A.W. MacGregor

A cereal haemoglobin gene is expressed in seed and root tissues under anaerobic conditions

Legumes, and a very few non-legume plant species, are known to possess functioning haemoglobin genes. We describe here the characterization of a haemoglobin cDNA isolated from barley. The deduced amino acid sequence shows 71% amino acid identity with a non-legume haemoglobin gene, a further 16% of the residues being conservative replacements. The barley cDNA also hybridizes to genomic sequences in rye, maize and wheat. The demonstration of a gene from a monocotyledon with close sequence homology to the known non-legume plant haemoglobins fills a major gap in the known distribution of haemoglobin genes in the plant kingdom. The expression of the gene is induced in isolated barley aleurone layers exposed to anaerobic conditions, and the roots of flooding-stressed barley plants. The expression of the RNA under anoxic conditions is similar to that of a known anaerobic response gene, alcohol dehydrogenase. Our results suggest that the increased expression of haemoglobin RNA is an integral part of the normal anaerobic response in barley. The findings are discussed in the light of current theories of haemoglobin function and evolution.

The structure and protein composition of vitreous, piebald and starchy durum wheat kernels

The structure and composition of hand-picked starchy, piebald and vitreous kernels from Wakooma and Wascana durum wheat were compared. The two cultivars exhibited similar trends with degree of vitreosity. Vitreous kernels were higher in protein content and harder than starchy kernels. Starchy durum wheat kernels were as hard as hard common wheats. Piebald kernels were intermediate in protein content, but comparable in hardness to vitreous kernels. Scanning electron micrographs of endosperm of vitreous kernels and vitreous zones within piebald kernels revealed a tight compacted structure, whereas the endosperm of starchy kernels and starchy zones within piebald kernels exhibited a less compacted structure with numerous open spaces. Starchy and vitreous zones within the endosperm of piebald kernels occurred in sharply defined areas. At the interface of starchy and vitreous zones adjacent endosperm cells appeared totally starchy or totally vitreous. The gluten proteins of vitreous kernels contained a greater proportion of gliadins than did gluten proteins of starchy kernels. Piebald kernels contained a lower total amount of gluten protein than vitreous kernels, but the proportion of gliadins present in piebald kernels was similar to that found in vitreous kernels. Reversed-phase HPLC and PAGE failed to detect qualitative differences in gluten proteins attributable to vitreosity. Despite striking differences in structure between the starchy and vitreous zones within the endosperm of piebald kernels, the two zones were similar in protein content and protein composition.

The action of germinated barley alpha-amylases on linear maltodextrins

Abstract The actions of barley alpha-amylase isozymes 1 and 2 (EC 3.2.1.1) on malto-oligosaccharides and their p-nitrophenyl glycosides were similar, but not identical. For each isozyme, transglycosylation occurred with small substrates that were hydrolysed with difficulty, whereas the rates of hydrolysis increased with increase in the size of the substrate for both the malto-oligosaccharides and the p-nitrophenyl glycosides. A p-nitrophenyl group was found to mimic a glucose residue to a large extent. The differences in action of the isozymes are believed to be caused by differences at more than one subsite of the active site. A lysine-arginine substitution is postulated to account for some of the observed variations.

Models for the action of barley alpha-amylase isozymes on linear substrates

Abstract The formation of maltodextrins, G 1 to G 12 , during the hydrolysis of amylose by alpha-amylases 1 and 2 from barley malt was followed by HPLC. Similar, but not identical, distributions of products were obtained with the two alpha-amylase components. Maltose, G 6 , and G 7 were major products, but G 7 was degraded as hydrolysis proceeded. alpha-Amylase 1 produced more G 1 and G 3 than did alpha-amylase 2 at all stages of hydrolysis. Products formed during the hydrolysis of G 9 , G 10 , G 11 , and G 12 by the two alpha-amylase were also determined. A different spectrum of products was observed with each substrate and small differences were observed in the action pattern of the two alpha-amylases, e.g., G 3 and G 7 were the major products formed during the hydrolysis of G 10 by alpha-amylase 1, whereas G 2 and G 8 were the major products formed by alpha-amylase 2 on the same substrate. These results were used to develop a model of the active site of barley malt alpha-amylases. This site contains ten contiguous subsites with the catalytic site situated between subsites 7 and 8. The model can be used to predict hydrolysis patterns of amylose and maltodextrins by cereal alpha-amylases.

A model for the action of cereal alpha amylases on amylose

Abstract A model is proposed to explain the action of cereal alpha amylases (EC 3.2.1.1) on such linear substrates as amylose. It is suggested that, at the active site of the enzyme, there are nine contiguous subsites, each capable of interacting with a glucose residue. An estimate is made of the energies involved in subsite-glucose interaction, and values obtained for the binding energies are used to predict the distributions of small oligosaccharides to be expected at intermediate stages of amylose hydrolysis catalyzed by cereal alpha amylases.

Determination of specific activities of malt α-amylases

Highly purified samples of α-amylases 1 and 2 were prepared from malted barley using ion exchange chromatography on carboxymethyl cellulose and affinity chromatography on cyclohepta-amylose epoxy Sepharose 6B. Specific activities of the two enzymes on amylose were determined by using end-group analysis of the products formed. Per unit of protein, the specific activity of α-amylase 2 was 2·3 times higher than that of α-amylase 1.

Effect of an α-amylase inhibitor from barley kernels on the formation of products during the hydrolysis of amylose and starch granules by α-amylase II from malted barley

High performance thin layer chromatography was used to analyze the products formed during degradation of amylose and starch granules by α-amylase II from malted barley in the presence and absence of an α-amylase inhibitor from barley kernels. A 40-fold molar excess of inhibitor reduced significantly the rate of α-amylolysis of amylose, but the composition of the dextrin mixture formed was not altered by the inhibitor. Significant inhibition of starch granule hydrolysis by α-amylase II was obtained in the presence of a 6-fold molar excess of inhibitor over enzyme. The composition of the low molecular weight products obtained during α-amylolysis of starch granules appeared to be slightly altered by the inhibitor. It appears unlikely that the inhibitor decreases the rate of starch granule hydrolysis by preventing adsorption of α-amylase II to the starch granules.

Influence of pH on the hydrolysis of p-nitrophenyl maltodextrins by alpha-amylase 2 from malted barley

Abstract The action of barley malt alpha-amylase 2 (E.C. 3.2.1.1) on p-nitrophenyl derivatives of maltodextrins was studied at pH 4.8, 6.0 and 7.8. Distributions of products from any one substrate changed little with pH, but a difference in behaviour was observed at pH 4.8 between longer and shorter substrates. With long substrates, such as p-nitrophenyl alpha-malto-octaoside, the enzyme showed high activity at pH 4.8 and 6.0, while for shorter substrates, enzyme activity was much less at pH 4.8 than at 6.0.

Models for the action of barley alpha-amylase isozymes on linear substrates: Carbohydrate Research

The formation of maltodextrins, G1 to G12, during the hydrolysis of amylose by alpha-amylases 1 and 2 from barley malt was followed by HPLC. Similar, but not identical, distributions of products were obtained with the two alpha-amylase components. Maltose, G6, and G7 were major products, but G7 was degraded as hydrolysis proceeded. alpha-Amylase 1 produced more G1 and G3 than did alpha-amylase 2 at all stages of hydrolysis. Products formed during the hydrolysis of G9, G10, G11, and G12 by the two alpha-amylases were also determined. A different spectrum of products was observed with each substrate and small differences were observed in the action pattern of the two alpha-amylases, e.g., G3 and G7 were the major products formed during the hydrolysis of G10 by alpha-amylase 1, whereas G2 and G8 were the major products formed by alpha-amylase 2 on the same substrate. These results were used to develop a model of the active site of barley malt alpha-amylases. This site contains ten contiguous subsites with the catalytic site situated between subsites 7 and 8. The model can be used to predict hydrolysis patterns of amylose and maltodextrins by cereal alpha-amylases.