Subcellular Sites of Polyphosphoinositide Metabolism in Rat Liver

Abstract

Although the polyphosphoinositides, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5 bisphosphate (PIP2), have often been proposed as important components of biological membranes, the subcellular distribution of PIP, PIP2, and the enzymes involved in their metabolism have not been fully characterized. We have therefore prepared various subcellular organelles from rat liver and determined their PI kinase and PIP kinase activities. Polyphosphoinositide-degrading enzymes have also been assayed using [32P] PI P and [32P]PIP2 as substrates. Plasma membranes exhibit the highest specific activity for PIP kinase, but PI kinase appears to be fairly evenly distributed throughout the cell. The plasma membrane expressed the highest hydrolytic activity toward PIP2 while similar rates of PIP hydrolysis were observed in all the fractions analyzed. Rates of incorporation of 32P into PIP and PIP2 in subcellular organelles were also determined after fractionation of isolated rat hepatocytes incubated with 32Pi. [32P]PIP2 synthesis is confined almost exclusively to the plasma membrane, while [32P]PIP is found in other subcellular fractions as well. These studies suggest that the primary site of PIP2 metabolism is at the plasma membrane, and that PIP is metabolized throughout the cell.