Electrophoretic characterization of posttranslational modifications of human parotid salivary alpha-amylase.
Human salivary alpha-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 mobility. 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 electrophoresis 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.
Bank
R A
RA
Institute of Human Genetics, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands.
Hettema
E H
EH
Arwert
F
F
Amerongen
A V
AV
Pronk
J C
JC
D000595:Amino Acid Sequence; D003720:Densitometry; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007834:Lasers; D008969:Molecular Sequence Data; D010306:Parotid Gland / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D012463:Saliva / Q000201:enzymology*; D013194:Staining and Labeling; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics
D007527:Isoenzymes; D000516:alpha-Amylases