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Purification and Characteristics of an Endogenous -Amylase Inhibitor from Barley Kernels ¹

Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba R3C 3G8 Canada, Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada, Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada

An inhibitor of malted barley (Hordeum vulgare cv Conquest) -amylase II was purified 125-fold from a crude extract of barley kernels by (NH₄)₂SO₄ fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the -amylase II inhibitor and trypsin inhibitor from barley. Complex formation between -amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over -amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition.

Paper No. 522 of the Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba R3C 3G8 Canada. Paper No. 648 of the Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada.