Activation of α-Amylase by Protein and Detergents1

Abstract

Optimal salt concentration for α-amylase hydrolysis of the PHADEBAS starch substrate was 50 mmol/1 NaCl, and 50 mmol/1 phosphate buffer (pH 7.0). Under these conditions, 7 μmol/l albumin increased the activity of the enzyme 1.3-fold. The degree of activation was shown to depend on pH. Activation was greatest in the more acid region. At pH 5.4 with 7 μmol/1 albumin, there was a 6-fold increase in activity over the control. The degree of activation fell to 2-fold at pH 6.0. A similar trend in activation versus pH was shown with the bile salt sodium taurodeoxycholate, the neutral detergent Triton X-100 and the polymer polyethylene glycol. The degree of activation, however, was less than with protein. With 1.25 mmol/1 bile salt, the activation was 2.5-fold at pH 5.4, falling to 1.4-fold at pH 6.0. Albumin caused an increase in Vmax accompanied by an apparent decrease in K(m). With taurodeoxycholate, V(max) was increased without change in the apparent K(m). The possible mechanism of activation is discussed.

Effect of Free and Conjugated Bile Salts on α-Amylase Activity

“…It can be seen that Vmax is increased (1.43-fold), with no apparent effect on Km. A similar result was obtained with taurodeoxycholate [14]. a-Amylase hydrolysis of soluble starch was not stimulated by these bile salts.…”

“…All reagents were Analar except the following : cholic acid, deoxycholic acid, taurochenodeoxycholic acid, chenodeoxycholic acid, lithocholic acid and bovine albumin (fraction V), which were purchased from Sigma Chemical Co., London. Sodium taurocholate, purchased from Hopkins and Williams, was purified by the method of Norman [12], Phadebas tablets, obtained from Pharmacia, London, were used as substrate, having been washed free of buffer salts as previously described [14], The starch fraction (molecular weight 4,800) was prepared from soluble starch 'nach Zulkowsky' (E. Merck, Darmstadt) by dialysis and fractional ultrafiltration on Amicon membranes. The molecular size was estimated on a calibrated Sephadex G-100 column.…”

“…Lineweaver-Burk plots throughout an 8-fold increase of substrate (blue starch) concentration were determined in the presence and absence of sodium cholate and chenodeoxycholate. The procedure was as previously described [14]. Due to the insoluble nature of the substrate in these experiments, the Michaelis constant obtained from the double reciprocal plot must be regarded as an apparent K Inhibition by chenodeoxycholate of a-amylase hydrolysis of a soluble starch fraction (molecular weight 4,800) was also studied at pH 6.0, throughout a 16-fold range of sub strate concentration (0.312-5.2 mmol/1).…”

“…Recent publications [13,14] have demonstrated that proteins and certain detergents, including taurodeoxycholate, enhance a-amylase hydrolysis of the insoluble Cibachron Blue starch [5]. The effect was more pronounced at acid pH.…”

Ethanol modifies the actin cytoskeleton in rat pancreatic acinar cells — Comparison with effects of CCK

“…Because of no ethanol-induced ·-amylase secretion via high-affinity receptors, a negativefeedback action of PKC on such a secretion was not possible and could not be prevented by PKC inhibition, contrary to suboptimal CCK-8 stimulation. Moreover, the two higher ethanol concentrations were not able to elicit an ·-amylase secretion in addition to that induced by PMA, whereas they induced significant secretions in the Siegmund/Lüthen/Kunert/Weber presence of only the PMA solvent DMSO (4 g/l ethanol elicited a significant secretion in this case, in contrast to experiments with GF, probably because of the shorter pre-incubation, a stabilizing effect of DMSO or/and a slight activation of ·-amylase by DMSO [44]). These findings agree with the results of Mironov and Hermann [22], who concluded from their experiments with rat hippocampal cells that ethanol and PMA utilize the same mechanism to act on stimulus-secretion coupling.…”