New dimeric inhibitor of heterologous alpha-amylases encoded by a duplicated gene in the short arm of chromosome 3B of wheat (Triticum aestivum L.)

A new wheat dimeric alpha-amylase inhibitor, designated WDAI-3, has been characterized. WDAI-3 is a homodimeric protein active against alpha-amylase from human saliva and from the insect Tenebrio molitor, but inactive against that from pig pancreas or against trypsin. Its N-terminal amino acid sequence is closer to those of the wheat dimeric inhibitors 0.19 and 0.53 (89-91% identical positions in 44 residues) than to that of the monomeric 0.28 inhibitor (69% identical positions). Iha-B1-2, the gene encoding the new inhibitor, is located in the short arm of chromosome 3B, where it is part of an intrachromosomal gene duplication that also codes for the 0.53 inhibitor.     

Molecular size and fidelity of DNA polymerase alpha from the regenerating liver of the rat

Thermal stabilization resulting from protein . protein association between two protein inhibitors (coded as 0.19, a dimer, and 0.28, a monomer) from wheat flour and the alpha-amylase from Tenebrio molitor L. (yellow mealworm) larvae was investigated by differential scanning calorimetry (heating rate 10 degrees C/min). Thermograms (plots of heat flow vs. temperature) for the two inhibitors showed broad endothermic peaks with the same extrema (denaturation temperatures) at 93 degrees C, and equal, small enthalpies of denaturation (2 cal/g). The amylase produced a sharp endotherm at 70.5 degrees C, but a larger enthalpy change on denaturation (6 cal/g). The amylase . inhibitor complexes differed in thermal stability, but both showed significant stabilization relative to free enzyme. The complex formed with monomeric inhibitor 0.28 showed a higher denaturation temperature (85.0 degrees C) than that formed with dimeric inhibitor 0.19 (80.5 degrees C). This order of stabilization agrees with the relative affinities of the inhibitors for the amylase. These thermograms are consistent with previous results which indicated that 1 mol of amylase binds 1 mol of inhibitor 0.19.     

小麦抗虫α-淀粉酶抑制因子成熟蛋白编码基因序列分析

对17份小麦和山羊草材料的小麦抗虫24kD-α-淀粉酶抑制因子成熟蛋白编码基因进行了分离克隆和序列分析。结果发现,在二倍体材料中α-淀粉酶抑制因子由单个基因编码,而在普通小麦中是以多拷贝的形式存在。从中得到17个24kD-α-淀粉酶抑制因子基因,其中2个来自普通小麦与1个来自粗山羊草的基因编码的抑制因子与WDAI-0-19的氨基酸序列完全相同,为同一蛋白。在普通小麦中得到1个编码蛋白质与WDAI-0-53十分相似的基因。序列分析表明,24kD-α-淀粉酶抑制因子成熟蛋白编码基因在序列大小与核酸组成上都十分相似,一致性达到91.2%。这说明小麦和山羊草中24kD-α-淀粉酶抑制因子基因可能起源于相同原始基因。     

Interaction of wheat monomeric and dimeric protein inhibitors with alpha-amylase from yellow mealworm (Tenebrio molitor L. larva).

The highly purified alpha-amylase from Tenebrio molitor L. larva (yellow mealworm) reversibly combines with two closely related homogeneous glycoprotein inhibitors, one dimeric (termed 'inhibitor 0.19') and one monomeric (termed 'inhibitor 0.28'), from wheat flour. As established by means of difference spectroscopy and kinetic studies, molar combining ratios for the amylase--inhibitor-0.19 and amylase-inhibitor-0.28 complexes were 1:1 and 1:2 respectively. Two amylase--inhibitor-0.19 complexes with slightly different retention volumes on Bio-Gel P-300 and only one amylase--inhibitor-0.28 complex were observed. Dissociation constants of the amylase--inhibitor-0.19 and amylase--inhibitor-0.28 complexes were 0.85 nM and 0.13 nM respectively. A strong tendency of both complexes to precipitate under an ultracentrifugal field was observed; the minimum molecular weight calculated for the two complexes under such conditions was approx. 95 000. The two complexes showed difference spectra indicating involvement of structurally related or identical tryptophyl side chains in the binding of inhibitors 0.28 and 0.19 to the amylase. A model summarizing the main features of the inhibition of the insect amylase by the two wheat protein inhibitors is proposed.     

Inhibitory effect of 0.19 alpha-amylase inhibitor from wheat kernel on the activity of porcine pancreas alpha-amylase and its thermal stability

The inhibitory effect of 0.19 alpha-amylase inhibitor (0.19 AI) from wheat kernel on the porcine pancreas alpha-amylase (PPA)-catalyzed hydrolysis of p-nitrophenyl-alpha-D-maltoside (pNP-G2) was examined. 0.19 AI is a homodimer of 26.6 kDa with 13.3-kDa subunits under the conditions used. The elution behaviors in gel filtration HPLC of PPA and 0.19 AI indicated that a PPA molecule bound with a 0.19 AI molecule (homodimer) at a molar ratio of 1:1. 0.19 AI inhibited PPA activity in a competitive manner with an inhibitor constant, K(i), of 57.3 nM at pH 6.9, 30 degrees C, and the binding between them was found to be endothermic and entropy-driven. The activation energy for the thermal inactivation of 0.19 AI was determined to be 87.0 kJ/mol, and the temperature, T(50), giving 50% inactivation in a 30-min incubation at pH 6.9 was 88.1 degrees C. The high inhibitory activity of 0.19 AI against PPA and its high thermal stability suggest its potential for use in the prevention and therapy of obesity and diabetes.     

Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities.

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect alpha-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human alpha-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.     

Phosphate content of muscle phosphofructokinase in the genetically diabetic mouse (C57BL/KsJ)

The amount of phosphofructokinase based on total soluble protein in extracts of skeletal muscle from genetically diabetic mice C57BL/KsJ (db/db) was about 30% lower than that of normal controls (db/m). Organic phosphate content of five control animals varied between 0.11 and 0.19 mol/mol protomer. On the other hand, the phosphate content of diabetic mice had a much broader range (0.11 to 0.39) with a mean content for five animals of 0.24 mol/mol enzyme protomer. Partial resolution of high- and low-phosphate forms can be achieved by ion-exchange chromatography. The more highly phosphorylated enzyme is slightly more sensitive to ATP inhibition than the low-phosphate enzyme.     

Purification and partial characterization of α-Amylase allozymes from the lesser grain borer,Rhyzopertha dominica

α-Amylase was purified from adults of the lesser grain borer, Rhyzopertha dominica (F.), by ammonium sulfate precipitation, glycogen complex formation, and gel filtration chromatography. Specific activity increased from 16 AU/mg protein in the crude extract to 705 AU/mg protein in the final sample (1 AU = 1 mg maltose hydrate/min at 30°C). Two major protein bands, active in starch zymograms, were present at R_m 0.71 and 0.79 when the sample was examined by polyacrylamide gel electrophoresis (PAGE) on 7.5% gels. In addition, several minor proteins that had α-amylase activity were also present. Molecular masses of the two major allozymes were estimated to be 57 and 55 kDa under dissociating conditions. Isoelectric points of the allozymes were at pH 3.4 and 3.5. The amylases were most active at pH 7 and the presence of 20 mM NaCl resulted in a 10.7-fold increase in V_max. K_m for soluble starch was 0.127%.Saline extracts of wheat (“Florida 302”) were 2- and 3-fold more inhibitory on a weight basis towards the amylases from R. dominica than were extracts prepared from two cultivars of triticale, “Morrison” and “CT-4161”, respectively. Interaction of purified α-amylase inhibitors from wheat, inhibitor-0.28 and a sample of the inhibitor-0.19 family of isoinhibitors, with the α-amylases from R. dominica was studied. Complex formation between the amylases and inhibitor-0.28 was demonstrated by PAGE, although the protein-protein complexes that formed were not completely stable during electrophoresis. K_i values were estimated to be 2.6 nM for inhibitor-0.28 and 2.9 nM for inhibitor-0.19. Binding of these inhibitors to α-amylases from R. dominica was not as tight compared with the interaction of these inhibitors with amylases from Sitophilus weevils and Tenebrio molitor.     

Monoclonal antibodies against an alpha-amylase inhibitor from wheat kernel

An alpha-amylase inhibitor (called the 0.53-inhibitor, Maeda, K., Takamori, Y. and Oka, O. (1982) Agric. Biol. Chem. 41, 2873-2875) and the carboxymethylated inhibitor were used to immunize mice (strain BALB/c) according to a procedure described earlier (McMaster, W.R. and Williams, A.F., (1979) Eur. J. Immunol. 9, 426-433). After fusion of spleen cells with NS-1 myeloma cells, three stable clones producing antibodies against the inhibitor were obtained. The binding characteristics of the monoclonal antibodies, AWAI-1, AWAI-2 and AWAI-3, to the inhibitor were analyzed by radioimmunoassay. Two of these monoclonal antibodies to the alpha-amylase inhibitor did not show any binding affinity towards carboxymethylated inhibitor, suggesting that the main antigenic determinant on the native inhibitor is tertiary-structure dependent. The monoclonal antibodies obtained cross-reacted with three other alpha-amylase inhibitors (the 0.19-, the 0.36- and the 0.38-inhibitor) in wheat and these were separated together with the 0.53-inhibitor from the rest of inhibitors by immunoaffinity chromatography. One stable clone producing antibody against the carboxymethylated inhibitor was also established, AWAI-4. The antigenic determinant to this antibody was found to be included in the region of Met(5)-Lys(25) on the carboxymethylated inhibitor.     

A dimeric inhibitor or insect alpha-amylase from barley. Cloning of the cDNA and identification of the protein

A cDNA clone, designated pUP-44, whose longest open reading frame codes for a protein that is homologous to the wheat alpha-amylase inhibitors, has been isolated from a library obtained from developing barley endosperm. The deduced sequence for the mature protein, which is 122 residues long, is preceded by a sequence of 30 residues which has the typical features of a signal peptide. A closely corresponding protein, designated BDAI-1, has been isolated from mature endosperm. BDAI-1 behaves as a dimer and inhibits the alpha-amylase from the insect Tenebrio molitor at concentrations that have no effect on salivary or pancreatic alpha-amylases.     

Further characterization studies of the alpha-amylase protein inhibitor of gel electrophoretic mobility 0.19 from the wheat kernel.

A highly purified amylase protein inhibitor from the kernels of hexaplois wheat, designated 0.19 according to its gel electrophoretic mobility, has been characterized according to its circular dichroism spectra determined at different pH values and in the presence or absence of dissociating and reducing agents. The 0.19 albumin has also been characterized according to the specificity with which it inhibits 21 alpha-amylases from different origins and according to its sensitivity to a number of chemical and enzymatic treatments of its inhibitory action on human saliva and Tenebrio molitor L. larval midgut alpha-amylases. Inhibitory activity of 0.19 toward human saliva amylase significantly increased when the inhibitor was incubated with the enzyme before the addition of starch, but it was not affected by the preincubation of 0.19 with starch. Maltose reversed the inhibition of human saliva by 0.19 and showed some inhibitory activity toward the enzyme. However, maltose concentrations that only slightly affected amylase activity were very effective in restoring the amylase activity inhibited by 0.19. The inhibitory action of 0.19 on human saliva and T. molitor L. amylases were equally resistant to trypsin and thermal treatments, but 0.19 was readily inactivated by incubation with pepsin or by reduction of disulfide bonds. The inhibition of the mammalian amylase by 0.19 was adversely affected by a treatment with CNBr (1:100 ratio of methionine residues to CNBr) whereas the inhibition of the insect amylase was not. As shown by circular dichroism measurements in the far ultraviolet, 0.19 is a protein with about 50% of ordered structure. Significant and largely reversible changes have been observed in the aromatic CD spectrum of 0.19 at alkaline pH values or in the presence of sodium dodecyl sulfate. These changes, which were associated with a partial loss of inhibitory activity, indicate that ionizable tyrosine groups contribute significantly to the ellipticity bands of 0.19 in the near ultraviolet.     

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.     

Proteomic analysis of wheat proteins recognized by IgE antibodies of allergic patients

Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQ(DECA) nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as alpha-amylase inhibitors, beta-amylase, profilin, serpin, beta-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and alpha-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against alpha-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.     

Detection of single nucleotide polymorphisms in 24 kDa dimeric alpha-amylase inhibitors from cultivated wheat and its diploid putative progenitors

Seventeen new genes encoding 24 kDa family dimeric alpha-amylase inhibitors had been characterized from cultivated wheat and its diploid putative progenitors. And the different alpha-amylase inhibitors in this family, which were determined by coding regions single nucleotide polymorphisms (cSNPs) of their genes, were investigated. The amino acid sequences of 24 kDa alpha-amylase inhibitors shared very high coherence (91.2%). It indicated that the dimeric alpha-amylase inhibitors in the 24 kDa family were derived from common ancestral genes by phylogenetic analysis. Eight alpha-amylase inhibitor genes were characterized from one hexaploid wheat variety, and clustered into four subgroups, indicating that the 24 kDa dimeric alpha-amylase inhibitors in cultivated wheat were encoded by multi-gene. Forty-five cSNPs, including 35 transitions and 10 transversions, were found, and resulted in a total of ten amino acid changes. The cSNPs at the first site of a codon cause much more nonsynonymous (92.9%) than synonymous mutations, while nonsynonymous and synonymous mutations were almost equal when the cSNPs were at the third site. It was observed that there was Ile105 instead of Val105 at the active region Val104-Val105-Asp106-Ala107 of the alpha-amylase inhibitor by cSNPs in some inhibitors from Aegilops speltoides, diploid and hexaploid wheats.     

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

Plant alpha-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 alpha-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 alpha-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 alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Some aspects of the mechanism of complexation of red kidney bean alpha-amylase inhibitor and alpha-amylase

Bovine pancreatic alpha-amylase binds 1 mol of acarbose (a carbohydrate alpha-amylase inhibitor) per mol at the active site and also binds acarbose nonspecifically. The red kidney bean alpha-amylase inhibitor-bovine pancreatic alpha-amylase complex retained nonspecific binding for acarbose only. Binding of p-nitrophenyl alpha-D-maltoside to the final complex of red kidney bean alpha-amylase inhibitor and bovine pancreatic alpha-amylase has a beta Ks (Ks') value that is 3.4-fold greater than the Ks (16 mM) of alpha-amylase for p-nitrophenyl alpha-D-maltoside alone. The initial complex of alpha-amylase and inhibitor apparently hydrolyzes this substrate as rapidly as alpha-amylase alone. The complex retains affinity for substrates and competitive inhibitors, which, when present in high concentrations, cause dissociation of the complex. Maltose (0.5 M), a competitive inhibitor of alpha-amylase, caused dissociation of the red kidney bean alpha-amylase inhibitor--alpha-amylase complex. Interaction between red kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor and porcine pancreatic alpha-amylase proceeds through two steps. The first step has a Keq of 3.1 X 10(-5) M. The second step (unimolecular; first order) has a forward rate constant of 3.05 min-1 at pH 6.9 and 30 degrees C. alpha-Amylase inhibitor combines with alpha-amylase, in the presence of p-nitrophenyl alpha-D-maltoside, noncompetitively. On the basis of the data presented, it is likely that alpha-amylase is inactivated by the alpha-amylase inhibitor through a conformational change. A kinetic model, in the presence and absence of substrate, is described for noncompetitive, slow, tight-binding inhibitors that proceed through two steps.     

Purification and characterization of Streptomyces griseus metalloendopeptidases I and II.

Two metalloendopeptidases, designated as Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37,000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease inhibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).     

Assignment of the five disulfide bridges in an alpha-amylase inhibitor from wheat kernel by fast-atom-bombardment mass spectrometry and Edman degradation.

The assignment of the five disulfide bridges in an alpha-amylase monomeric inhibitor from wheat kernel (coded 0.28) was achieved by combining fast-atom-bombardment mass spectrometry (FAB-MS) and automatic sequencing based on Edman degradation. Direct FAB-MS analysis of the native and reduced enzymatic digests of the protein allowed the assignment of three disulfide bridges out of five, including those involving two adjacent cysteine residues. The remaining two disulfide bridges were assigned by sequencing automatically the peptide clusters purified from the tryptic digest of the native protein.     

Pig plasma protease inhibitor gene complex: isolation and partial characterization of three inhibitors

1. Pig plasma alpha-protease inhibitors (protease inhibitor-1, PI1; protease inhibitor-2, PI2; postalbumin-1A, PO1A; postalbumin-1B, PO1B), all encoded by one gene complex (gene cluster), were isolated by rivanol-ammonium sulphate fractionation and double-one dimensional IPG-PAGE. The proteins were recovered from the polyacrylamide gel by a combination of electrophoresis and isoelectric focusing. 2. Molecular wt estimated by SDS-PAGE under reducing conditions was 63,000 each for PI1 and PI2 and 60,000 each for PO1A and PO1B. The two main components of a genetic variant of PI2 differed in mol. wt by approx. 1000. 3. PO1A, PO1B and PI2 were shown to be glycoproteins. The major component of both PO1A and PO1B contained about 15% carbohydrate and the two components of PI2 had about 24 per cent and 21 per cent carbohydrate, respectively. 4. Neuraminidase treatment showed that the main component of PO1A had 8 sialic acid residues and fast and slow components of PI2 had respectively 11 and 10 residues. 5. Amino acid compositions of PO1A, PO1B and PI2 were very similar to one another, indicating that the genes for these proteins have evolved by regional duplications of a common ancestral gene. 6. The results (mol. wt, amino acid and carbohydrate compositions) confirm that pig PI2 is homologous to human plasma alpha 1-antichymotrypsin.     

Polyacrylamide Gel Electrophoresis of Plasteins

A peptic hydrolysate of soybean protein was filtered with Sephadex G–25 and was separated approximately into four fractions (I, II, II, and IV in the order of mol. wt.). Fraction II (av. mol. wt: 1043) and III (av. mol. Wt.: 685) were more plastein-productive than others. When plastein produced from Fraction II with Nagarse was investigated by plate electrophoresis using 7.5% polyacrylamide-gel, the upper limit of the molecular weight was found to be about 25,000. A similar result was obtained also with Fraction III. The increase of molecular weight in the course of the plastein formation with the mixture (substrate) of Fractions II and III was shown that the final product lay mainly in a position between cytochrome c (mol. wt.: 11,700) and Nagarse (mol wt.: 27,600). In addition, the gel-electrophoretic experiments revealed that the most favorable condition for the plastein synthesis were pH 6.5 and 35% in substrate concentration.