Western Blotting And Amido Black Characterization Of The Glycoprotein Nature Of A Melanoplus differentialis Lectin: GHA

Abstract

The carbohydrate nature ofthe lectin GHA was studied using Western Blotting (Concanavalin A as a carbohydrate indicator) coupled with Amido Black staining (as a protein indicator). GHA is composed of two molecules: GHA la and GHA lb used in Western Blotting and Amido Black staining. In the presence of Concanavalin A, non-deglycosylated GHA la and lb produce bands. Upon cleavage by PNGase F, GHA la and lb show an Amido Black band and the absence of a Concanavalin A-sensitive band. These results show GHA to be an N-linked glycoprotein whose carbohydrate has a mass of ~ 2500 Da (~10 hexose units). Unexpected results indicated that reduced GHA la and lb contain a carbohydrate-rich doublet. This Concanavalin A-sensitive doublet disappeared upon deglycosylation, implying covalent linkage. Amido Black does not stain the doublet. Peak 3 and Pre a are not related to the GHA la and lb doublet. Peak 2 showed a carbohydrate-rich doublet that runs faster than the GHA la and lb doublet. The doublet found for Peak 2 is also not sensitive to Amido Black staining. Further data are needed to determine the source of the carbohydrate-rich GHA la and lb doublet and its effects.