Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes.

Abstract

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.