Barley α-amylase/subtilisin inhibitor. I. Isolation and characterization

Abstract

A protein inhibitor of endogenous α-amylase 2 has been isolated from germinated barley by glycogen precipitation followed by cation-exchange chromatography. Preliminary kinetic analysis showed a mixed type mechanism of inhibition with an apparent Ki of 4×10-8M. The inhibitor formed well-defined complexes with barley malt α-amylase 2 and co-purified with the α-amylase by cycloheptaamylose affinity chromatography of glycogen precipitates. The inhibitor was inactive towards α-amylases from sorghum malt, hog pancreas, Aspergillus oryzae, and Bacillus subtilis. The amino acid composition and molecular weight near 21,000 were found to be the same as those of both "band-2 protein" previously identified in preparations of barley malt α-amylase and a specific subtilisin inhibitor from barley. Inhibition experiments confirmed that the malt α-amylase inhibitor is a strong inhibitor of subtilisin Carlsberg. Measurements of α-amylase activity in the presence of equimolar amounts of inhibitor and subtilisin showed that the inhibitor is "double headed". The inhibitory activity towards α-amylase was lost after treatment of the inhibitor at 70 °C for 15 min. Isoelectric focusing patterns confirmed that the partially heat-labile, basic α-amylase isozymes (pI=6.6) of barley malt are complexes of α-amylase 2 (pI=6.2) and the inhibitor (pI=7.2). Evidence is presented to suggest that proteins with properties similar to those of the barley inhibitor are present in other cereals including wheat and rye. Possible in vivo functions and some practical aspects of the barley inhibitor are discussed. © 1983 Carlsberg Laboratory.