Identification of a membrane associated, phosphatidylinositol binding protein in mammalian cells

The capacity of mammalian cells to transfer lipids between membranes and generate lipid second messengers has been shown to be dependent on lipid binding and transfer proteins. The objective of the current study was to identify novel proteins which bind and/or transfer arachidonate (AA)-containing glycerolipids. Neutrophils, THP-1 monocytes or HL60s were pulse-labeled with [3H]-AA for 5-min., washed and further incubated for 5 min. Proteins from microsomal and cytosolic fractions of these cells were then precipitated and extracted (5x) with organic solvents and then separated by gel filtration. One major labeled protein was identified at 24 kDa by gel filtration in the solubilized microsomal fraction of all three cell types. Further analysis of the lipid bound to this protein reveal it to be predominantly phosphatidylinositol (PI) containing labeled AA. Stimulation of neutrophils with A23187 induced an increase in the quantity of labeled AA-containing PI associated with this protein when compared to unstimulated cells. The labeled protein was purified over 4000-fold utilizing ion exchange, gel filtration and reverse phase HPLC procedures. These data demonstrate the partial purification of a membrane bound protein that binds AA-containing PI. Further purification, sequencing, and cloning will be necessary to determine whether this protein is distinct from the previously-described cytosolic PI transfer protein.