Two major forms of phospholipase D (PLD) activity, solubilized from rat brain membranes with Triton X-100, were separated by HPLC on a heparin-SPW column with buffer containing octyl glucoside. One form was completely dependent on sodium oleate for activity. The other, which was dramatically activated by the addition of ADPribosylation factor (ARF) 1 and guanine 5' [y-thio] (14,15). The cytosolic factors were purified and identified as ADP-ribosylation factor 1 (ARF1) and/or ARF3, which stimulated PLD activity from HL-60 cell membranes in the presence of GTP[yS] (20,24). These ARFs are ==20-kDa proteins initially identified as activators of cholera toxin ADP-ribosyltransferase activity (25) (27).

Unilamellar vesicles of varying and reasonably uniform size were prepared from 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) by the extrusion procedure and sonication. Quasi-elastic light scattering was used to show that different vesicle preparations had mean (Z-averaged) diameters of 1340, 900, 770, 630, and 358 A (sonicated). Bilayer-phase behavior as detected by differential scanning calorimetry was consistent with the existence of essentially uniform vesicle populations of different sizes. The response of these different vesicles to treatment with poly(ethylene glycol) (PEG) was monitored using fluorescence assays for lipid transfer, contents leakage, and contents mixing, as well as quasi-elastic light scattering. No fusion, as judged by vesicle contents mixing and change in vesicle size, was detected for vesicles of diameter greater than 770 A. The diameters of smaller vesicles increased dramatically when treated with high concentrations of PEG, although mixing of their contents could not be detected both because of their small trapped volumes and because of the extensive leakage induced in small vesicles by high concentrations of PEG. Lipid transfer was detected between vesicles of all sizes. We conclude the high bilayer curvature does encourage fusion of closely juxtaposed membrane bilayers but that highly curved vesicles appear also to rupture and form larger structures when diluted from high PEG concentration, a process that can be confused with fusion. Despite the failure of PEG to induce fusion of large, uncurved vesicles composed of a single phosphatidylcholine, these vesicles can be induced to fuse when they contain small amounts of certain amphiphathic compounds thought to play a role in cellular fusion processes. Thus, vesicles which contained 0.5 mol % L-alpha-lysopalmitoylphosphatidylcholine, 5 mol % platelet activating factor, or 0.5 mol % palmitic acid fused in the presence of 30%, 25%, and 20% (w/w) PEG, respectively. However, vesicles containing 1,2-dipalmitoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, 1-oleoyl-2-acetyl-sn-glycerol, or monooleoyl-rac-glycerol at surface concentrations up to 5 mol % did not fuse in the presence or absence of PEG. There was no correlation between the abilities of these amphipaths to induce phase separation or nonlamellar phases and their abilities to support fusion of pure DPPC unilamellar vesicles in the presence of high concentrations of PEG. The results are discussed in terms of the type of disrupted lipid packing that could be expected to favor PEG-mediated fusion.

We have examined the effect of poly(ethylene glycol) (PEG) on stable large unilamellar vesicles formed by a rapid extrusion technique and composed of pure synthetic phosphatidylcholines. The lipid systems studied were the saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the monounsaturated 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). PEG at all concentrations (3.8-40 wt %) induced lipid mixing between large vesicles composed of these phosphatidylcholines. Extensive leakage of internal contents also occurred at high PEG concentrations. However, in contrast to our previous report [Parente, R. A., & Lentz, B. R. (1986) Biochemistry 25, 6678], we could detect no mixing of internal contents indicative of fusion. This discrepancy is due to environmental factors that affect the behavior of 8-amino-naphthalene-1,3,6-trisulfonic acid (ANTS), the fluorophore used in the assay for contents mixing and leakage [McIntyre, Parks, Massenburg, & Lentz (1991) (submitted)]. In agreement with the results of the fusion assay, quasielastic light-scattering measurements revealed no increase in vesicle size following treatment with PEG. These results emphasize the importance of using assays for both membrane mixing and contents mixing to demonstrate fusion, since significant lipid mixing occurred in the absence of fusion. We conclude that large vesicles composed of pure phosphatidylcholine do not fuse in the presence of even high concentrations of PEG. However, DOPC vesicles containing a small amount of an amphipathic "impurity" have been shown to fuse in the presence of PEG at 23 degrees C. These results are discussed in terms of their implications for the mechanism of PEG-induced membrane fusion.

High concentrations (> or = 20 wt %) of poly(ethylene glycol) (PEG) induce large, unilamellar, dipalmitoylphosphatidylcholine model membrane vesicles to fuse when the bilayers contain small amounts of amphipathic peturbant molecules. In addition to fusion, similar concentrations of PEG induce these vesicles to leak their contents. In this paper, we have asked if fusion could occur independently of leakage or if fusion might be described as local bilayer rupture followed by resealing. By following the release of MW 10,000 fluoresceinated dextran trapped inside vesicles, it was determined that PEG-induced leakage was the result of major membrane disruption and not small-pore formation. Fusion of vesicles containing 0.5 mol % palmitic acid was clearly observed at 20 wt % PEG, while 25 wt % was needed to cause rupture. On the other hand, vesicles containing 0.5 mol % lysophosphatidylcholine ruptured at roughly the same concentration needed to induce rupture. Two methods were developed for removing PEG so that fusion products could be characterized. Quasi-elastic light scattering demonstrated that fusing vesicles grew in size and that nonfusing vesicles did not. Moreover, PEG concentrations that induced rupture led to the appearance of species with mean diameters much larger than those of fused vesicles. High-resolution nuclear magnetic resonance showed that the population of large vesicles that correlated with rupture was composed of multilamellar vesicles while the population resulting from fusion alone remained unilamellar. We conclude that, upon incubation with and subsequent removal of PEG, vesicles were either unaffected, or fused to form larger, unilamellar vesicles, or ruptured to form larger, nonunilamellar species.