A Fluorescence-based Assay of Phospholipid Scramblase Activity.
Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin's scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin's scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.
Department of Biochemistry, Weill Cornell Medical College.
Birgit
B
Ploier
Department of Biochemistry, Weill Cornell Medical College; akm2003@med.cornell.edu.
Anant K
AK
Menon
D001692:Biological Transport; D061848:Optical Imaging; D010715:Phosphatidylglycerols; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D012160:Retina; D012243:Rhodopsin
D010715:Phosphatidylglycerols; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D012243:Rhodopsin
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