Soy ß-Conglycinin Peptide Attenuates Obesity and Lipid Abnormalities in Obese Model OLETF Rats.

We previously reported that soy ß-conglycinin (ßCG) improves obesity-induced metabolic abnormalities, but not obesity, in obese model Otsuka Long-Evans Tokushima fatty (OLETF) rats. In the present study, we aimed to investigate the effects of ßCG-derived peptide consumption on obesity and lipid abnormality in OLETF rats. To this end, wild-type Long-Evans Tokushima Otsuka and OLETF rats were provided a normal diet containing 20% casein for four weeks as a control. In addition, we prepared ßCG peptide by enzymatic hydrolysis, and OLETF rats were fed a diet in which half of the casein was replaced by ßCG peptide (ßCG peptide group). Consequently, rats in the ßCG peptide group showed decreased abdominal white adipose tissue weight and lipid content (serum and liver triglycerides, and serum and liver cholesterol) compared to control OLETF rats. Further analysis demonstrated that ßCG peptide consumption decreased lipogenic enzyme activity and increased lipolytic enzyme activity in the liver of OLETF rats. In addition, suppressive effects on both synthesis and absorption of cholesterol were observed in ßCG peptide-fed OLETF rats. These findings suggest that peptidization of ßCG enhanced the anti-obese and hypolipidemic effects of ßCG.

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

Purification and biophysical characterization of an 11S globulin from Wrightia tinctoria exhibiting hemagglutinating activity.

Wrightia tinctoria globulin (WTG), one of the major seed storage proteins, was isolated for the first time from seeds of the medicinal plant. WTG was extracted and purified to homogeneity in two steps using anion-exchange and size-exclusion chromatographies. On an SDS-PAGE gel under non-reducing conditions, a major band of ~56 kDa was observed; under reducing conditions, however, two major polypeptides, one with molecular weight ~32-34 kDa and the other with molecular weight ~22-26 kDa were observed. Intact mass determination by MALDI-TOF supported this observation. The N-terminal amino acid sequence of WTG matched in NCBI database with an expressed sequence tag obtained from the c-DNA of developing embryo m-RNA of Wrightia tinctoria. The EST sequence was further substantiated by partial de novo internal sequencing using MALDI-TOF/TOF. The high sequence homology with seed storage protein 11S globulin confirmed that WTG is a type of 11S globulin. Circular dichroism analysis showed that the secondary structure of WTG consists predominantly of ß-sheets (44.2%) and moderate content of α-helices (10.3%). WTG showed hemagglutinating property indicating that the protein may possess lectin-like activity. WTG was crystallized at 20 Å°C by the vapour diffusion method using PEG 400 as precipitant. The crystals belonged to the orthorhombic space group P212121 with cell dimensions of a=109.9Å, b=113.2Å and c=202.2Å with six molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.2Å under cryocondition. Preliminary structure solution of WTG indicated the possibility of a hexameric assembly in its asymmetric unit.

Amino acid profiles and quality from lotus seed proteins.

BACKGROUND: Protein composition, amino acid profile and nutritional value of the lotus seed and its Osborne fractions were investigated. The seed was rich in protein with 19.85%, and showed well balanced amino acid composition compared with FAO/WHO pattern, Its nutritive properties were similar to those observed in the reference soybean protein. Phenylalanine, tyrosine, leucine and lysine were the limiting amino acids in the seed proteins. The albumin and globulin were the main protein fraction, the amino acid profile and nutritional value were close to the seed protein. RESULTS: Changes in transition temperature and thermal stability were observed through different solvent extractions. Albumin possessed the predominant thermal stability (81.4 °C) followed by globulin (74.49 °C), prolamin (69 °C) and glutelin (65.6 °C). So, solvent compositions influence the profile of AAs and their nutritive value, and aqueous solvent with 0.1 mol L⁻¹ NaCl was an efficient protein solubiliser. CONCLUSION: The results indicated that the extraction processes influenced the lotus seed protein quality and thermal stability. Overall, the study revealed that the lotus seed protein was nutritionally well-balanced protein and might be of significant importance in the formulation of diets for humans.

Isolation of glycinin (11S) from lipid-reduced soybean flour: effect of processing conditions on yields and purity.

Defatted soybean flour was treated with hexane and ethanol to reduce lipid content and heated to inactivate lipoxygenase (LOX, linoleate:oxygen reductase; EC 1.13.11.12) to obtain lipid-reduced soybean flour (LRSF). The effects of processing conditions such as pH, reducing agent and storage time on yields and purity of glycinin (11S) were evaluated in the fractionation of soybean glycinin isolated from LRSF. Adjusting the pH of protein extract from 6.2 to 6.6, the yield of glycinin decreased by 16.71%, while the purity of the protein increased by 4.60%. Sulfhydryl and disulfide content of proteins increased by degrees with increasing pH. Compared with dithiothreitol (DTT) or ß-mercaptoethanol (ME) as reducing agent, the yield of glycinin was the highest when sodium bisulfite (SBS) was added to the protein extract at pH 6.4. The effect of DTT on yields of glycinin was the lowest of the three kinds of reducing agent. The purity of glycinin was similar when the three kinds of reducing agent were used. These results showed that SBS was the best choice for the isolation of 11S-rich fraction. Prolonging storage time in the precipitation stage, 10 h was the best for yields and purity of glycinin in the experiment, while there was no significant difference at P ≥ 0.05 for total sulfhydryl and disulfide content. The decreased free sulfhydryl content of glycinin indicated that the oxidation of free sulfhydryls and the formation of disulfide bonds occurred when the extraction time was prolonged.

Soybean glycinin improves HDL-C and suppresses the effects of rosuvastatin on hypercholesterolemic rats.

BACKGROUND: This study was an investigation of the effects of ingesting a daily dose of isolated glycinin soy protein (11S globulin), in association with rosuvastatin, on the control of hypercholesterolemia in experimental animals. METHODS: Male Wistar rats were kept in individual cages under appropriate controlled conditions of temperature, light and humidity. The animals were divided into five groups (n = 9): 1) standard (STD): fed on casein as protein source; 2) hypercholesterolemic (HC): STD plus 1% cholesterol and 0.5% cholic acid; 3) HC+11S: hypercholesterolemic + glycinin (300 mg/kg/day); 4) HC+ROS: hypercholesterolemic + rosuvastatin (10 mg/kg/day); 5) HC+11S+ROS: HC diet, the 11S protein and the drug in the doses given in (3) and (4). The protein and the drug were administered by gavage for 28 days. The results indicated that the addition of 1% cholesterol and 0.5% cholic acid induced hypercholesterolemia in the animals without interfering with their weight gain. RESULTS: A single daily dose of glycinin contributed an additional 2.8% of dietary protein intake and demonstrated its functional role, particularly in raising HDL-C, decreasing triglycerides in the liver and improving the atherogenic index in animals exposed to a hypercholesterolemic diet. CONCLUSION: Most of the beneficial effects of the isolated treatments disappeared when the drug (rosuvastatin) and the protein (glycinin) were taken simultaneously. The association was shown not to interact additively, as noted in the plasma levels of total cholesterol and non-HDL cholesterol, and in the significant increase of cholesterol in the liver. Studies are in progress to identify the effects of peptides derived from the 11S globulin and their role in cholesterol metabolism.

Effects of calcium and pressure treatment on thermal gelation of soybean protein.

The effect of calcium and high-pressure (HP) treatment on the heat gelation of soybean proteins was investigated. In the presence of calcium (2 to 25 mM), the gelation of dispersions of soybean protein isolate (SPI), a beta-conglycinin-enriched fraction (7SEF), and a glycinin-enriched fraction (11SEF) started with protein having a lower degree of denaturation. The gels from these dispersions had greater stiffness than the samples without added calcium. HP treatment had different effects on heat-induced gelation depending on the presence of calcium and on the nature of the proteins. In the absence of calcium, gels with low stiffness were formed after HP treatment, compared with untreated samples, and regardless of the sample type (SPI, 7SEF, 11SEF). In the presence of calcium, gel stiffness was increased after HP treatment of dispersions containing beta-conglycinin (SPI and 7SEF), while the opposite effect was observed for 11SEF. In the presence of calcium, HP treatment promoted a greater contribution of hydrophobic interactions in SPI and 7SEF. In the dispersions containing beta-conglycinin, these conditions also promoted the appearance of a heterogeneous distribution of molecular sizes, from enormous aggregates to dissociated species. Our results suggest that, in the presence of calcium, HP treatment has an opposite effect on the ability of glycinin and beta-conglycinin to participate in the formation of a 3-dimensional network upon heating.

Extraction, fractionation and characterization of bitter melon seed proteins.

Protein fractions (water-soluble/albumin, salt-soluble/globulin, alkali-soluble/glutelin, and alcohol-soluble/prolamin) were extracted from defatted ripe bitter melon seed (Momordica charantia) using water, 1 M sodium chloride solution, alkali/pH 11.0, and 70% ethanol, sequentially. The main protein fraction was albumin (49.3%), followed by globulin (29.3%) and glutelin (3.1%). No prolamin was detected, and 18.3% of the protein was nonextractable. The surface hydrophobicities of albumin, globulin, and glutelin were 757, 1,034, and 292, respectively. The molecular sizes of all the fractions were mostly about 45 and 55 kDa. The denaturation temperatures of albumin, globulin, and glutelin were 111.9, 117.3, and 133.6 degrees C, respectively. The levels of all essential amino acids in the bitter melon protein fractions met the minimum requirements for preschool children (FAO/WHO/UNU) with the exception of Thr. Bitter melon protein fractions with unique protein profiles and higher denaturation temperatures could impart novel characteristics when used as food ingredients.

Effect of protein hydrolysates from germinated soybean on cancerous cells of the human cervix: an in vitro study.

Consumption of soybeans can reduce the risk of different types of cancer. Little is known about the effect of germination on the anticancer properties of soya. This study was done to determine if germination improves the anticancer properties of soybean protein through generation of amino acids or bioactive peptides. Soybean was germinated for 0, 2, 3, 4, 5, and 6 days and proteins were isolated from the seeds. Isolates with and without ethanol-soluble phytochemicals were hydrolyzed with digestive enzymes and their effect on growth in HeLa and C-33 (epidermoid cervical carcinoma) and HaCaT (non-cancerous human keratinocytes) cells were evaluated with the Alamar Blue method. Germination induced degradation of the alpha and alpha' fractions of beta-conglycinin and acid fraction of glycinin, generating low molecular weight peptides. Degrees of hydrolysis ranged from 73-77%. Hydrolysates inhibited the growth of HeLa cells and C-33 at concentrations exceeding 1.25 mg/ml. Major inhibition was observed with the hydrolysate germinated for 2 days and containing ethanolsoluble phytochemicals (IC(50) 2.15 and 2.27 mg/ml for HeLa and C-33, respectively). Interestingly, hydrolysate cytoxicity for normal cells was minimal in comparison to cancer cells.

Purification, crystallization and initial crystallographic characterization of the Ginkgo biloba 11S seed globulin ginnacin.

Ginkgo biloba, a well known ;living fossil' native to China, is grown worldwide as an ornamental shade plant. Medicinal and nutritional uses of G. biloba in Asia have a long history. However, ginkgo seed proteins have not been well studied at the biochemical and molecular level. In this study, the G. biloba 11S seed storage protein ginnacin was purified by sequential anion-exchange and gel-filtration chromatography. A crystallization screen was performed and well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained. There are six protomers in an asymmetric unit. Structure refinement is currently in progress.

Mungbean [Vigna radiata (L.) Wilczek] globulins: purification and characterization.

Vicilin type (8S) and basic 7S globulins and legumin type (11S) globulins were isolated from mungbean [Vigna radiata (L.) Wilczek]. The native molecular weights of the different globulin types were 360000 for legumin, 200000 for vicilin, and 135000 for basic 7S. Some of the 8S globulin apparently complexed and coeluted with the 11S on gel filtration. On SDS-PAGE, 11S was composed of two bands of 40000 and 24000, 8S was composed of 60000, 48000, 32000, and 26000 bands, and basic 7S was composed of 28000 and 16000 bands. The percent composition of total globulins was estimated to be as follow: 8S, 89%; basic 7S, 3.4%; and 11S, 7.6%. The basic 7S and 11S but not the 8S globulins were found to have disulfide bonds. The presence of carbohydrates by conjugated peroxidase reaction was observed in all bands of 8S, the acidic polypeptide of basic 7S, and its complex but not in 11S. The 28000 basic 7S band and its 42000 complex and the first three major bands of 8S cross-reacted with antibodies to all types of soybean conglycinin subunits (alpha, alpha', and beta), whereas the fourth band cross-reacted only with the anti-beta subunit. None of the mungbean globulins cross-reacted with anti-soybean glycinin. Basic 7S was found to be easily extracted with 0.15 M NaCl, 11S was extracted with 0.35 M NaCl,and 8S was extracted over a wide range of NaCl concentrations. The N-terminal sequences of the different subunits/fragments of the globulins were determined and found to have strong homology with storage proteins of other legumes and crops.

Isolation and characterization of the genes up-regulated in isolated neurons by aged garlic extract (AGE).

Aged garlic extract (AGE) produces neurotrophic effects on cultured fetal rat hippocampal neurons. These studies examined the molecular events triggered by AGE that might account for a suppression of neuronal cell death. Genes differentially expressed by the addition of AGE in primary cultured hippocampal neurons isolated from fetal rat brain were screened using mRNA differential display. Four cDNA clones were significantly enhanced at their transcriptional level; they were designated as #24, #110, #153 and #155. Quantitative reverse transcription-polymerase chain reaction (RT-PCR), as well as dot-blot hybridization combined with RT-PCR, confirmed that the transcription from these four genes was elevated at least twofold, particularly the mRNA of #153, which was increased >20 times 72 h after the addition of AGE. A homology search of the respective cDNA sequences in the DNA database revealed that #153 is an alpha 2-microglobulin-related protein (alpha 2MRP) gene. The others genes were not identified. Induction of the alpha 2MRP gene expression occurred within 24 h after addition of AGE. These findings suggest a possible mechanism by which AGE may regulate gene expression and bring about a neurotrophic effect. Further, our results suggest that alpha 2MRP may function at the initial step of the molecular events triggered by AGE and play an important role in the survival of hippocampal neurons.

Use of a single method in the extraction of the seed storage globulins from several legume species. Application to analyse structural comparisons within the major classes of globulins.

In this study, a single, improved methodology was used to extract, fractionate and purify the 11S (legumin-type or related to the alpha-conglutin from Lupinus albus L.), 7S (vicilin-type or related to the beta-conglutin from L. albus) and 2S (related to the gamma-conglutin from L. albus) families of proteins from eight legume species: L. albus, Glycine max (L.) Merr., Pisum sativum L., Vicia faba L., Cicer arietinum L., Phaseolus vulgaris L., Lens culinaris Med. and Arachis hypogaea L. The sedimentation coefficients obtained varied from 1.9 to 8.1 for the gamma-conglutin-related proteins, from 5.1 to 10.5 for the beta-conglutin-related proteins and from 12.0 to 14.9 for the alpha-conglutin-related globulins. The gamma-conglutin-related proteins is the most heterogeneous group. Antibodies produced against each type of gamma-conglutin polypeptide chain recognize the other polypeptide chain as well as other polypeptides in the corresponding globulins from all species examined. The 7S globulins are typically composed of a large number of polypeptides, covering a wide range of molecular masses (10 to 70 kD). The presence of disulphide bonds is apparently absent and the occurrence of glycopolypeptides is not widespread. Finally, the 11S globulins are characteristically formed by a limited number of polypeptides that may be divided into a lighter group (20-25 kD) and a heavier group (35-50 kD). The presence of disulphide bonds is apparently widespread but the occurrence of glycopolypeptides seems to be relatively rare. Both the 7S family and the 11S globulins studied by immunoblotting exhibit a low level of structural similarity.

Purification, characterization, and crystallization of single molecular species of beta-conglycinin from soybean seeds.

Four major molecular species of beta-conglycinin, alpha 3, alpha 2 beta, alpha beta 2, and beta 3, were isolated and purified from seeds of an alpha' subunit-deficient strain of soybeans (Glycine max). All components were found to be homogeneous by high pressure liquid chromatography, SDS-polyacrylamide gel electrophoresis, and amino acid and amino terminal sequence analyses. The amino acid compositions of the alpha 3 and beta 3 components agreed fairly well with the compositions deduced from the cDNA sequences, and all of the components were highly glycosylated. The alpha 3 and beta 3 components were compared regarding their secondary structures. The secondary structure of the alpha 3 component deduced from CD measurements showed a higher alpha-helix content than that of the beta 3 component. The beta 3 component was crystallized by decreasing the ionic strength from 0.5 to 0.14 in phosphate buffer, pH 7.3, and the crystals grew to a size (1.0 mm x 0.2 mm x 0.2 mm) suitable for X-ray crystallographic analysis. A preliminary X-ray analysis showed that the crystal belonged to an orthorhombic crystal system having the space group P2(1)2(1)2(1) and unit cell dimensions of a = 185.1 A, b = 107.9 A, and c = 97.6 A.

A corm-specific gene encodes tarin, a major globulin of taro (Colocasia esculenta L. Schott).

A gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (G1, ca. 28 kDa) was isolated from a lambda Charon 35 library, using a cDNA derived from a highly abundant corm-specific mRNA, as probe. The gene, named tar1, and the corresponding cDNA were characterized and compared. No introns were found. The major transcription start site was determined by primer extension analysis. The gene has an open reading frame (ORF) of 765 bp, and the deduced amino acid sequence indicated a precursor polypeptide of 255 residues that is post-translationally processed into two subunits of about 12.5 kDa each. The deduced protein is 45% homologous to curculin, a sweet-tasting protein found in the fruit pulp of Curculigo latifolia and 40% homologous to a mannose-binding lectin from Galanthus nivalis. Significant similarity was also found at the nucleic acid sequence level with genes encoding lectins from plant species of the Amaryllidaceae and Lilliaceae families.

Narbonin, a novel 2S protein from Vicia narbonensis L. seeds: cDNA, gene structure and developmentally regulated formation.

cDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced. The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (M(r) ca. 33 100) and an initiation methionine. The deduced amino acid sequence lacks a transient N-terminal signal peptide. The genomic clones do not contain any intron. No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries. The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins. However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein. Southern analysis suggests the existence of a small narbonin gene family. Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max. In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds.

Identification of soybean protein components that modulate the action of insulin in vitro.

We have reported that a 37-kDa protein from a soybean isolate shows an affinity for bile acids and modulates insulin action on fat decomposition in vitro [Makino et al., Agric. Biol. Chem., 52, 803 (1988)]. In this study, the major components of the protein were identified as the acidic A1a and A2 subunits of glycinin, via amino-terminal sequence analyses of purified proteins and examination of their effects on fat decomposition in rat adipose cells. The most hydrophobic region of the subunits was found to be responsible for the bile acid-binding ability, and the binding region probably does not contribute to the insulin-modulating activity. These bile acid-binding and insulin-modulating properties were also noted in a 40-kDa protein from pea seeds, probably acidic subunits of legumin, suggesting that these characteristics may be common to legume proteins.

Studies on groundnut proteins. VI. Isolation, characterisation and hydrogen ion titration of conarachin II.

A method is described for isolating conarachin II in a homogeneous form by the techniques of DEAE-cellulose chromatography, polyacrylamide gel electrophoresis and sedimentation velocity. The protein contains 0.72% carbohydrate and no phosphate. Hydrogen ion titration curve indicated that the sidechain carboxyl, imidazol and epsilon-amino groups titrated with normal pK Int values and their number agreed with the analytical values obtained from amino acid analysis. However, tyrosine phenolic groups had abnormal pK Int of 10.5.

The isolation of allergens from the green pea.

The aqueous extract of green peas was separated into 3 fractions (albumin, legumin, and vicilin) by dialysis against distilled water and isoelectric precipitation. The major antigenic and all of the allergenic activity of the pea extract was associated with the albumin fraction. The albumin fraction retains its allergenicity upon heating at 60 degrees C for 30 min or boiling at 100 degrees C for 5 min, but becomes partially inactivated by autoclaving at 120 degrees C for 15 min. The allergenic determinant expressed by the albumin fraction appears to be common to several other members of the legume family. In addition, the pea dialysate fraction was shown to specifically inhibit precipitin and passive cutaneous anaphylaxis (PCA) reactions involving rabbit antipea serum and the pea albumin fraction, and histamine release from passively sensitized monkey lung tissue using the serum of pea-sensitive patients.