| Summary: | GM$ sb2$ ganglioside hydrolysis requires the formation of a ternary complex consisting of substrate, enzyme ($ beta$-N-acetylhexosaminidase A = Hex A), and the GM$ sb2$ activator protein. In order to study the interaction between Hex A and GM$ sb2$ activator, the human GM$ sb2$ activator cDNA was cloned into the p-FLAG vector. The fusion protein (FLAG-AP) was expressed in E. coli, and purified to homogeneity using immunoaffinity chromatography. === A retardation assay was designed using the immunoaffinity column to detect transient interactions between FLAG-AP and Hex A. Hex A and Hex S are retarded by the column, but not Hex B or unrelated proteins. Hex A retardation is absolutely dependent upon the presence of immobilized FLAG-AP, but does not require the presence of GM$ sb2$ ganglioside. Interaction of GM$ sb2$ activator and Hex A does not involve the enzyme's active site, but does appear to depend upon hydrophobic interactions between the two proteins.
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