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.