Cautionary note on the use of the B subunit of cholera toxin as a ganglioside GM1 probe: detection of cholera toxin A subunit in B subunit preparations by a sensitive adenylate cyclase assay.
The use of the B subunit of cholera toxin, a protein that binds specifically to ganglioside GM1, has provided a new paradigm for studying physiological functions of ganglioside GM1. The B subunit inhibited the growth of rat glioma C6 cells that had been pretreated with ganglioside GM1. In some preparations of the B subunit, the inhibition was independent of adenylate cyclase activation and was due to the binding of the B subunit to ganglioside GM1 inserted onto the cell surface. However, in other preparations of the B subunit, there was an additional inhibitory effect due to small contaminations with the A subunit, which caused increases in intracellular cyclic adenosine monophosphate (cAMP) levels and concomitant growth inhibition. This vanishingly small contamination with the A subunit could not be detected by conventional protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis but could be measured utilizing a sensitive adenylate cyclase activation assay. Thus caution must be used to ensure that any biological effects of the B subunit are not due to contaminating A subunit and are due solely to the binding of the B subunit to ganglioside GM1 exposed on the cell surface. This is especially important in cyclic nucleotide-sensitive systems.
Spiegel
S
S
Department of Biochemistry, Georgetown University Medical Center, Washington, DC 20007.
D000262:Adenylyl Cyclases / Q000032:analysis*; D000818:Animals; D002772:Cholera Toxin / Q000032:analysis*; D000242:Cyclic AMP / Q000032:analysis; D004273:DNA, Neoplasm / Q000096:biosynthesis; D005677:G(M1) Ganglioside / Q000032:analysis*; D005910:Glioma / Q000032:analysis*; D046911:Macromolecular Substances; D015335:Molecular Probes; D051381:Rats; D014407:Tumor Cells, Cultured / Q000032:analysis
D004273:DNA, Neoplasm; D046911:Macromolecular Substances; D015335:Molecular Probes; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases