Divergence in properties of two closely related α-amylase inhibitors of barley

The only inhibitor of human salivary α-amylase identified so far in Hordeum has been isolated from barley cv. Bomi endosperm. This protein has the same N-terminal sequence (23 residues), molecular mass, and isoelectric point as one of the subunits of the barley tetrameric inhibitor previously characterized. However, enzymatic cleavage of both proteins with endoproteinase Glu-C revealed that they are products of different genes. The two isoforms have diverged in their aggregative and inhibitory properties. Thus, the subunit previously characterized forms, along with two other subunits, a tetramer active towards insect but not human salivary α-amylase, while the isoform reported here behaves as a homodimer effective against the human enzyme. These results are discussed in the context of the evolution of the cereal α-amylase inhibitor family.     

A tetrameric inhibitor of insect α-amylase from barley

A tetrameric inhibitor that is active against α-amylase from the larvae of the insect Tenebrio molitor, but inactive against the enzyme from human saliva and against the endogenous one, has been described in barley endosperm. The subunits of the inhibitor have been identified as the previously characterized proteins CMa, CMb and CMd, of which only CMa was inhibitory by itself.     

Repression of α-Amylase Activity by Anoxia in Grains of Barley is Independent of Ethanol Toxicity or Action of Abscisic Acid

Abstract: We studied the effects of anoxia on α-amylase induction, comparing rice ( Oryza sativa L.) and barley ( Hordeum vulgare L.) grains. While gibberellic acid (GA₃) induces α-amylase in rice half-grains under either aerobic or anaerobic conditions, barley half-grains are insensitive to this hormone when applied under anoxia. The possible repressive role of ethanol and abscisic acid (ABA) was investigated. Exogenously added ethanol at concentrations mirroring those found in anaerobically treated tissues was unable to repress α-amylase. The level of ABA in anoxic tissues was found to be much lower than the threshold for α-amylase repression. Overall, the results indicated that these two compounds cannot be held responsible for the failure of barley grains to respond to gibberellic acid. Furthermore, anoxia repressed the induction of α-amylase downstream of the slender mutation, indicating that the repression is independent of effects related to gibberellin perception. Overall, the results suggested that the ability of rice to respond to gibberellins under anoxia is an adaptative trait, independent of known negative regulators of α-amylase induction.     

A major barley allergen associated with baker's asthma disease is a glycosylated monomeric inhibitor of insect α-amylase: cDNA cloning and chromosomal location of the gene

A 14.5 kDa barley endosperm protein that is a major allergen in baker's asthma disease, as previously shown by both in vitro (IgE binding) and in vivo tests, has been identified as a glycosylated monomeric member of the multigene family of inhibitors of -amylase/trypsin from cereals. A cDNA encoding this allergen (renamed BMAI-1) has been isolated and characterized. The deduced sequence for the mature protein, which is 132 residues long, is identical in its N-terminal end to the 20 amino acid partial sequence previously determined from the purified allergen, and fully confirms that it is a member of the multigene family of cereal inhibitors. Southern-blot analysis of wheat/barley addition lines using the insert in the BMAI-1 cDNA clone as a probe, has led to the location of the allergen gene (Iam1) in barley chromosome 2, while another related member of this protein family, the barley dimeric -amylase inhibitor BDAI-1 gene (Iad1) has been located in chromosome 6. Iam1 is the first member of this inhibitor family in cereals to be assigned to chromosome group 2, thus extending the dispersion of genes in the family to five out of the seven homology groups of chromosomes in wheat and barley (chromosome 2, 3, 4, 6 and 7).     

Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds

An α-amylase inhibitor was purified to homogeneity by acid extraction, ammonium sulphate fractionation, chromatography on carboxymethyl-cellulose, diethylaminoethyl-cellulose and Sephadex G-100 from proso grains (Panicium miliaceum). The calculated molecular weight was 14000. The inhibitor was fairly heat stable and stable under acidic and neutral conditions. The factor was more effective by two orders of magnitude in its action on human pancreatic amylase than on human salivary amylase. It did not inhibit onA. oryzae,B. subtilis and porcine pancreatic amylases. Pepsin rapidly inactivated the inhibitor. Chemical modification studies revealed that amino and guanido groups are essential for the action of the inhibitor. The inhibitor was found to protect both human salivary and pancreatic amylases against inactivation by acid. The mode of inhibition was found to be uncompetitive     

Heat shock proteins do not provide thermoprotection to normal cellular protein synthesis, α-amylase mRNA and endoplasmic reticulum lamellae in barley aleurone layers

Heat shock in barley ( Hordeum vulgare L. cv. Himalaya) aleurone layers induces the synthesis of heat shock proteins (hsps) and suppresses the synthesis and secretion of α-amylase, the principal secretory protein. This is accompanied by the destabilization of α-amylase mRNA and a concomitant dissociation of ER lamellae. In the absence of heat shock α-amylase mRNA is extremely stable (Belanger et al. 1986. Proc. Natl. Acad. Sci. USA 83: 1354–1358). In most organisms there is a direct correlation between the synthesis of hsps and thermotolerance. The ability of hsps to provide thermoprotection to secretory protein synthesis, α-amylase mRNA and ER lamellae was analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of pulse-chased, [³⁵S]-methionine-labeled proteins revealed that the half-life of hsps in barley aleurone cells recovering from heat shock was approximately 12 h. Within approximately 6 h, there was a recovery of α-amylase mRNA and a reformation of ER lamellae. Heat shock protein synthesis was induced by either heat shock (40°C) or arsenite, the cells were allowed to recover for 8 h, then were re-exposed to heat shock. Results from SDS-PAGE showed that, despite the presence of hsps, α-amylase synthesis was suppressed. Northern blot hybridizations showed that α-amylase mRNA levels were reduced in heat-shocked tissues. Transmission electron microscopy demonstrated that ER lamellar structures were dissociated. The synthesis of hsps did not enable barley aleurone cells to sustain the synthesis of any proteins at lethal temperature. In contrast, similar conditions established thermotolerance and provided thermoprotection to protein synthesis in germinating barley embryos. Our findings suggest that the aleurone layer does not become thermotolerant following the induction of hsp synthesis.     

Purification, biochemical characterisation and partial primary structure of a new α-amylase inhibitor from Secale cereale (rye)

Plant α-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the α-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13 756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional α-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus α-amylases was observed. The inhibitor is more effective against insect α-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional α-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Trypsin and α-amylase inhibitors are differentially induced in leaves of amaranth (Amaranthus hypochondriacus) in response to biotic and abiotic stress

Seeds of Amaranthus hypochondriacus L. are known to accumulate a trypsin-inhibitor (ATI) member of the potato-I inhibitor family and an α -amylase inhibitor (AAI), possessing a knottin-like fold. They are believed to have a defensive role due to their inhibition of trypsin-like enzymes and α -amylases of insect pests. In this work, both inhibitory activities were found in leaves of young A. hypochondriacus plants. High constitutive levels of foliar inhibitory activity against bovine trypsin and insect α -amylases were detected in in vitro assays. Trypsin inhibitory activity was further increased by exposure to diverse treatments, particularly water stress. Salt stress, insect herbivory and treatment with exogenous methyl jasmonate (MeJA) or abscisic acid (ABA) also induced trypsin inhibitor activity accumulation, although to a lesser degree. In gel and immunoblot analyses showed that foliar trypsin inhibitor activity was constituted by at least three different inhibitors of approximately 29, 8 (including ATI) and 3 kDa, respectively. These inhibitors showed differing patterns of accumulation in response to diverse treatments. On the other hand, significant increases in α -amylase inhibitor activity and AAI levels were detected in leaves of insect-damaged, MeJA- and ABA-treated A. hypochodriacus plantlets, but not in those subjected to water- or salt-stress. A differential induction of trypsin inhibitor activity and α -amylase inhibitor accumulation in response to insect herbivory by two related species of lepidopterous larvae was observed, whereas mechanical wounding failed to induce either inhibitor. The overall results suggest that trypsin and α -amylase inhibitors could protect A. hypochondriacus against multiple types of stress.     

Characterization of cDNA clones of the family of trypsin/α-amylase inhibitors (CM-proteins) in barley (Hordeum vulgare L.)

Summary Recombinants encoding members of the trypsin/-amylase inhibitors family (also designated CM-proteins) were selected from a cDNA library prepared from developing barley endosperm. Inserts in two of the clones, pUP-13 and pUP-38, were sequenced and found to encode proteins which clearly belong to this family, as judged from the extensive homology of the deduced sequences with that of the barley trypsin inhibitor CMe, the only member of the group for which a complete amino acid sequence has been obtained by direct protein sequencing. These results, together with previously obtained N-terminal sequences of purified CM-proteins, imply that there are at least six different members of this dispersed gene family in barley. The relationship of this protein family to the B-3 hordein and to reserve prolamins from related species is discussed in terms of their genome structure and evolution.     

A proteinaceous thermo labile α-amylase inhibitor from Albizia lebbeck with inhibitory potential toward insect amylases

Insects feeding on stored grains cause considerable damage to harvested cereals and legumes every year. The use of α-amylase inhibitors to interfere with the pest’s digestion process has become an interesting alternative biocontrolling agent. In this study, we have studied the interactions of α-amylase inhibitors from Albizia lebbeck seeds with the amylases of coleopteran and lepidopteran insect pests. We isolated and purified the α-amylase inhibitor using acetone precipitation and gel filtration chromatography. Two prominent activity bands of α-amylase inhibitors were detected in electrophoretic analysis using 8% starch PAGE. We found that the α-amylase inhibitor, isolated as a monomer, had a molecular weight of 14.4 kDa. The α-amylase inhibitor was purified 36.15-fold with gel filtration chromatography. Its specific activity was determined at 14.4 U/mg/min. Feeding analysis of Tribolium confusum larvae on a diet containing purified α-amylase inhibitor from Albizia lebbeck revealed that survival of the larvae was severely affected, with the highest mortality rate occurring on the fifth day of feeding. We found that the isolated α-amylase inhibitor inhibits T. confusum and Helicoverpa armigera α-amylases in electrophoretic analysis as well as in solution assays. The isolated α-amylase inhibitor was found to be resistant to commercial protease as well as T. confusum and H. armigera digestive proteinases. The isolated α-amylase inhibitor was degraded by heating above 60°C. Our results suggest that A. lebbeck α-amylase inhibitor could be a useful future biocontrolling agent.     

Nucleotide sequence of a cDNA coding for the barley seed protein CMa: an inhibitor of insect α-amylase

The primary structure of the insect -amylase inhibitor CMa of barley seeds was deduced from a full-length cDNA clone pc43F6. Analysis of RNA from barley endosperm shows high levels 15 and 20 days after flowering. The cDNA predicts an amino acid sequence of 119 residues preceded by a signal peptide of 25 amino acids. Ala and Leu account for 55% of the signal peptide. CMa is 60–85% identical with -amylase inhibitors of wheat, but shows less than 50% identity to trypsin inhibitors of barley and wheat. The 10 Cys residues are located in identical positions compared to the cereal inhibitor family with a Pro-X-Cys motif present in all.     

Novel isoforms of proteinaceous α-amylase inhibitor (α-AI) from seed extract of Albizia lebbeck

Proteinaceous inhibitors of digestive α-amylase occur naturally in leguminous seeds and find applications in agriculture and clinical studies. We have detected and isolated eight novel α-amylase inhibitor isoforms in the seed extract of Albizia lebbeck. They are designated as AL-αAI-1 to AL-αAI-8. These isoforms specifically inhibit human salivary α-amylase and porcine pancreatic α-amylase. The occurrence and profile of α-amylase inhibitor isoforms were revealed by 7 % native-PAGE containing 0.1 % starch. The apparent molecular weights of native bands of AL-αAIs were 97.4, 68.6, 61.0, 57.2, 56.0, 54.7, 51.1, and 47.7 kDa, respectively. Partial purification of potent α-amylase inhibitor was achieved using ammonium sulfate fractionation and gel filtration chromatography on G-100 Sephadex column followed by preparative gel electrophoresis. SDS-PAGE analysis of partially purified AL-αAI showed two polypeptide bands of ~35.8 and ~32.6 kDa. All these isoforms showed effective resistance to in vitro proteolysis by pepsin, trypsin, and chymotrypsin. These inhibitors are stable over a wide range of pH and temperature and have optimum activity at pH 7 and at 37 °C. The finding and information obtained in the present investigation about novel isoforms of α-amylase inhibitors from A. lebbeck could be important and may find applications in clinical studies to modulate starch digestion and glycemic index.     

Taxonomy of the Streptomyces strain ZG0656 that produces acarviostatin α-amylase inhibitors and analysis of their effects on blood glucose levels in mammalian systems

Aims:  To clarify the taxonomic status of strain ZG0656 and analyse the effects of its acarviostatin products on blood glucose levels in mammalian systems.
Methods and Results:  Our program to screen for new α-amylase inhibitors led to the isolation of strain ZG0656. The polyphasic taxonomic study revealed that strain ZG0656 represents a novel variation of Streptomyces coelicoflavus , for which we propose the name S. coelicoflavus var. nankaiensis . Four chemically distinct α-amylase inhibitors, acarviostatins I03, II03, III03 and IV03, were isolated from strain ZG0656. Acarviostatins III03 and IV03 are both novel oligomers. All four acarviostatins are mixed noncompetitive porcine pancreas α-amylase inhibitors. Acarviostatin III03 is the most potent α-amylase inhibitor known to date. Moreover, in the in vitro and in vivo experiments, acarviostatins III03 showed significant inhibition of starch hydrolysis and glucose transfer to blood.
Conclusions:  Strain ZG0656 is a novel variation of S. coelicoflavus, whose products are novel effective α-amylase inhibitors. Among the products, acarviostatins III03 could significantly depress blood glucose levels in mammalian systems and be developed towards a possible therapeutic agent for diabetes.
Significance and Impact of the Study:  Acarviostatin III03 is the most potent α-amylase inhibitor known to date. The oligomer will benefit the research on the relationship between α-amylase and various inhibitors and will offer more choices in diabetes treatments.     

Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A plant-based expression system for matching cDNA clones and isozymes

Isozymes of barley α-amylase were matched to cDNAs that encode them using transient expression in oat aleurone layers. Four cDNAs, including two that are previously unpublished, were inserted into oat aleurone cells by microparticle bombardment. The cDNAs were under the control of theAct1 promoter of rice. Expression levels were sufficient for in-gel detection of enzyme activity following isoelectric focusing of aleurone homogenates. The system has also proved useful in characterizing a hybrid β-glucanase gene.     

Salivary Hsp72 does not track exercise stress and caffeine-stimulated plasma Hsp72 responses in humans

Heat shock protein 72 (Hsp72) has been detected within saliva, and its presence may contribute to oral defence. It is currently unknown how physiological stress affects salivary Hsp72 or if salivary Hsp72 concentrations reflect plasma Hsp72 concentrations. We studied the effect of exercise upon salivary Hsp72 expression, and using caffeine administration, investigated the role of sympathetic stimulation upon salivary Hsp72 expression. Six healthy males performed two treadmill running exercise bouts in hot conditions (30°C) separated by 1 week in a randomized cross-over design, one with caffeine supplementation (CAF) the other with placebo (PLA). Plasma and saliva samples were collected prior to, during and post-exercise and assayed for Hsp72 concentration by ELISA. Exercise significantly increased plasma Hsp72, but not salivary Hsp72 concentration. Mean salivary Hsp72 concentration (5.1 ± 0.8 ng/ml) was significantly greater than plasma Hsp72 concentration (1.8 ± 0.1 ng/ml), and concentrations of salivary and plasma Hsp72 were unrelated. Caffeine supplementation and exercise increased the concentration of catecholamines, salivary α-amylase and total protein, whilst the salivary Hsp72:α-amylase ratio was lower in CAF. Salivary Hsp72 was not altered by exercise stress nor caffeine supplementation, and concentrations did not track plasma Hsp72 concentration.     

Protein secretion in Bacillus subtilis as influenced by the combination of signal sequence and the following mature portion

Abstract We have constructed secretion vector plasmids that have the signal sequence of the Bacillus licheniformis penicillinase gene ( penP ) or the Bacillus stearothermophilus α-amylase gene ( amyT ). We have also constructed penP, amyT and hsa (human salivary α-amylase gene) cartridges. Each of these cartridges was cloned on secretion vectors in Bacillus subtilis , and enzyme production was examined. When amyT vector was used, nearly the same efficiency of enzyme secretion was observed for amyT and penP cartridges. When penP vector was used, enzyme secretion for amyT decreased to about 3% of that for penP cartridges. The eukaryotic gene hsa was hardly expressed in any secretion vectors in B. subtilis .