Phosphatidylserine decarboxylase: generation of asymmetric vesicles and determination of the transbilayer distribution of fluorescent phosphatidylserine in model membrane systems.
Large unilamellar vesicles (LUV) that contained a fluorescent analog of phosphatidylserine (NBD-PS) were used in model systems to determine the feasibility of employing phosphatidylserine decarboxylase (PS-decarboxylase) to generate asymmetric vesicles and to determine the transbilayer distribution of PS. PS-decarboxylase prepared by sonication of Escherichia coli JA 200 pLC 8-47 was found to be stable in detergent-free buffers and catalyzed the conversion of NBD-PS to NBD-phosphatidylethanolamine (NBD-PE). PS-decarboxylase was capable of decarboxylating virtually all of the NBD-PS present in the outer leaflet of LUV containing a symmetric or asymmetric (outside only) distribution of NBD-PS, but not NBD-PS present in the inner leaflet of the vesicles. The ability of PS-decarboxylase to decarboxylate only NBD-PS located in the outer leaflet of the vesicles was independently verified by resonance energy transfer (between NBD-PS and (lissamine) rhodamine B-labeled phosphatidylethanolamine) and by derivatization with trinitrobenzenesulfonic acid (TNBS). These techniques revealed that the exchangeable pool (the fraction of NBD-PS on the outer leaflet) and the respective fraction of Tnp-(NBD-PS) formed were equivalent to the extent of PS-decarboxylase-mediated decarboxylation of NBD-PS to NBD-PE. These results show that PS-decarboxylase can be used to generate asymmetric vesicles (i.e., PS inside, PE outside) and determine the intrabilayer distribution of PS in model membranes.
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D002262:Carboxy-Lyases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008051:Lipid Bilayers; D010718:Phosphatidylserines / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D008051:Lipid Bilayers; C034296:N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine; D010718:Phosphatidylserines; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase; D009327:4-Chloro-7-nitrobenzofurazan