GUSTIN FROM HUMAN PAROTID-SALIVA IS CARBONIC-ANHYDRASE-VI

Gustin, a zinc-metalloprotein constituting about 3% of human parotid s aliva protein was previously isolated and characterized as a single po lypeptide chain of 37kDa with one mole of zinc tightly bound to the pr otein. It exhibited biological activity activating calmodulin dependen t bovine brain cAMP phosphodiesterase and was decreased in saliva of p atients with loss of taste in whom taste buds showed a specific pathol ogical morphology. Determination of its primary structure by amino aci d sequence revealed it was identical with carbonic anhydrase (CA) [EC 4.2.1.1] VI and had two N-linked glycosylation sites. Analysis by reve rse phase HPLC and SDS-PAGE before and after deglycosylation confirmed a single peak with molecular weight of the purified protein being 37k Da, the deglycosylated protein, 33kDa. N-linked carbohydrate chains co ntained N-acetyl glucosamine, galactose, mannose, and fucose interior to di, tri and tetra sialyated termini. By isoelectric focusing five i ncreasingly acidic pi values were determined consistent with addition of sialic acid as the terminal carbohydrate residue on the N-linked gl ycoforms of the protein. Gustin was found to exhibit CA activity but w as inhibited by known CA inhibitors in a different manner than CA I or II. These findings, consistent with analysis of previous investigator s, indicate that parotid saliva gustin is CA VI. (C) 1998 Academic Pre ss.