Identification of phosphatidylinositol kinase in rat liver lysosomal membranes

Liver lysosomes from Triton-injected or normal rats were found to rapidly incorporate 32P from [gamma-32P]ATP into a lipid component of the membrane, in vitro. The lipid was identified as phosphatidylinositol 4-phosphate based on its chromatographic behavior on Silica Gel H thin layer plates as compared with standard phosphoinositides. The deacylation product, glyceryl-phosphorylinositol phosphate, was compared with standards in chromatographic and electrophoretic systems to further substantiate the identification of the radioactive material. A trace of phosphatidylinositol 4,5-bisphosphate was also found. The properties of the lysosomal membrane phosphatidylinositol kinase were examined using both endogenous lipid and exogenous phosphatidylinositol as substrate. The enzyme was active at neutral pH in the presence of 20 mM MgCl2. The addition of 0.4% Triton X-100 stimulated the enzyme activity toward endogenous substrate, and the highest activity was observed in the presence of detergent and 1 mM phosphatidylinositol. Degradation of the product was seen only in the presence of Triton X-100. The specific activity of the lysosomal phosphatidylinositol kinase is comparable to the detergent-stimulated activity of liver microsomes and plasma membrane, the previously recognized sources of this enzyme in the liver cell.