Analysis of transmembrane dynamics of cholera toxin using photoreactive probes

✍ Scribed by Wisnieski, B. J. ;Shiflett, M. A. ;Mekalanos, J. ;Bramhall, J. S.

• Publisher: Wiley (John Wiley & Sons)
• Year: 1979
• Tongue: English
• Weight: 421 KB
• Volume: 10
• Category: Article
• ISSN: 0091-7419
• DOI: 10.1002/jss.400100209

✦ Synopsis

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, we have shown that lZ5 I-labeled cholera toxin binds to Newcastle disease virus. Pretreatment of Newcastle disease virus with "cold" cholera toxin (at 37°C for 30 minutes) inhibits the binding of 1251-labeled toxin in a subsequent incubation (at 37OC for 30 minutes). These results suggest that cholera toxin binds to Newcastle disease virus in a specific manner. The precise receptor for toxin is unknown in Newcastle disease virus but it is presumed t o be the ganglioside GMl . We have previously shown that the photoreactive probe 12-(4-azido-2-nitrophenoxy)stearoylgucosamine[ 1-14C] labels the membrane proteins of Newcastle disease virus. Since the reactive group of the probe, ie, N3, resides within the membrane bilayer, studies were initiated t o determine which, if any, of the subunits of cholera toxin cross the membrane of Newcastle disease virus and become radioactively labeled upon photoactivation of the probe at 360 nm. After a 15-minute incubation of cholera toxin with Newcastle disease virus containing the photoreactive probe, irradiation effected the l4 C-labeling of the active A l subunit of cholera toxin. Irradiation of cholera toxin in solution with an equivalent amount of probe but without virus resulted in no labeling of toxin subunits.