in a 25-fold stimulation of basal ATPase activity. Phosphatidylinositol 4,5-bisphosphate, in the presence of RNA, was also able to stimulate ATPase activity but to a lower extent than that of PIP. Therefore, the nuclear envelope-associated ATPase appears to require interaction with a polynucleotide and PIP to express full activity. PIP, which is actively metabolized in the nuclear envelope, may therefore be involved in the regulation of the activity of this enzyme.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D008099:Liver / Q000648:ultrastructure; D008297:Male; D009685:Nuclear Envelope / Q000201:enzymology*; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000494:pharmacology*; D010743:Phospholipids / Q000494:pharmacology; D012313:RNA / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D003902:Detergents; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010743:Phospholipids; D012313:RNA; D000251:Adenosine Triphosphatases