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

• Published in: Biophysical journal Vol. 67; no. 3; pp. 1117 - 1125
• Main Authors: Mancheño, J.M., Gasset, M., Lacadena, J., Ramón, F., Martínez del Pozo, A., Oñaderra, M., Gavilanes, J.G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.1994; The Biophysical Society
• Subjects: Animals; Biophysical Phenomena; Biophysics; Cattle; Endoribonucleases; Energy Transfer; Fluorescence; Fungal Proteins - pharmacology; In Vitro Techniques; Kinetics; Light; Lipid Bilayers - chemistry; Liposomes - chemistry; Membrane Fusion - drug effects; Phosphatidylglycerols - chemistry; Phosphatidylserines - chemistry; Ribosomes - drug effects; Scattering, Radiation
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(94)80578-8

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed 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 mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles.