ACE inhibitory tetrapeptides from Amaranthus hypochondriacus 11S globulin

Amaranth seed is a valuable source of dietary protein with very high nutritional quality, and recently its potential as a nutraceutical has been proposed. The aim of this work was to provide experimental evidence for the presence of anti-hypertensive peptides in globulin 11S, one of the major constituents of the seed, by means of an in-silico based peptide library screening method. A three-dimensional model of globulin 11S was built, upon which anti-hypertensive peptides were mapped via a database-driven method. Solvent accessibility was evaluated for each potential peptide, and two potent and exposed tripeptides were detected: IKP and LEP. An N-terminal extension of these two peptides was built using the globulin 11S primary sequence information, and ACE inhibitory behaviour was simulated by automated ligand-protein docking. The occurrence of two inhibitory tetrapeptides, ALEP and VIKP, was predicted and experimentally validated by an in vitro ACE inhibition assay that showed IC50 values of 6.32 mM and 175 μM, respectively. This study is the first to provide experimental proof of the anti-hypertensive value of Amaranth. Furthermore, this is the first time that a peptide docking approach is used to find ACE-inhibitory peptides from a food protein source.     

Small-angle X-ray scattering studies of 7S and 11S globulins from pea (Pisum sativum)

Small-angle X-ray scattering studies have been conducted on solutions of 11S and 7S globulins isolated from peas (Pisum sativum cv. Filby), and the radii of gyration and molecular weights determined. The general features of the scattering curves were similar to those reported for other seed storage proteins.     

In vitro Culture of Immature Cotyledons of Soya Bean (Glycine max L. Merr.)

An in vitro procedure promoting the rapid growth and proteinincrease of soya bean cotyledons has been developed. The amountof protein synthesized varied greatly depending on the nitrogen(N) source provided. Glutamine was the most effective N source,while inorganic forms of N were ineffective. Growth and proteinsynthesis were both more rapid in vitro than in vivo. Underthe best conditions, soya bean cotyledons increased 8-fold bothin dry weight and in protein in 6 days. The formation of the7S and 11S storage proteins in vitro was similar to that invivo. Hence, this in vitro culture method is appropriate forstudying legume seed storage protein synthesis under controlledconditions.     

Crystal structure of soybean proglycinin A1aB1b homotrimer

Soybean glycinin is a member of the 11 S globulin family. The crystal structure of proglycinin was determined by X-ray crystallography at 2.8 A resolution with an R-factor of 0.199 and a free R-factor of 0.250. A trimer molecule was found in an asymmetric unit of crystals. The trimer model contains three A1aB1b subunits and comprises 1128 amino acid residues and 34 water molecules. The constituent protomers of the homo-trimeric protein are arranged around a 3-fold symmetry axis with dimensions of 95 Ax95 Ax40 A. The protomer model is composed of five fragments which correspond roughly to conserved regions based on the sequence alignment of various 11 S globulins. The core of the protomer consists of two jelly-roll beta-barrels and two extended helix domains. This structure of proglycinin is similar to those of canavalin and phaseolin belonging to the 7 S globulin family, strongly supporting the hypothesis that both 7 S and 11 S globulins are derived from a common ancestor. The inter and intra-chain disulfide bonds conserved in the 11 S globulin family are clearly observed. It is found that the face with the inter-chain disulfide bond (IE face) contains more hydrophobic residues than that with the intra-chain disulfide bond. This suggests that a mature hexamer is formed by the interaction between the IE faces after processing.     

Experience in the use of synchrotron radiation for the x-ray study of biopolymers]

The results of methodical work, carried out on the sources of synchrotron radiation (SR) with the aim of using SR as a powerful source of X-rays for studying biopolymer structure, are presented. The questions of monochromatization are considered. The technique designed for photoregistration of diffraction patterns within the wide range of scattering angles is described. X-ray diffraction patterns of feather ceratin, collagen and striated muscle are obtained with exposure periods ten times less than those in the case of X-ray tubes. The high resolution of diffraction lines, the absence of parasitic phone and the presence of reflections within the wide range of scattering angles are characteristic of these patterns.     

Soya food intake and risk of endometrial cancer among Chinese women in Shanghai: population based case-control study

Objective To evaluate the association of intake of soya food, a rich source of phytoestrogens, with the risk of endometrial cancer.Design Population based case-control study, with detailed information on usual soya food intake over the past five years collected by face to face interview using a food frequency questionnaire.Setting Urban Shanghai, China.Participants 832 incident cases of endometrial cancer in women aged of 30 to 69 years diagnosed during 1997-2001 and identified from the Shanghai Cancer Registry; 846 control women frequency matched to cases on age and randomly selected from the Shanghai Residential Registry.Main outcome measures Odds ratios for risk of endometrial cancer in women with different intakes of soya foods.Results Regular consumption of soya foods, measured as amount of either soya protein or soya isoflavones, was inversely associated with the risk of endometrial cancer. Compared with women with the lowest quarter of intake, the adjusted odds ratio of endometrial cancer was reduced from 0.93 to 0.85 and 0.67 with increasing quarter of soya protein intake (P for trend 0.01). A similar inverse association was observed for soya isoflavones and soya fibre intake. The inverse association seemed to be more pronounced among women with high body mass index and waist:hip ratio.Conclusion Regular intake of soya foods is associated with a reduced risk of endometrial cancer.     

Structure of 7S seed globulin from Phaseolus vulgaris L. in solution

The structure of the 7S globulin from Phaseoulus vulgaris L in dilatue solutions has been studied by small angle X-ray scattering (SAXS), by quasi-elastic light scattering (Q ELS), by circular dichroism spectroscopy (c.d.), and by precise density measurements. The molar mass, the radius of gyration, the volume, the maximum dimension and the diffusion coefficient were determined as M = 1.45 × 10⁵ g mol⁻¹, R_G = 4.05 nm, V = 300- nm³, L = 13.0 nm and D_20,w⁰ = 4.5 × 10⁻⁷ cm² s⁻¹, respectively. The molecule has an asymmetrical shape with the dimensions 12.5 × 12.5 × 3.75 nm. The secondary structure of the 7S globulin is characterized by a small portion of -helical structure (14%) and a marked content of β-structure (18%).     

发酵法精制大豆低聚糖的研究

研究了三种面包酵母对大豆低聚糖碳源利用的选择性。结果表明 :面包酵母C可选择性地利用蔗糖 ,而水苏糖和棉子糖的保留率大于 96 %;通过添加酵母膏 ,经过 3 6h培养 ,面包酵母C可全部利用大豆低聚糖中的蔗糖。进一步研究表明 ,以大豆乳清废糖浆为原料直接发酵再经下游处理 ,可得到蔗糖含量低于 1 3 %的精制大豆低聚糖干粉。     

蛋白酶抑制剂对含水生菜种子耐冻性的影响

选取生菜(Lactuca sativa)种子作为试材,外源添加蛋白酶抑制剂2-硝基苯甲酸[5,5'-Dithiobis-(2-nitrobenzoic acid),DTNB]对种子吸胀处理,通过程序降温,分析2-硝基苯甲酸对低温下种子发芽率及生理活性的影响。结果表明,低温下含水生菜种子的致死温度为–20 ℃;外源添加2-硝基苯甲酸2 mmol·L–1时种子发芽率最高,即对种子活性的保护效果显著;在此浓度下种子内SOD活性比对照提高1.38倍,羟基自由基清除能力提高1.17倍;与对照组相比产生两种新的蛋白11 S种子贮藏球蛋白Jug r4和11 S种子贮藏球蛋白2,均属于球蛋白家族,可提高含水种子的耐冻性;低温下种子内积累更小分子量的球蛋白多肽,对种子具有低温保护效果。综上,低温条件下生菜种子产生一定的抗冷反应,外源添加2 mmol·L–1 2-硝基苯甲酸可提高含水种子发芽率及生理活性,产生抗冻蛋白,积累更小分子量的球蛋白多肽进而提高种子抗冻性。     

Production of Yeast Cells from Methanol

Using immunochemical technique thermal denaturation of soybean 11S globulin, dissolved in different ionic strength solutions (µ=0~4.0) and heated at 100°C for 5 min, has been quantitatively studied. The curves of the percentage of antigenicity remaining were obtained as a function of salt concentration. The 11S globulin became strongly resistant to thermal denaturation with increasing both KCl and potassium phosphate. The stabilizing effect (in terms of percent antigenicity) was separated into three regions. At ionic strength below 0.7, potassium phosphate had no stabilizing effect while KCl had aslightly effect. The rise in stabilizing effect up to about 50%, near 1.0~1.5 µ, represented a second transition to a different denatured state which retains undissociated molecule. At rises up to 75~95%, near 2.5~3.5µ, a different conformational state resulted in which thermally denatured 11S globulin maintained almost intact native conformation after heating. The selection of an adequate ionic strength of protein solution has enabled preparation of thermally denatured 11S globulins which have desired-residual amounts of structured regions.     

Identification of the 7S and 11S globulins as percutaneously sensitizing soybean allergens as demonstrated through epidermal application of crude soybean extract

Cutaneous exposure to food allergens can predispose individuals to food allergies. Soybean, a major allergenic food, is an ingredient in various cosmetic products. However, the types of soybean proteins that are percutaneously sensitizing in humans or animal models remain unknown. In this study, BALB/c mice were dorsally shaved and epicutaneously exposed to a crude soybean extract including sodium dodecyl sulfate or distilled water alone. Specific IgEs secreted in response to 7S globulin (Gly m 5), 11S globulin (Gly m 6), Gly m 3, and Gly m 4 were measured using enzyme-linked immunosorbent assays or immunoblots. Exposure to soybean extract elicited the secretion of soybean-specific IgEs. Of the soybean proteins, 7S and 11S globulins acted as percutaneous sensitizers in 6/9 mice (67%). Additionally, IgE bound specifically and preferentially to the 7S globulin β subunit. In conclusion, this is the first report to identify percutaneously sensitizing soybean allergens in a mouse model.     

X-ray scattering by single-headed heavy meromyosin. Cleavage of the myosin head from the rod does not change its shape

Low angle X-ray scattering from heavy meromyosin (HMM) and from single-headed heavy meromyosin (sHMM) have been examined to determine if the heads of myosin change shape when cleaved from the rod to form subfragment 1 (S1). The scattering intensities of intact HMM and sHMM were compared with those of their chymotryptic digestion products, S1 and subfragment 2 (S2). As the data with HMM were complicated by scattering between the two heads, the more extensive analysis was done with sHMM. Pseudo-Guinier plots of intact and digested sHMM, over the angular range used previously for S1, were linear and showed a difference in apparent radius of gyration (Rg) of only 0.07 +/- 0.04 nm. The absolute apparent Rg value of sHMM was 3.2 +/- 0.2 nm, which is comparable to the radius of gyration reported previously for S1 alone. A plot of the fractional differences in scattering intensities of intact and digested sHMM was flat to a reciprocal spacing of at least 1/3.5 nm-1. These results indicate that the head portions of sHMM and S1 have very similar structures at low resolution. Scattering curves for various models of sHMM and mixtures of S1 and S2 were calculated and the fractional difference plots of scattering intensities were made to determine how sensitive this type of analysis is to changes in the shape of the head. Changes in Rg of 0.1 nm or greater gave detectably non-flat difference plots. Thus, the X-ray scattering of sHMM (and HMM) demonstrated that differences in structure between the head of myosin and isolated S1 are likely to be small. Current controversies over myosin head structure are discussed in light of this result.     

Acetylation of Amino Groups and Its Effect on the Structure of Soybean Glycinin

Purified soybean glycinin (11S globulin) was acetylated at three degrees; low (21-51%), middle (60-81%) and high (90-92%) acetylation in lysine residues. With increasing the acetyl content, the β-structure gradually decreased and the random structure increased resulting in the exposure of tyrosine residue. These were determined from the results of optical rotatory dispersion, intrinsic viscosity, ultraviolet and fluorescence measurement. Gel filtration, ultracentrifugation and gel electrophoresis studies showed drastic conformational changes of highly acetylated 11S (over 90%), in which most of the modified protein (75%) polymerized, and the other dissociated into 3S protein. The close relation between the conformation of acetylated 11S and its emulsifying properties was discussed.     

Emergence of Proteases in Germinating Cucumber Cotyledons and Their Roles in the Two-Step Degradation of Storage Protein

The degradation of storage protein in germinating cucumber seedswas shown to proceed via two distinct steps. First, severalproteases with acidic isoelectric points (pIs) were involvedin solubilization and partial degradation of 11S globulin. Treatmentof seedlings with cycloheximide inhibited this step and theexpression of these proteases. Thus, the first step appearedto be governed by these proteases, which were synthesized denovo after imbibition. The first step was observed in dark-growncotyledons, but the complete degradation of 11S globulin didnot occur in the absence of illumination. An additional protease,with a pI of 4.5, was induced by illumination, and it was involvedin the further cleavage of the partially degraded products ofIIS globulin. Thus, the complete degradation of the storageprotein proceeded via a two-step process in illuminated germinatingseedlings. Light is needed to induce the second step in thedegradation of 11S globulin that supplies the nitrogen requiredfor development of the photosynthetic apparatus in the greeningcotyledon. (Received November 9, 1995; Accepted January 31, 1996)     

Small-angle X-ray scattering study of ribosomal proteins S3, S4 S7 and s20.

Ribosomal proteins S3, S4, S7, S20 from Escherichia coli have been studied by small-angle X-ray scattering techniques. The molecular weights found by X-ray scattering agree with other determinations. The large values of the radii of gyration indicate anisometric particles. A more detailed morphological analysis is hindered by low solubility. An interpretation of the experimental results is given in terms of compact objects of simple shape. Yet the possibility is envisaged that these proteins may be less rigid and compact than other proteins.     

Isolation of legumin-like protein from Phaseolus aureus and Phaseolus vulgaris

An 11S seed globulin has been isolated from Phaseolus aureus and P. vulgaris by zonal isoelectric precipitation and the MWs of the constituent subunits determined. The protein of P. vulgaris occurs in the protein body fraction and its chemical composition, including the N-terminal amino acids and amino acid composition has been determined. The similarity between the 11S globulin of the two Phaseolus spp. and legumin from other leguines is discussed.     

Imaging plate and its application to X-ray diffraction of muscle

The excellent performance of the IP as an integrating X-ray area detector makes it well suited to X-ray diffraction and scattering experiments using synchrotron radiation. The IP is particularly useful for biological specimens for which the shortest exposure time or the smallest amount of X-ray dose possible is required. It is also useful for time-resolved measurements of an X-ray diffraction pattern, to complement its uses in static measurements.A combination of two powerful tools, synchrotron radiation and imaging plates, has mutually enhanced the potentials of both. The IP may replace conventional X-ray film and some other X-ray detectors which have been conventionally used in many of the application fields of synchrotron radiation. As examples, it has recently proved to be very promising in experiments of X-ray diffraction under high pressure and high temperature (12) DEXAFS and X-ray diffuse scattering. X-ray microscopy will also benefit from the IP when the spatial resolution is improved to a few tens of microns in FWHM.When more intense X-rays are available from insertion devices installed in planned 6–8 GeV storage rings, the IP system will play a more important role as one of the best X-ray area detectors because of its high DQE and the lack of any instantaneous count-rate limitations.     

Structural studies on the 30 S ribosomal subunit from Escherichia coli

Small-angle X-ray scattering curves computed for various 30 S subunit structures have been compared with the experimental scattering curve. The curve from the 30 S subunit is best approximated by that calculated for a 1:3.6:3.6 ellipsoidal structure. The rather prolate ellipsoidal model suggested by recent electron microscope studies gives a scattering curve considerably different from the 30 S curve, suggesting that the electron microscope model is not that found in solution. Analysis of the more extended portions of the experimental scattering curve suggests some internal structure. A model is proposed that contains RNA and protein in positions such that the calculated scattering curve shows more extensive, yet similar internal structure. Resultant constraints on the structure of the 30 S subunit in solution are given.     

Rapid production of ethanol in high concentration by immobilized cells of Saccharomyces cerevisiae through soya flour supplementation

Summary Ethanol concentration and fermentation productivities were substantially increased when soya extract was added to the fermentation medium using immobilized cells of a locally isolated strain of S. cerevisiae. Very high concentrations, 152 and 162 g/l of ethanol, were obtained from a medium containing 300 and 350 g/l sugars respectively by supplementing the medium with soya extract. The fermentation time was also reduced by more than 50%.     

An investigation of the dissociation and denaturation of legumin by salts using laser light scattering and circular dichroism spectroscopy

The dissociation of legumin, a 12 S seed storage globulin from Pisum sativum, has been studied by laser light scattering and circular dichroism spectroscopy. Salts from the Hofmeister series, in particular sodium perchlorate, were used as dissociating agents. The Mr 360,000 hexameric protein was found to dissociate first to trimers and further to monomers and the number of amino acids involved in the trimer-trimer interaction estimated to be 23(+/-4). Native legumin appears to be more strongly bound together than some analogous seed storage globulins from other plant species such as Arachis hypogaea or Sesamum indicum and the dissociation process was accompanied by some changes in conformation.