Wheat protein inhibitors of α-amylase

The location in the seed, molecular properties and biological role of protein α-amylase inhibitors from wheat are discussed. Inhibition specificity of albumin inhibitors and structural features essential for interaction with inhibited amylases are also examined. The possible significance of these naturally occurring inhibitors in relation to their presence in foods in active form is described. Finally, genetic aspects of the albumin inhibitor production and the possibility of improving nutritional value and insect re     

Comparison of wheat albumin inhibitors of α-amylase and trypsin

Wheat albumins were extracted from whole wheat flour with 150 mM sodium chloride solution and precipitated between 0·4 and 1·8 M ammonium sulphate. The albumin precipitate was separated by gel filtration on Sephadex G100 into five peaks. Three peaks (II, III, and IV), whose MWs were 60 000, 24 000 and 12 500 daltons respectively, were active toward several insect α-amylases, whereas only peak III inhibited human saliva and pancreatic α-amylases. Peaks III and IV also inhibited trypsin. In each active peak, we found several α-amylase inhibitors slightly different in their electrophoretic mobilities in a Tris—glycine buffer system (pH 8·5), whereas only one major trypsin inhibitor was present in peaks III and IV. In contrast to α-amylase inhibitors that were all anodic, trypsin inhibitors migrated to the cathode under our experimental conditions. From a quantitative standpoint, wheat albumins that inhibit trypsin are negligible, whereas about 2/3 of the total albumin inhibits amylases from different origins. All inhibitor components of peak III were active toward both insect and mammalian α-amylases. Moreover, they reversibly dissociated in the presence of 6 M guanidine hydrochloride giving two similar subunits.     

The phylogenesis of protein α-amylase inhibitors from wheat seed and the speciation of polyploid wheats

Summary Protein -amylase inhibitors extracted with water from seeds of a number of Triticum and Aegilops species were characterized according to their molecular weights and action specificities towards human salivary and Tenebrio molitor L. -amylases. Four inhibitor peaks, with molecular weights 60000, 44000, 22000 and 11000, active towards the two amylases have been detected. Another inhibitor peak with molecular weight 11000, only active towards the insect -amylase, has been found in several species tested. Triticum urartu showed only the 22000 inhibitor peak, while other diploid Triticum species did not exhibit any inhibitory activity. All the diploid Aegilops species tested contained -amylase inhibitors and the inhibitor patterns differed greatly even for closely related species. In general, tetraploid Triticum species (turgidun and timopheevi) exhibited amylase inhibitor patterns of higher complexity than diploid Triticum and Aegilops species.The relationships existing among the amylase inhibitor patterns of the Triticinae species tested are consistent with the hypothesis of the polyphyletic origin of tetraploid wheats by Sarkar and Stebbins (1956) and suggest that the amylase inhibitors from diploid species all derive from common ancestral genes.     

A model for the interaction of wheat monomeric and dimeric protein inhibitors with α-amylase

Summary The amylase-protein amylase inhibitor system offers a unique model of specific and reversible protein-protein interaction. The monomeric and dimeric inhibitors, exhibiting closely related properties and interacting with the same amylase, also provide a convenient test to compare effects of monomer-monomer and monomerdimer interactions between enzyme and inhibitor proteins.TmL amylase, Tenebrio molitor L. larval -amylase; CP amylase, chicken pancreatic -amylase; 0.19, -amylase protein inhibitor from wheat kernel with gel electrophoretic mobility 0.19; 0.28, -amylase protein inhibitor from wheat kernel with gel electrophoretic mobility 0.28.     

Effects of bean and wheat α-amylase inhibitors on α-amylase activity and growth of stored product insect pests

Insect α-amylase inhibiting and/or growth inhibiting activities of proteinaceous inhibitors from red kidney bean (Phaseolus vulgaris) and hard red winter wheat (Triticum aestivum) were examined. The bean inhibitor was most effectivein vitro against α-amylases from the red flour beetle (Tribolium castaneum) and the confused flour beetle (T. confusum), followed by those from the rice weevil (Sitophilus oryzae) and yellow mealworm (Tenebrio molitor). The insect enzymes were from two- to 50-fold more susceptible than human salivary α-amylase. When the inhibitors were added at a 1% level to a wheat flour plus germ diet, the growth of red flour beetle larvae was slowed relative to that of the control group of larvae, with the bean inhibitor being more effective than the wheat inhibitor. Development of both the red flour beetle and flat grain beetle (Cryptolestes pusillus) was delayed by 1% bean inhibitor, but development of the sawtoothed grain beetle (Oryzaephilus surinamensis) and lesser grain borer (Rhyzopertha dominica) was not affected by either the bean or wheat inhibitor at the 1% level. Rice weevil adults fed a diet containing 1% bean or wheat inhibitor exhibited more mortality than weevils fed the control diet. When the wheat amylase inhibitor was combined with a cysteine protease inhibitor, E-64, and fed to red flour beetle larvae, a reduction in the growth rate and an increase in the time required for adult eclosion occurred relative to larvae fed either of the inhibitors separately. The bean inhibitor was just as effective alone as when it was combined with the protease inhibitor. These results demonstrate that plant inhibitors of insect digestive enzymes act as growth inhibitors of insects and possibly as plant defense proteins, and open the way to the use of the genes of these inhibitors for genetically improving the resistance of cereals to storage pests. Cooperative investigation between the Agricultural Research Service, the University of California, San Diego, and the Kansas Agricultural Experiment Station (Contribution no. 94-416-J). Supported in part by a grant from the Ministry of Education and Science, Spain-Fulbright Program to J.J.P. Mention of a proprietary product does not constitute a recommendation or endorsement by the USDA. The USDA is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Expression and secretion of wheat α-amylase in Kluyveromyces lactis

The plasmid pCR1 has been constructed to express a wheat -amylase enzyme in Kluyveromyces lactis strains. The contruct is based on the vector pCXJ-kan1, which has been derived from pDK1, a native plasmid of K. lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Contruct pCR1 produces an -amylase by DNA isolated from a wheat cDNA clone and is controlled by a Saccharomyces cerevisia PGK promoter. Correspondence to: C. Russell     

Purification and properties of an α-amylase inhibitor from wheat

Four inhibitors of α-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-change chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60–90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.     

Effect of triterpenoid glycosides on α- and β-amylase activity and total protein content in wheat seedlings

Influence of the aleanolic acid glycosides from Silphium perfoliatum L. (silphioside B, C, E and G) and their progenins on the amylase activity and total protein content in wheat seedlings was studied. Treatment of the Triticum aestivum L. seeds with 1–10 μM water solutions of mono- and diglycosides (mono- and bisdesmosines) elevated the α-amylase and total amylase activities in seedlings. Silphioside E containing three glucose moieties in its molecule did not change α-amylase activity, but it did if bis-triglycoside acetylated carbohydrate (as in silphioside C). Effects of 5–10 μM solutions of the active glycosides was comparable with that of exogenous gibberellin A₃ and 6-benzylaminopurine.     

SNP and haplotype identification of the wheat monomeric α-amylase inhibitor genes

Seventy-three gene sequences encoding monomeric α-amylase inhibitors were characterized from cultivated wheat “Chinese Spring”, group 6 nullisomic-tetrasomic lines of “Chinese Spring” and diploid putative progenitors of common wheat. The monomeric α-amylase inhibitors from the different sources shared very high homology (99.54%). The different α-amylase inhibitors, which were determined by the 24 single nucleotide polymorphisms (SNPs) of their gene sequences, were investigated. A total of 15 haplotypes were defined by sequence alignment, among which 9 haplotypes were found with only one single sequence sample. Haplotype H02 was found to be the main haplotype occurring in 83 WMAI sequence samples, followed by haplotype H11. The median-joining network for the 15 haplotypes of monomeric α-amylase inhibitor gene sequences from hexaploid wheats was star like, and at least two subclusters emerged. Furthermore evidence of homologous recombination was found between the haplotypes. The relationship between nucleotide substitutions and the amino acid changes in WMAI of hexaploid wheats was summarized. It was clear that only five polymorphic sites in the nucleotide sequence of WMAI resulted in amino acid variations, and that should be the reason for different structure and function of inhibitors. However, little evidence could be found that there were WMAI genes in the A genome of hexaploid wheat, whereas it could conclude from our results that the A genome diploid wheat had WMAI genes. The overall information on the monomeric α-amylase inhibitors from wheat and Aegilops strongly support the view that these inhibitors have evolved from a common ancestral gene through duplication and mutation. Ji-Rui Wang and Yu-Ming Wei are contributed equally to this paper.     

Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Polymorphism and chromosomal location of endogenous α-amylase inhibitor genes in common wheat

Summary Polymorphism of an endogenous -amylase inhibitor in wheat was studied using iso-electric focusing followed by monoclonal antibody — based immunoblotting. Ten isoforms of the inhibitor detected in common wheat and its wild counterparts were assigned to five homoeologous loci. Three -amylase inhibitor loci (Isa-1) were identified in common wheat and located on the long arms of chromosomes 2A, 2B and 2D. In a sample of 27 bread wheats, eight durum wheats, and 12 diploid wheat relatives, amphiploids and triticales, a high resolution isoelectric-focusing separation demonstrated two active and one null allele at the Isa-A1, two alleles at the Isa-B1, one allele at the Isa-D1, four alleles at the Isa-S1, and one allele at the Isa-G1 locus. The most frequent electrophoretic pattern of common wheat cultivars consisted of two isoforms, encoded respectively by the Isa-B1b, Isa-D1 a alleles and the Isa-Alnull allele. All the durum wheats had only one inhibitor form controlled by allele Isa-B1b, which was accompanied by the null allele at the Isa-A1 locus.Contribution No. 210 of the Food Science Department, University of Manitoba     

Isolation and partial characterisation of α-amylase components evolved during early wheat germination

The development of α-amylase (EC 3.2.1.1) activity in wheat was followed during 4 days of germination. The enzyme was purified and separated by gel chromotography into two distinct entities (α-amylase I and α-amylase II), with different molecular weights and isoelectric points. α-Amylase I contained a much higher content of sugars than α-amylase II, which decreased as the germination proceeded. The time sequence analysis of the starch degradation pattern showed that on the 4th day of germination, 15% of the total activity was present in α-amylase I and the rest in a-amylase II. Similarly, differences in the relative rates of synthesis of their isoenzymes were observed. α-Amylase I was resolved on the 4th day of germination, only into 3 isoenzymes, whereas α-amylase II could separate into 4 isoenzymes. The enzyme activity was however maximal in the most electropositive isoenzyme in both the components.     

Gibberellic-acid-regulated expression of α-amylase and six other genes in wheat aleurone layers

The effect of gibberellic acid (GA₃) on gene expression in wheat aleurone cells has been characterised. In-vitro translation of polyadenylated RNA indicated that α-amylase and other messenger-RNA (mRNA) species increase in relative concentration in GA₃-treated tissue. At least one mRNA species declines in relative level in response to GA₃. There is also a GA₃-dependent, four-fold increase in the level of polyadenylated RNA. This effect is largely the result of increased levels of many mRNA species which are also present in untreated tissue. Seven GA₃-induced polyadenylated RNA species including the Amyl α-amylase gene product have been cloned as complementary DNA in the plasmid pBR322. These cloned DNAs have been used as hybridisation probes to show that the GA₃-induced increase in α-amylase mRNA is more prolonged than the accumulation of the other GA₃-regulated mRNA species. A polyadenylated-RNA sequence showing reduced concentration in GA₃-treated tissue has also been cloned.     

Sugars as repressors of gibberellin-induced synthesis of α-amylase in wheat

In germinating cereal caryopses, α-amylase is synthesized in the aleurone layer and scutellum epithelium. Produced enzyme is released into the endosperm, where starch is hydrolyzed. We investigated the effect of sugars on gibberellic acid (GA)-induced synthesis of this enzyme in both tissues of wheat (Triticum aestivum L.) seeds. α-Amylase synthesis in the embryo was much more sensitive to sugars, and their inhibitory effect was observed at the lower concentrations (10–20 mM), whereas in the aleurone layer the enzyme was only inhibited at a relatively high (above 100 mM) concentration of sugars in the medium. These results point to a specific (repressive) influence of sugars on embryonic α-amylase and probably to its nonspecific (osmotic) effect on the cells of the aleurone layer. It was found that phosphorylated sugars were more effective repressors of α-amylase than nonphosphorylated sugars.     

Bifunctional inhibitor of α-amylase/trypsin from wheat grain

A trypsin inhibitor, isolated from whole-wheat grain (Triticum aestivum L.) by the method of biospecific chromatography on trypsin-Sepharose, was potent in inhibiting human salivary α-amylase. The bifunctional α-amylase/trypsin inhibitor was characterized by a narrow specificity for other α-amylases and proteinases. The high thermostability of the inhibitor was lost in the presence of SH group-reducing agents. The inhibitor-trypsin complex retained its activity against α-amylase. The inhibitor—α-amylase complex was active against trypsin. Studies of the enzyme kinetics demonstrated that the inhibition of α-amylase and trypsin was noncompetitive. Our results suggest the existence of two independent active sites responsible for the interaction with the enzymes.     

Expression of α-amylase and other gibberellin-regulated genes in aleurone tissue of developing wheat grains

Aleurone tissue from freshly harvested immature wheat grains (Triticum aestivum L. cv. Sappo) which is normally unresponsive to gibberellic acid can be made responsive by subjecting the tissue to a pre-incubation treatment in a simple buffered medium prior to the addition of the growth substance. The effectiveness of this treatment is dependent on grain age, with grains less than 15–20 days post anthesis failing to become converted to a responsive state whilst tissue from grains older than this become increasingly susceptible. Tissue from grains of a certain age (approx. 25–28 days post anthesis) produce small amounts of -amylase following this treatment even in the absence of exogenously applied growth substance. Using different ³²-labelled complementary-DNA probes for -amylase in wheat it was demonstrated that the failure of freshly harvested tissue to produce -amylase was correlated with the absence of the appropriate mRNA species. Inability to accumulate -amylase mRNA in response to gibberellic acid was removed by the pre-iccubation treatment and also by enforced drying. The gibberellin-regulated expression of other unidentified genes also responds to pre-incubation or drying. Induction of gibberellin-responsiveness in immature aleurone cells did not extend to the secretion of acid phosphatase, protease and ribonuclease.Abbreviations cDNA complementary DNA - dpa days post anthesis - GA gibberellin - GA₃ gibberellic acid     

Isolation and characterization of brown algal polyphenols as inhibitors of α-amylase,lipase and trypsin

Extracts ofAscophyllum nodosum, Fucus serratus, F. vesiculosus andPelvetia canaliculata contain inhibitors of α-amylase, lipase and trypsin. The inhibitors were isolated and identified by¹H NMR spectroscopy as polyphenols which have apparent molecular weights in the range from 30 000 to 100 000 daltons, as determined by ultra-filtration with Amicon membranes. These polyphenols account for the whole of the inhibitory activity in crude algal extracts. The compounds inhibit α-amylase and trypsin in an apparently non-competitive manner, when preincubated with the enzymes, and the inhibition is directly proportional to the concentration of the inhibitor. Starch protects α-amylase when added to the enzyme together with the inhibitors. Under this condition the effectiveness of the inhibitors is reduced ten-fold.     

Variation of seed α-amylase inhibitors in the common bean

Variation of seed -amylase inhibitors was investigated in 1 154 cultivated and 726 non-cultivated (wild and weedy) accessions of the common bean, Phaseolus vulgaris L. Four -amylase inhibitor types were recognized based on the inhibtion by seed extracts of the activities of porcine pancreatic -amylase and larval -amylase and larval -amylase of the Mexican bean weevil, Zabrotes subfasciatus Boheman. Of the 1 880 accessions examined most (1 734) were able to inhibit porcine pancreatic -amylase activity, but were inactive against the Z. subfasciatus larval -amylase; 41 inhibited only the larval -amylase activity, 52 inhibited the activities of the two -amylases, and 53 did not inhibit the activity of either of the -amylases. The four different inhibitor types were designated as AI-1, AI2, AI-3, and AI-0, respectively. These four inhibitor types were identified by the banding patterns of seed glycoproteins in the range of 14–20 kDa by using SDSpolyacrylamide gel electrophoresis. Additionally, four different banding patterns were recognized in accessions with AI-1, and were designated as AI-1a, 1b, 1c, and 1d. Two different patterns of the accessions lacking an -amylase inhibitory activity were identified and designated as AI-0a and AI-0b. The largest diversity for seed -amylase inhibitors was observed in non-cultivated accessions collected from Mexico where all eight inhibitor types were detected. The possible relationships between the variation of seed -amylase inhibitors and bruchid resistance are discussed.