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 stim ulate phospholipase D (PLD) in HL60 cells acutely permeabilized with s teptolysin O. The ability of GTP[S] to stimulate PLD is impaired if th e cells are previously permeabilized such that the majority of the cyt osol has leaked out. Rat brain and HL60 cytosols were both found to re store GTP[S]-stimulated PLD activity in a reconstitution assay consist ing of previously permeabilized HL60 cells. Rat brain cytosol was frac tionated on heparin agarose and assayed for reconstitution of GTP[S]-s timulated PLD activity. The active fractions were pooled, concentrated and chromatographed on gel filtration to assess its molecular mass. T he 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 P LD 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.