KINETIC-STUDY OF THE AGGREGATION AND LIPID MIXING PRODUCED BY ALPHA-SARCIN ON PHOSPHATIDYLGLYCEROL AND PHOSPHATIDYLSERINE VESICLES - STOPPED-FLOW LIGHT-SCATTERING AND FLUORESCENCE ENERGY-TRANSFER MEASUREMENTS

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukary otic ribosomes. A kinetic study of the aggregation and lipid mixing pr omoted by this protein on phosphatidylglycerol (PG) and phosphatidylse rine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, d imyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been cons idered. The initial rates of the vesicle aggregation induced by the pr otein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process w as deduced from the second-order dependence of the initial rates on ph ospholipid concentration. The highest alpha-sarcin concentration studi ed did not inhibit the vesicle aggregation, indicating that many prote in molecules are involved in the vesicle cross-linking. These are comm on characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. H owever, the kinetics of the scattering values revealed that more compl ex changes occurred in the later steps of the aggregation process of e gg PG and brain PS vesicles than in those of their synthetic counterpa rts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG o r DMPS, which was measured by fluorescence resonance energy transfer a ssays. A delay in the onset of the process, dependent on the protein c oncentration, was observed. Measurement of the rates of lipid mixing r evealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mi xing in heterogeneous systems composed of egg PG + DMPG or brain PS DMPS vesicles. The dilution of the fluorescence probes was faster when these were incorporated into the bilayers made of synthetic phospholi pids than were present in those made of natural phospholipids. The bil ayer destabilization produced by the protein in the vesicles composed of the dimyristoyl-phospholipids should be transmitted to the more sta ble ones made of natural phospholipids. The obtained results are inter preted in terms of lipid mixing occurring within vesicle aggregates la rger than dimer.