Physicochemical Properties and Specific Structural Features of Protein–Lipid Composites of Increased Nutritive Value

Fractional and component compositions of protein–lipid composites with increased nutritive value (compared to the protein preparations from which they were produced) were studied based on solubility and electrophoretic behavior. Differences in the fractional compositions of proteins and the amounts of hydrogen, ionic, and hydrophobic bonds were found. It was demonstrated that the water-, salt-, and alkali-soluble fractions of proteins changed during the manufacturing of the composites with soybean and wheat bran flour; the water- and alkali-soluble fractions, with protein concentrate from bran. Heterogeneity of the compositions and specific conformational features of composite proteins resulting from disulfide bonds were found. It was demonstrated that, during the manufacturing of composites, proteins of soybean flour aggregated (with the involvement of disulfide bonds), whereas protein products from wheat bran disaggregated. Breaks of interchain (wheat) or intrachain (concentrate) disulphide bonds accompanied the disaggregation. Overall the properties and specific structural features of the protein–lipid composites studied depended on the nature of the protein (soybean or wheat), type of initial preparations (flour or concentrate), and method of their production (emulsifying or drying).     

Isolation of 3-Hydroxy-β-ionol from Burley Tobacco

Foxtail millet (Setaria italica) proteins were fractionated into five fractions, i. e., water-, salt-, ethanol-, sodium dodecyl sulfate (SDS)- and 2-mercaptoethanol (ME)-soluble fractions, by successive extraction with various solvents from millet flour. The proportion in each fraction was 7.2, 5.6, 40, 25 and 20% respectively, of total flour nitrogen. The proteins of the ethanol- and SDS-soluble proteins were similar in amino acid composition and molecular weight distribution. More than 15 different molecular weight classes of proteins ranging from 11,000 to 150,000 were distinguished by SDS-polyacrylamide gel electrophoresis without prior reduction of their disulfide bonds. These major protein bands in the gel were estimated to be homo-olygomers (monomer, dimer, trimer, etc.) of subunit A or subunit B. The molecular weights of subunits A and B were 12,000 and 17,000, respectively. Subunits A and B were also different in amino acid composition: subunit A had higher content of methionine.     

Effectiveness of enzyme preparations of fodder in the degradation of nonstarch polysaccharides from grain substrates

The effects of commercial and laboratory preparations were compared in the course of treatment of components of compound fodder. The most potent preparations were selected for the treatment of soybean flower, sunflower meal, and wheat and barley flour. Preparation 181-1008, which had a high proteinase activity, provided the highest yield of protein from soybean flour and sunflower meal. Preparations aGA, AG20X, and VR, characterized by high activities of pectinase and alpha-galactosidase, as well as laboratory preparation B2000Mix with a high activity of alpha-galactosidase, provided the highest yield of sugars from soybean flour. Preparations with high alpha-galactosidase activity were the most potent in hydrolyzing soluble carbohydrates from soybean flour. The highest yield of reducing sugars was observed after treatment of wheat and barley flour with preparations B2000Mix and aGa. Xylanase activity of these preparations was lower than that of preparations 3.130.2 and TG20X. Preparations 3.130.2 and TG20X were the most potent in hydrolyzing wheat middlings.     

Effectiveness of enzyme preparations of fodder in the degradation of nonstarch polysaccharides from grain substrates

The effects of commercial and laboratory preparations were compared in the course of treatment of components of compound fodder. The most potent preparations were selected for the treatment of soybean flower, sunflower meal, and wheat and barley flour. Preparation 181–1008, which had a high proteinase activity, provided the highest yield of protein from soybean flour and sunflower meal. Preparations aGA, AG20X, and VR, characterized by high activities of pectinase and α-galactosidase, as well as laboratory preparation B2000Mix with a high activity of α-galactosidase, provided the highest yield of sugars from soybean flour. Preparations with high α-galactosidase activity were the most potent in hydrolyzing soluble carbohydrates from soybean flour. The highest yield of reducing sugars was observed after treatment of wheat and barley flour with preparations B2000Mix and aGA. Xylanase activity of these preparations was lower than that of preparations 3.130.2 and TG20X. Preparations 3.130.2 and TG20X were the most potent in hydrolyzing wheat middlings.     

Agarose gel filtration of Triticum vulgare proteins in dissociating solvents

Certain wheat proteins (glutenins emerged from 4% agarose columns at the void volumes even in the presence of 6 M guanidine hydrochloride, 4 M urea with or without 1% sodium dodecyl sulphate, and 8 M urea. These proteins were of considerably greater molecular size than bovine thyroglobulin (sub-unit MW 335000). Urea plus sodium dodecyl sulphate was the most effective dissociating solvent. Low MW wheat flour proteins, which had been covalently labelled with a fluorescein derivative, were not incorporated through formation of new disulphide bonds into higher MW fractions during acidic extraction of flour. Limited incorporation through non-covalent association was observed. The results do not support the contention that glutenin is an artifact of extraction. It has been confirmed that all the protein of wheat flour is not extractable with water followed by 2 M urea.     

Immunoblotting detection of lectins in gluten and white rice flour

The gluten lectin was isolated by affinity chromatography, separated by sodium dodecyl sulphate-gel electrophoresis together with purified wheat germ agglutinin (WGA) and electrotransferred to nitrocellulose filters. The binding pattern of anti-WGA to the blotted filters confirmed the presence of WGA in gluten. A lectin from rice bran and white rice flour, respectively, was isolated by affinity chromatography. Both lectins reacted with anti-WA in immunoblotting. As patients with coeliac disease are known to tolerate rice flour, the finding of a WGA-like lectin questioned the suggestion that WGA in gluten is involved in the pathogenesis of coeliac disease. A second lectin was also isolated from rice flour which reacted only with antibodies against soybean lectin on immunoblots. This may indicate a contamination of soybean proteins in rice flour.     

Identity of SDS-insoluble Proteins with Purified Glutenin from Wheat Flour

Sodium dodecyl sulfate (SDS)-insoluble proteins from wheat flour were solubilized by the reduction of their disulfide linkages with 2-meracaptoethanol. The polypeptide compositions of the reduced SDS-insoluble proteins were compared with those of the reduced glutenin by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and amino acid analysis. SDS-polyacrylamide gel electrophoretic patterns of the reduced SDS-insoluble proteins almost coincided with those of the reduced glutenin. Seven major bands (Band 1–7) were obtained from both samples of the reduced proteins. These protein bands were subjected to analysis of amino acid compositions and isoelectric focusing, and similarities between polypeptides of the SDS-insoluble proteins and the glutenin were observed in their amino acid compositions and isoelectric focusing patterns. The results obtained suggested that the preparation of the reduced SDS-insoluble proteins might be used as a simple and rapid method to obtain the glutenin subunits.     

Molecular cloning and characterization of two soybean protein disulfide isomerases as molecular chaperones for seed storage proteins

Protein disulfide isomerase family proteins play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL-1 and GmPDIL-2, respectively. Recombinant versions of GmPDIL-1 and GmPDIL-2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL-1 and GmPDIL-2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon-intron structure across plant species. The promoter sequences of GmPDIL-1 apparently contain a cis-acting regulatory element functionally linked to unfolded protein response. GmPDIL-1, but not GmPDIL-2, expression was induced under endoplasmic reticulum-stress conditions. GmPDIL-1 and GmPDIL-2 promoters contain some predicted regulatory motifs for seed-specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with proglycinin, a precursor of the seed storage protein glycinin, and the alpha'-subunit of beta-conglycinin, a seed storage protein found in cotyledon cells under conditions that disrupt the folding of glycinin or beta-conglycinin, suggesting that GmPDIL-1 and GmPDIL-2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones.     

响应曲面法优化灵芝廉价型深层发酵培养基的研究

为了获得生产用廉价型灵芝发酵培养基,采用中心组合旋转设计法和响应曲面法对低成本培养基组分进行了优化。优化的四个组分为玉米粉(x1)、麸皮粉(x2)、豆饼粉(x3)和蔗糖(x4)。结果表明,灵芝菌体发酵和多糖发酵的培养基预测模型分别为:Y1=15.1–0.31x1–0.34x2+0.36x3–0.44x4–1.26x12–1.98x22–0.85x32–1.15x42–0.59x2x3和Y2=2.0–0.08x1–0.08x2+0.04x3–0.09x4–1.13x12–0.33x22–0.08x32–0.16x42–0.16x2x3–0.10x1x4。从中获得菌体发酵的最优配方为:玉米粉19.7g/L,麸皮粉11.3g/L,豆饼粉6.3g/L,蔗糖19.5g/L;多糖发酵的最优配方为:玉米粉19.6g/L,麸皮粉11.0g/L,豆饼粉6.7g/L,蔗糖19.1g/L。150L发酵罐中试放大结果表明,灵芝菌体的产量为16.92g/L,多糖产量为1.86g/L。所得培养基为灵芝产品的高效低成本生产提供了基础。     

Disulfide bonds of purothionine, a lethal toxin for yeasts.

Purothionin isolated from commercial wheat flour contained several components and two of them (A-I and A-II) were isolated in pure form by CM-52 column chromatography. Each component contained 45 amino acid residues with a 4 disulfide bonds. Purothionin A-II was digested with trypsin and thermolysin to isolate cystine peptides. These were separated and purified by chromatography on an SP-Sephadex column, and paper electrophoresis and chromatography. A peptide containing a -Cys-Cys- sequence was hydrolyzed with 10 N sulfuric acid. Amino acid compositions and partial sequence studies of the cystine peptides and their performic acid-oxidized peptides revealed the positions of all 4 disulfide bonds in purothionin A-II. They were formed between residues 3 and 39, 4 and 31, 12 and 29, and 16 and 25. The results of a partial study of purothionin A-I are also presented.     

Intermediatry steps in Acetobacter xylinum cellulose synthesis: studies with whole cells and cell-free preparations of the wild type and a celluloseless mutant.

Intermediatry steps in cellulose synthesis in Acetobacter xylinum were studied with resting cells and particulate-membranous preparations of the wild-type strain and of a celluloseless mutant. Exogenously supplied [1-14C]glucose was rapidly converted by resting cells of both types into glucose 6-phosphate, glucose 1-phosphate, and uridine glucose 5'-diphosphate (UDP)-glucose and incorporated into lipid-, water-, and alkali-soluble cellular fractions. The decrease in the level of labeled hexose-phosphates and UDP-glucose upon depletion of the exogenous substrate was accounted for by a continuous incorporation of [14C]glucose into cellulose in the wild type and into the above-mentioned cellular components in the mutant. [14C]glucose retained in the alkali- and water-soluble fractions of pulse-labeled wild-type cells was quantitatively chased into cellulose. Sonic extracts of both strains catalyzed the transfer of glucose from UDP-glucose into lipid-, water-, and alkali-soluble materials, as well as into an alkali-insoluble cellulosic beta-1,4-glucan. The results strongly support the sequence glucose leads to glucose 6-phosphate leads to glucose 1-phosphate leads to UDP-glucose leads to cellulose and indicate that lipid- and protein-linked cellodextrins may function as intermediates between UDP-glucose and cellulose in A. xylinum.     

Stable expression of <Emphasis Type="Italic">1Dx5</Emphasis> and <Emphasis Type="Italic">1Dy10</Emphasis> high-molecular-weight glutenin subunit genes in transgenic rye drastically increases the polymeric glutelin fraction in rye flour

We generated and characterized transgenic rye synthesizing substantial amounts of high-molecular-weight glutenin subunits (HMW-GS) from wheat. The unique bread-making characteristic of wheat flour is closely related to the elasticity and extensibility of the gluten proteins stored in the starchy endosperm, particularly the HMW-GS. Rye flour has poor bread-making quality, despite the extensive sequence and structure similarities of wheat and rye HMW-GS. The HMW-GS 1Dx5 and 1Dy10 genes from wheat, known to be associated with good bread-making quality were introduced into a homozygous rye inbred line by the biolistic gene transfer. The transgenic plants, regenerated from immature embryo derived callus cultures were normal, fertile, and transmitted the transgenes stably to the sexual progeny, as shown by Southern blot and SDS-PAGE analysis. Flour proteins were extracted by means of a modified Osborne fractionation from wildtype (L22) as well as transgenic rye expressing 1Dy10 (L26) or 1Dx5 and 1Dy10 (L8) and were quantified by RP-HPLC and GP-HPLC. The amount of transgenic HMW-GS in homozygous rye seeds represented 5.1% (L26) or 16.3% (L8) of the total extracted protein and 17% (L26) or 29% (L8) of the extracted glutelin fraction. The amount of polymerized glutelins was significantly increased in transgenic rye (L26) and more than tripled in transgenic rye (L8) compared to wildtype (L22). Gel permeation HPLC of the un-polymerized fractions revealed that the transgenic rye flours contained a significantly lower proportion of alcohol-soluble oligomeric proteins compared with the non-transgenic flour. The quantitative data indicate that the expression of wheat HMW-GS in rye leads to a high degree of polymerization of transgenic and native storage proteins, probably by formation of intermolecular disulfide bonds. Even -40k secalins, which occur in non-transgenic rye as monomers, are incorporated into these polymeric structures. The combination 1Dx5 + 1Dy10 showed stronger effects than 1Dy10 alone. Our results are the first example of genetic engineering to significantly alter the polymerization and composition of storage proteins in rye. This may be an important step towards improving bread-making properties of rye whilst conserving its superior stress resistance.     

Distinct Distribution of Deoxynivalenol,Nivalenol, and Ergosterol in <Emphasis Type="Italic">Fusarium</Emphasis>-infected Japanese Soft Red Winter Wheat Milling Fractions

The occurrence of mycotoxins in small grain cereals and their retention in final products are serious concerns for food safety. Previously, we investigated the fate of deoxynivalenol and nivalenol in a Japanese soft red winter wheat cultivar during milling and we found that deoxynivalenol and/or nivalenol was readily distributed among flours for human consumption. In the present study, we analyzed the ergosterol concentrations in the milling fractions as an index of fungal biomass to elucidate the relationship between deoxynivalenol/nivalenol accumulation and fungal invasion into the grain, after the in-house validation of an analytical method for quantifying ergosterol in the resulting milling fractions (patent flour, low-grade flour, bran, and shorts). Using three samples with different levels of deoxynivalenol and/or nivalenol contamination, the contents of deoxynivalenol/nivalenol and ergosterol in the resulting milling fractions were evaluated. The concentration of ergosterol was always lowest in patent flour and highest in bran or shorts, indicating that most of the fungi is retained in the outer layers of grain (bran and shorts) even in highly contaminated grain. On the other hand, the concentrations of deoxynivalenol and nivalenol were similar in the low-grade and patent flours and only slightly lower than in the medium-level and high-level contaminated grains. Moreover, the percentage distribution of ergosterol was higher in bran than in other fractions in all cases, which differed from that of deoxynivalenol/nivalenol. This result indicates the diffusion of deoxynivalenol/nivalenol inside the grain that is independent of fungal invasion.     

Accumulation of β-conglycinin in soybean cotyledon through the formation of disulfide bonds between α'- and α-subunits

β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α'- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α'/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions.     

Purification, characterization, and intracellular localization of glycosylated protein disulfide isomerase from wheat grains.

Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably upregulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.     

基因枪法转化小麦谷蛋白基因研究进展

小麦面粉品质的优劣主要取决于麦谷蛋白多聚体结构的组成,谷蛋白多聚体由高分子量谷蛋白亚基(HMW-GS)、低分子量谷蛋白亚基(LMW-GS)和醇溶蛋白以二硫键相互交联构成,其数量和结构特征直接影响面团的粘弹性,所以通过基因工程方法转化优质谷蛋白基因,增加谷蛋白数量,改善谷蛋白多聚体结构组成,进而改良面粉品质的研究逐渐引起国内外的重视,并在近年来取得了重要进展。基因枪法是目前利用基因工程改良小麦品质的主要途径,自1992年以来已在多个研究室取得了较为瞩目的成果,显示了基因工程改良小麦品质的可能性及前景。综述了迄今为止国内外利用基因枪法转化谷蛋白基因改良小麦品质的研究进展,并在受体材料的选择等方面的研究现状作了较为详细的阐述。     

Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein

Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress. The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes, tomato, Arabidopsis, wheat, and cotton. These proteins are involved in signal transduction or stress response systems. Most of the Cys residues in these proteins are conserved, suggesting that they share similar tertiary structures. Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity, implying that posttranslational modifications are important for its function. Therefore, we investigated the posttranslational modifications in EDGP using mass spectrometry. As the result, six disulfide bonds in EDGP were identified: Cys(70)-Cys(158), Cys(84)-Cys(89), Cys(97)-Cys(113), Cys(100)-Cys(108), Cys(201)-Cys(426), and Cys(332)-Cys(378). In addition, the N-terminal glutamine was cyclized into pyroglutamic acid. All four putative glycosylation sites were occupied by N-linked glycans, which have similar masses of m/z 1171. Finally, measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI 9.5) involved only disulfide bonds, N-terminal modification, and glycosylation.     

Structural heterogeneity of chromatin preparations at the level of DNA topology

The structural heterogeneity of calf thymus chromatin preparations was studied at the level of DNA topology by analysing the influence of ethidium bromide on the chromatin viscosity in deproteinizing medium. In 0.7 M NaCl the chromatin was separated into the fractions with linear DNA (3--36% in various preparations) and with supercoiled circular DNA (scc DNA), which differ from each other in their adhesive properties. Reduction of disulfide bonds in residual chromatin protein with 5% mercaptoethanol linearized scc DNA, present in chromatin preparations as nuclear matrix subunits containing some loops of scc DNA on the protein globule.     

Formation of disulfide bonds in proteins and peptides

For many proteins and peptides, disulfide bridges are prerequisite for their proper biological function. Many commercialized proteins are crosslinked by disulfide bridges that increase their resistance to destructive effects of extreme environment used in industrial processes or protect protein-based therapeutics from rapid proteolytic degradation. Manufacturing of these products must take into account oxidative refolding--a formation of native disulfide bonds by specific pairs of cysteines located throughout a sequence of linear protein. This review describes basic and practical aspects of oxidative folding that should be considered while designing and optimizing manufacturing of proteins using chemical synthesis, semi-synthesis and a recombinant expression.     

Effect of cleaning, milling, and baking on deoxynivalenol in wheat

Samples of wheat naturally infected by Fusarium graminearum Schwabe were obtained from mills in Oklahoma, Missouri, Kansas, and Minnesota and fields in Nebraska and Kansas in 1982; they were analyzed for deoxynivalenol (DON). The wheat was milled, and DON was found throughout all the milling fractions (bran, shorts, reduction flour, and break flour). The DON recoveries for each mill run ranged from 90 to 98%. These samples, regardless of DON concentration, also gave similar fractional distributions of DON. The greatest (21 ppm [21 micrograms/g]) concentration of DON was found in the bran, and the smallest (1 ppm) was found in the break flour. Cleaning and milling were not effective in removing DON; DON was not destroyed in the bread baked from the naturally contaminated whole wheat flour, but the effect on its concentration in the samples analyzed varied, the reduction ranging from 19 to 69%. The percent reduction found in the cleaned wheat ranged from 6 to 19%. DON concentrations in the following commercially made breads, caraway rye, seedless rye, and pumpernickel, were 45 ppb (ng/g), 39 ppb, and 0 ppb, respectively. The limits of detection by gas chromatography-mass spectrometry and high-pressure liquid chromatography for DON were 0.5 and 10 ng, respectively.