Human Salivary Alpha-amylase Research Articles

The action pattern of human salivary alpha-amylase in the vicinity of the branch points of amylopectin

Salivary alpha-amylase hydrolyses amylopectin in stages. At the end of the so-called second stage, there are present glucose, maltose, and a series of α-limit dextrins containing (1→4)- and (1→6)-α- d-glucosidic bonds. The structures of the limit dextrins containing a single (1→6)-bond were examined. Six such dextrins were found. Of these, two were capable of being further hydrolysed by alpha-amylase, whereas the remaining four were true, amylase-resistant α-limit dextrins. The structures of the limit dextrins afforded information about those (1→4)-α- d-glucosidic bonds of amylopectin that are capable of being cleaved by salivary alpha-amylase and those that are resistant. In order to define further the action of alpha-amylase, the alpha-amylolytic products of 6-α-maltotriosyl- d-glucose, 6 3-α-maltotriosylmaltotriose, and 6 3-α-maltotriosylmaltotetraose were examined.

Effects of cations on the activation of salivary amylase: I. Na+, K+, Ca2+ and Mg+.

Human salivary alpha-amylase (EC 3.2.1.1,a-l, 4-glucan-4-glucanohydrolase) requires chloride ion for activity. Most investigators activate this enzyme with NaCI, despite the fact that human saliva is saturated with calcium and relatively rich in K+, compared with Na+. Clifford (Biochem J 30: 2049-2053, 1936) and the following research demonstrate that there are significant differences in the degree of activation by different chlorides because of the cation component. Saliva was collected from healthy adults by expectoration. The sample was centrifuged and dialyzed at 4C against sufficient changes of deionized water to make free chloride concentrati-ations insignificant. Dialysis was completed within two days and saliva older than one week was not used. Purified pancreatic amylase (Porcine) was used as purchased after dialysis. The analytic matrix was composed of 14 pairs of test tubes. Ten pairs contained the five concentrations of the two salts to be compared. The four remaining pairs contained a maltose standard, a water blank, a blank containing substrate and water, and an enzyme blank containing dialyzed saliva, starch, and water (no salt) . Thus, for each individual comparison, enzyme concentration and other spurious variables were internally consistent; therefore, differences in enzyme activity reflect only differences in cation composition. Amylase activity was determined by the method of Bernfeld (Methods in Enzymology, vol I, 1955). The reaction was initiated by adding 1 ml dialyzed saliva (1:500 dilution) or pancreatic amylase (1:1000) to 2 ml of the reaction mixture containing 1 ml chloridefree starch solution (1% in distilled water) and 1 ml of salt solution of the requisite strength. The data summarized in the illustration demonstrate that differences in amylase activity depend on the cationic component. At concentrations of 10-4 to 10-1 M chloride, activation by K exceeds that by Na by a significant amount. At a 1 M chloride concentration there was no significant difference. Salivary amylase showed a maximum at about 10- M and concentrations greater than 10-' M were inhibitory. For pancreatic amylase, similar results were obtained, with the exception that no maximum was observed. Calcium chloride activated salivary amylase to a much greater extent than equimolar chloride concentrations of NaCl. At a concentration of up to 0.1 M chloride, the Ca showed no indication of inhibition, as did the Na, K curves. At chloride concentrations ranging from 10-4 to 10-2 M, MgCl2 was less effective than NaCl in activating salivary amylase.

Substrate differentiation of human pancreatic and salivary alpha-amylases.

To increase specificity in the clinical determination of amylase, the differential activities of human pancreatic and salivary amylases toward two different substrates—a Lintner soluble starch (LSS) and an insoluble, dyed starch, Amylose Azure (AA)—were investigated. A ratio of initial-velocity activities of Lintner soluble starch to Amylose Azure (LSS/AA) for salivary amylase was at least twice as great as the LSS/AA ratio obtained for pancreatic amylase. Statistical analysis of paired saliva samples and secretin-pancreozymin stimulated pancreatic juices revealed a significant (p < 0.01) difference between the mean LSS/AA activity ratios of these two biologic fluids. Even greater differences were found between the ratios for crystalline salivary amylase compared to the amylase contained in purified preparations from human duodenal fluid and pancreatic homogenates. A method to differentiate elevated serum amylase resulting from parotitis or pancreatitis is demonstrated.