Electrophoretic characterization of posttranslational modifications of human parotid salivary α‐amylase

Abstract

Human salivary α‐amylase displays multiple bands upon native polyacrylamide gel electrophoresis. In fresh saliva, due to posttranslational modifications, a pattern of 5–6 isozymes is observed. The isozymes are designated 1–6, in the order of increasing anodal mobilty. As a result of the development of a rapid and sensitive electrophoresis system, with markedly higher resolution than previously reported, we concluded that a previously proposed model (Karn et al., Biochem. Genet. 1973, 10, 341–350) is inadequate to explain the origin of the various bands. We propose an alternative model that fits in with our new and previously made observations. According to this model, band 2 is the primary gene product and band 1 is its glycosylated counterpart with only one neutral oligosaccharide present on each molecule. Band 3 originates from band 1 by the transialidase‐catalyzed incorporation of sialic acid into the biantennary chain. Bands 4 and 6 originate from bands 2 and 4, respectively, by deamidation; band 5 is the deamidation product of amylase with an acidic oligosaccharide (band 3). Only a minor part of band 3 consists of the deamidation product of band 1. Peptide Asn‐Gly‐Ser (residues 427–429) is the most probable candidate for glycosylation; literature data suggests that deamidation occurs in the stretch Glu‐Asn‐Gly‐Lys‐Asp (residues 364–368) and Asn‐Gly‐Asn‐Cys (residues 474–477). Both glycosylation and deamidation might play a role in the clearance of amylase from the systemic circulation. The elecroporesis system described is a powerful tool to determine amylase isozyme distributions in health and disease, especially for the screening of alterations seen in ectopically produced amylase.