The isoenzymes of human parotid amylase

Abstract The number and proportion of isoenzymes of α-amylase in the parotid saliva of a single subject have been studied. Five amylolytic bands were observed on disc-gel electrophoresis of samples containing approximately 120 μg amylase. Two additional bands were revealed at higher amylase concentrations. Limited storage of parotid saliva at 4 ° did not alter the banding pattern, nor did treatment of saliva and gel with 5 m m EDTA. Photocatalyzed polymerization was not a factor in isoenzyme formation. Densitometric scans of gels indicate that the relative proportions of amylase isoenzymes remain constant in a single individual over an extended period of time. Prolonged storage of human parotid saliva at 4 ° resulted in quantitative differences in the proportion of isoenzymes. Crystalline amylase was prepared from human parotid saliva, and was found to contain the same number of enzymatic forms present in freshly collected saliva. No evidence of dimers or higher polymers was gained on Bio-Gel filtration columns. All of the isoenzymes were retarded well beyond the elution volume expected for molecules of their reported size. Bio-Gel P-100 fractionates crystalline preparations into two families; the A family, containing isoenzymes 5, 3, and 1 and the B family, containing isoenzymes 4, 2, and two forms designated “z.” Exploratory experiments have been carried out with the respective families including comparison of their amino acid composition, free sulfhydryl groups, and sugar content. Peptide maps of pepsin hydrolysates have been compared. No differences were observed in the amino acid composition or in the number of free sulfhydryl groups per molecule. The major difference observed was in the carbohydrate content. The A family appears to contain 8 moles of neutral sugar and 4 moles of glucosamine per mole of enzyme whereas the B family contained no detectable carbohydrate. Only minor differences were noted in the peptide maps of the respective families. Attempts to fractionate the isoenzymes of the B family on QAE-Sephadex appear to confirm the observations made with whole parotid saliva that storage at pH 8–9 promoted the conversion of isoenzyme 4 to 2 and z.