Characterisation of chicken pancreas alpha-amylase isozymes and interaction with protein inhibitors from wheat kernel.

Abstract

AbstractHomogeneous chicken pancreas α‐amylase consists of two main isozymes which have been isolated by ion exchange chromatography; they show similar kinetic behaviour and inhibition patterns by two protein inhibitors from wheat, one monomeric (termed ‘inhibitor 0.28’) and one dimeric (termed ‘inhibitor 0.19’). Inhibitor 0.28 is the wheat kernel amylase inhibitor of Mr 12000 with a gel‐electrophoretic mobility relative to bromophenol blue of 0.28, while inhibitor 0.19 is the wheat kernel inhibitor of Mr 24000 with a relative mobility of 0.19. The chicken pancreatic enzyme was more effectively inhibited by the dimeric (Ki 3.7 nM) than monomeric inhibitor (Ki 570 nM); with both, the inhibition is dependent on pH, temperature and time of preincubation. Gel filtration, difference spectroscopy and kinetic studies have shown that molar combining ratios for amylase‐inhibitor 0.19 and amylase‐inhibitor 0.28 complexes are 1:1 and 1:2 respectively. Difference spectra of both amylase‐inhibitor complexes indicate that structurally related tryptophanyl side chains are involved in the binding of the two inhibitors to the enzyme. In conclusion, the interaction between chicken pancreas α‐amylase and inhibitor 0.19 or 0.28 fits the model proposed for amylase‐wheat inhibitor interactions.