We report that guanosine 5'-[y-thioltriphosphate (GTP [S]) can stimulate phospholipase D (PLD) in HL60 cells acutely permeabilized with steptolysin 0. 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+ (pCa5), MgATP and MgC1, enhanced GTP[S]-dependent reconstitution of PLD activity in the previously perrneabilized HL60 cells. These results demonstrate the presence in rat brain cytosol of a factor which is an activator of GTP[S]-stimulated PLD.Phosphatidylcholine (PtdCho) is the major phospholipid in mammalian cells. It has long been thought to be a structural lipid but during the last decade, as for the other phospholipids, it has been shown to be hydrolysed in response to extracellular signals. Phospholipases A?, D and, more recently, C, which are known to hydrolyse PtdCho, have been demonstrated to be activated in response to receptor coupling (see reviews [l -31. Both PtdCho-specific phospholipases C and D are important in cell regulation as either directly before or after dephosphorylation of phosphatidic acid (PtdOH), respectively, they will generate a second messenger, diacylglycerol. This molecule has been shown to play an important role in modulating the activity of protein kinase C (see reviews [4, 51). In addition, it has been postulated that PtdOH may be a second messenger [6].In the human promyelocytic cell line, HL-60, the hydrolysis of PtdCho via the activation of phospholipase D (PLD) has been reported by different authors including us ated-activation of PLD by ATP, which binds to P, purinergic receptors, and by the chemotactic factor, formyl-methionylleucyl-phenylalanine, has been demonstrated [11 -131. It has been shown that in HL-60 cells, this enzyme is regulated via protein kinase C [14, 15, 91 and also via a guanine-nucleotide-binding-regulatory (G)-protein [7-9, 12, 161. Moreover our results [9] and those of others [7, 171 indicate that the activation of PLD is dependent on the presence of cytosolic factors.The subcellular localization of PLD in mammalian cells has been reported to be the membranes [18-261. However, this enzyme has also been found in the cytosol [27, 281. As PLD has not been purified to homogeneity, its subcellular localization remains to be resolved, but it is highly possible that the enzyme may be present in the membranes as well as in the cytosol as shown for the phosphoinositol-lipid-specific phospholipases (see review [29]).The present wo...