Fluorescence analysis of the interaction between ganglioside GM1-containing phospholipid vesicles and the B subunit of cholera toxin.
Binding of cholera toxin B protomer (CT-B) to a pyrene-labeled analogue of its ganglioside GM1 receptor (pyrene-GM1) in the absence and presence of phosphatidylcholine vesicles was monitored using steady-state fluorescence spectroscopy. CT-B association with pyrene-GM1 micelles induces changes in the fluorescence properties of this ganglioside analogue that are consistent with its conversion from an excimer to a monomer form. Incubation of pyrene-GM1 with preformed vesicles of phosphatidylcholine (PC) results in complete conversion of pyrene-GM1 to its monomer form, however, unlike with CT-B binding, incorporation of pyrene-GM1 into PC vesicles occurs with a concomitant loss of fluorescence quenching by the small polar quenching agent acrylamide. Subsequent binding of CT-B to the PC-GM1 composite vesicles causes no further change in the pyrene fluorescence emission spectrum but does appear to increase acrylamide accessibility. These data lead to the conclusion that cholera toxin binding to a cell membrane alters membrane packing at the site of attachment. Furthermore, this phenomenon appears to be influenced by environmental conditions such as pH. A pH of about 4.0 or less causes acrylamide quenching to decrease to approximately the levels observed in the absence of CT-B. These results may be useful in describing the dynamics of the interaction between cholera toxin and target cell membranes. Moreover, these data could provide clues to the mechanism by which the toxic portion of CT is able to enter the cytoplasm of target cells.
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Department of Biology, Saint Louis University, MO 63103-2010.
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D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D002772:Cholera Toxin / Q000737:chemistry*; D005677:G(M1) Ganglioside / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D011721:Pyrenes / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D000178:Acrylamides; D010713:Phosphatidylcholines; D010743:Phospholipids; D011721:Pyrenes; D020106:Acrylamide; D005677:G(M1) Ganglioside; D002772:Cholera Toxin