Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation.

Incubation of nuclear envelopes isolated from normal rat liver with [gamma-32P]ATP resulted in the rapid labeling of chloroform-soluble products. These products were identified as phosphatidic acid (PA), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-bisphosphate (TPI) based on their chromatographic mobilities on Silica Gel H and cellulose thin layer plates, and by analysis of their deacylation products by high pressure liquid chromatography. The extent of phosphorylation of these products was dependent on the method used to isolate the nuclear envelopes. Relatively gentle isolation methods, such as lysis of purified nuclei with heparin or digestion with deoxyribonuclease I, produce nuclear envelopes which possess significantly higher lipid kinase activity than do envelopes isolated by sonication. Incorporation of 32P into PA, DPI, and TPI in 2 min under standard assay conditions was 13, 152, and 22 pmol/mg of membrane protein, respectively. Degradation of labeled DPI and TPI was evident after 2-5 min of incubation. Nuclear envelope-associated diacylglycerol kinase, phosphatidylinositol kinase, and DPI kinase are characterized with regard to their pH and Mg2+ requirements. The effects of metals, phospholipids, and sulfhydryl reagents on these kinase activities are also described.