Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels [Hordeum distichum]

• Published in: Plant physiology (Bethesda) Vol. 73; no. 4; pp. 1008 - 1012
• Main Authors: WESELAKE, R. J, MACGREGOR, A. W, HILL, R. D, DUCKWORTH, H. W
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01.12.1983
• Subjects: Acetates; Albumins; Amino acids; Analytical, structural and metabolic biochemistry; Barley; Biological and medical sciences; Chromatography; Enzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gels; Hydrolases; Proteins; Starches; Trypsin inhibitors; Monocotyledones; Vegetals; Purification; Hordeum vulgare; Gramineae; Enzyme; Angiospermae; Glycosidase; Spermatophyta; Inhibitor; Α-Amylase
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.73.4.1008

An inhibitor of malted barley (Hordeum vulgare cv Conquest) α-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 α-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between α-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 α-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.