RAT-BRAIN CYTOSOL CONTAINS A FACTOR WHICH RECONSTITUTES GUANINE-NUCLEOTIDE-BINDING-PROTEIN-REGULATED PHOSPHOLIPASE-D ACTIVATION IN HL-60 CELLS PREVIOUSLY PERMEABILIZED WITH STREPTOLYSIN-O

We report that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) can stimulate phospholipase D (PLD) in HL60 cells acutely permeabilized with steptolysin O. The ability of GTP[S] to stimulate PLD is impaired if the cells are previously permeabilized such that the majority of the cytosol has leaked out. Rat brain and HL60 cytosols were both found to restore GTP[S]-stimulated PLD activity in a reconstitution assay consisting of previously permeabilized HL60 cells. Rat brain cytosol was fractionated on heparin agarose and assayed for reconstitution of GTP[S]-stimulated PLD activity. The active fractions were pooled, concentrated and chromatographed on gel filtration to assess its molecular mass. The molecular mass of the reconstituting factor was found to be 16 kDa. Reconstitution by the cytosolic factor was dependent on GTP[S]. Ca2+ (pCa 5), MgATP and MgCl2 enhanced GTP[S]-dependent reconstitution of PLD activity in the previously permeabilized HL60 cells. These results demonstrate the presence in rat brain cytosol of a factor which is an activator of GTP[S]-stimulated PLD.