Opposite Contributions of Glycinin- and β-Conglycinin-Derived Peptides to the Aggregation Behavior of Soy Protein Isolate Hydrolysates

The aggregation behavior as a function of pH was studied for hydrolysates obtained by hydrolysis of soy protein isolate (SPI) and glycinin- and β-conglycinin-rich protein fractions with subtilisin Carlsberg. The substrates were hydrolyzed up to degrees of hydrolysis (DH) of 2.2% and 6.5%. Compared with nonhydrolyzed SPI, a decrease in solubility was observed for the hydrolysates of SPI [0.8% (w/v) protein, I = 0.03 M] around neutral pH. At pH 8.0, glycinin hydrolysates had a much lower solubility (∼43% and 60%, respectively, for DH 2.2% and 6.5%) than SPI and β-conglycinin-derived hydrolysates, which were almost completely soluble. Peptides that aggregated were all larger than 5 kDa, and as estimated by size-exclusion chromatography their composition was almost independent of the aggregation pH. The solubility of hydrolysates of SPIs with a varying glycinin and β-conglycinin composition showed that glycinin-derived peptides are the driving force for the lower solubility of SPI hydrolysates. The solubility of SPI hydrolysates at pH 8.0 was shown not to be the sum of that of glycinin and β-conglycinin hydrolysates. Assuming that the separate hydrolysis of glycinin and β-conglycinin did not differ from that in the mixture (SPI), this indicates that β-conglycinin-derived peptides have the ability to inhibit glycinin-derived peptide aggregation.     

Molecular analysis of glycinin genes in soybean mutants for development of gene-specific markers

Soybean mutant lines that differ in 11S glycinin and 7S β-conglycinin seed storage protein subunit compositions were developed. These proteins have significant influence on tofu quality. The molecular mechanisms underlying the mutant lines are unknown. In this study, gene-specific markers for five of the glycinin genes (Gy1 to Gy5) were developed using three 11S null lines, two A₄ null Japanese cultivars, Enrei and Raiden, and a control cultivar, Harovinton. Whereas gene-specific primers produced the appropriate products in the control cultivar for the Gy1, Gy2, Gy3 and Gy5 genes, they did not amplify in mutants missing the A_1aB₂, A₂B_1a, A_1b B_1b, and A₃B₄ subunits. However, ecotype targeting induced local lesions in genomes (EcoTILLING) and sequencing analysis revealed that the absence of the A₄ peptide in the mutants is due to the same point mutation as that in Enrei and Raiden. Selection efficiency of the gene-specific primer pairs was tested using a number of breeding lines segregating for the different subunits. Primer pairs specific to each of the Gy1, Gy2, Gy3, and Gy5 genes can be used to detect the presence or absence of amplification in normal or mutant lines. The Gy4 null allele can be selected for by temperature-switch PCR (TS-PCR) for identification of the A₄ (G4) null genotypes. In comparison to protein analysis by SDS-PAGE, gene-specific markers are easier, faster and more accurate for analysis, they do not have to use seed, and can be analyzed at any plant growth stage for marker-assisted selection.     

Subunit structure of γ-conglycinin in soybean seeds

Dissociated subunits of purified γ-conglycinin were isolated on a DEAE-Sephadex A-50 column. A single band was seen on two kinds of gel electrophoresis and isoleucine was shown as the only N-terminal amino acid. The isolated subunit reacted with antisera to the native γ-conglycinin. The M_r of the subunit was 51 000–51 500 estimated by urea-acetic acid and SDS-urea gel electrophoresis. A value of 50 000 was obtained by gel filtration with guanidine-hydrochloric acid on Sepharose CL-6B. The γ-conglycinin molecule was found to be made up of three subunits. This was determined by cross-linking the subunits and then submitting them to gel electrophoresis. Differences and similarities of subunit structure among γ-conglycinin, β-conglycinin and glycinin are discussed.     

Urea-Elicited Changes in Relative Electrophoretic Mobility of Certain Glycinin and beta-Conglycinin Subunits

Six molar urea in sodium dodecyl sulfate-polyacrylamide gels altered the relative electrophoretic mobility of several soybean protein subunits. Glycinin acidic polypeptide components A₃ and A₄ could be resolved from the other acidic polypeptides. A variant of the δ′ subunit of β-conglycinin was identified.     

Dynamic viscoelastic properties of glycinin and β-conglycinin gels from soybeans

The effects of heating temperature on gel properties and conformational changes were investigated in glycinin and β-conglycinin gels using Theological and Fourier transform ir (FTIR) methods. Solutions of 15 wt % glycinin or β-conglycinin in 35 mM phosphate buffer at pH 7.6 were heated at various temperatures for 30 min and rheological properties were measured at 20°C. The storage modulus G′ as a function of frequency changed from a monotonical decrease with decreasing frequency to a plateau in the range from 0.0018 to 40 Hz by heating at temperatures higher than 80°C for glycinin and 65°C for β-conglycinin. A band at 1618 cm^?1 (associated with the β-sheet structure) on ir spectra increased with the formation of heat-induced gels. The value of the storage modulus G′ correlated well with the increase in absorbance at 1618 cm^?1. These results suggest that the formation of a β-sheet structure may be closely related to the value of the storage modulus G′ for heat-induced gels in soybean proteins and that heat-induced gels of glycinin and β-conglycinin are formed by cross-links with intermolecular β-sheet structures. © 1994 John Wiley & Sons, Inc.     

Identification and characterization of DNA clones encoding group-II glycinin subunits

Summary DNA clones that encode the group-II subunits of soybean glycinin were identified and compared with clones for group-I subunits. The group-I clones hybridize weakly to those from group-II at low stringency, but fail to hybridize with them at moderate or high stringency. The genes for the group-II subunits are contained in 13 and 9 kb EcoRI fragments of genomic DNA in cultivar CX635-1-1-1. These fragments contain genes for subunits A₅A₄B₃ and A₃B₄, respectively. The larger size of mature group-II subunits compared with group-I subunits is correlated with a larger sized mRNA. However, the gross arrangement of introns and exons within the group-II coding regions appears to be the same as for the genes which encode group-I subunits. Messenger RNA for both groups of glycinin subunits appear in the seed at the same developmental interval, and their appearance lags slightly behind that of mRNAs for the a/a subunits of -conglycinin. These data indicate that the glycinin gene family is more complex than previously thought.Abbreviations bp base pairs - kb kilobase pairs - SDS sodium dodecyl sulfate Cooperative research between USDA/ARS and the Indiana Agric. Expt. Station. This work was supported in part by grants from the USDA Competitive Grants Program and the American Soybean Association Research Foundation. This is Journal Paper No. 10,078 from the Purdue Agricultural Experiment Station     

Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996     

Comparison of the aggregation behavior of soy and bovine whey protein hydrolysates

Soy-derived proteins (soy protein isolate, glycinin, and β-conglycinin) and bovine whey-derived proteins (whey protein isolate, -lactalbumin, β-lactoglobulin) were hydrolyzed using subtilisin Carlsberg, chymotrypsin, trypsin, bromelain, and papain. The (in)solubility of the hydrolysates obtained was studied as a function of pH. At neutral pH, all soy-derived protein hydrolysates, particularly those from glycinin, obtained by hydrolysis with subtilisin Carlsberg, chymotrypsin, bromelain, and papain showed a stronger aggregation compared to the non-hydrolyzed ones. This increase in aggregation was not observed upon hydrolysis by trypsin. None of the whey-derived protein hydrolysates exhibited an increase in aggregation at neutral pH. The high abundance of theoretical cleavage sites in the hydrophobic regions of glycinin probably explains the stronger exposure of hydrophobic groups than for the other proteins, which is suggested to be the driving force in the aggregate formation.     

Rheological properties and conformational states of β-conglycinin gels at acidic pH

The heat-induced gelation of β-conglycinin was investigated by dynamic viscoelastic measurements as a function of pH. At pH values below 5.5–6.0, the storage modulus G′ rapidly increased with decreasing pH. The conformational states of both sols and gels of β-conglycinin were investigated by Fourier transform ir (FTIR) and by differential scanning calorimetry (DSC). The degree of protonation of the carboxyl groups of β-conglycinin, when examined by FTIR indicated that, as would be expected, the degree of protonation increased with decreasing pH below pH 6.0. DSC measurements suggest that β-conglycinin tends toward thermal denaturation with decreasing pH (below pH 6.0). Second-derivative FTIR spectra for β-conglycinin gels show that a band at 1620 cm^?1 (associated with exposed β-strands) develops with decreasing pH. The results suggest that β-conglycinin undergoes denaturation with increasing protonation of its carboxyl groups, resulting in an increase in the amount of exposed β-strands. The exposed β-strands then intermolecularly bond to form gel networks. This process is promoted with decreasing pH, and as a result, rigid gels form at acidic pH values. © 1994 John Wiley & Sons, Inc.     

Glycinergic transmission

Inhibition in the mature central nervous system is mediated by activation of γ-aminobutyric acid (GABA_A) and glycine receptors. Both receptors belong to the same superfamily of ligand-gated ion channels and share common transmembrane topology and structural and functional features. Glycine receptors are pentameric ligand-gated anion channels composed of two different subunits, named α und β, that assemble with a fixed stoichiometric ratio of two α to three β subunits. Four genes encoding the α subunits exist, whereas only one gene encoding the β subunit has been detected. Ligand binding occurs at the interface of α and β subunits. The β subunit, which is unable to form homo-oligomeric receptors, is responsible for assembly and channel properties. Moreover, this subunit carries a binding motif for the cytoplasmic protein gephyrin, which is believed to mediate synaptic clustering and anchoring at inhibitory synapses by interacting with the subsynaptic cytoskeleton. Synaptic gephyrin appears to restrict the mobility of glycine receptors diffusing in the plane of the plasma membrane, thereby generating dynamic plasma membrane domains contributing to the plasticity of inhibitory synapses. Glycine receptors are well established as playing important roles in controlling motor functions and sensory signaling in vision and audition and those in the dorsal horn of the spinal cord are now considered to be new targets for pain therapies. Like GABA_A receptors, glycine receptors have been shown to be depolarizing during development. The functional meaning of the developmental switch from excitatory to inhibitory glycine receptor action remains to be elucidated.     

Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.     

Accumulation of Uranium by Chlorella Cells Grown under Autotrophic,Heterotrophic and Mixotrophic Culture Conditions

Subcellular localization of β-conglycinin and glycinin in developing soybean cotyledons was examined by sucrose density gradient ultracentrifugation (SDG-UC). Two major bands appeared at densities of 1.05 and 1.15 in the SDG-UC. They were the microbody fraction and rER fraction, respectively, from the distribution of marker enzymes, electron microscopic observation, and the effects of MgCl₂ on the profile of SDG-UC. In the soluble and microbody fractions, α and α′ subunits of β-conglycinin were major polypeptides. The amounts of β-conglycinin and glycinin associated with rER were roughly equal. β-Conglycinin was found in the soluble and microbody fractions as well as the rER fraction while glycinin was mostly localized in the rER fraction.     

Nickel Block of a Family of Neuronal Calcium Channels: Subtype- and Subunit-Dependent Action at Multiple Sites

Nickel ions have been reported to exhibit differential effects on distinct subtypes of voltage-activated calcium channels. To more precisely determine the effects of nickel, we have investigated the action of nickel on four classes of cloned neuronal calcium channels (α_1A, α_1B, α_1C, and α_1E) transiently expressed in Xenopus oocytes. Nickel caused two major effects: (i) block detected as a reduction of the maximum slope conductance and (ii) a shift in the current-voltage relation towards more depolarized potentials which was paralleled by a decrease in the slope of the activation-curve. Block followed 1:1 kinetics and was most pronounced for α_1C, followed by α_1E > α_1A > α_1B channels. In contrast, the change in activation-gating was most dramatic with α_1E, with the remaining channel subtypes significantly less affected. The current-voltage shift was well described by a simple model in which nickel binding to a saturable site resulted in altered gating behavior. The affinity for both the blocking site and the putative gating site were reduced with increasing concentration of external permeant ion. Replacement of barium with calcium reduced both the degree of nickel block and the maximal effect on gating for α_1A channels, but increased the nickel blocking affinity for α_1E channels. The coexpression of Ca channel β subunits was found to differentially influence nickel effects on α_1A, as coexpression with β_2a or with β₄ resulted in larger current-voltage shifts than those observed in the presence of β_1b, while elimination of the β subunit almost completely abolished the gating shifts. In contrast, block was similar for the three β subunits tested, while complete removal of the β subunit resulted in an increase in blocking affinity. Our data suggest that the effect of nickel on calcium channels is complex, cannot be described by a single site of action, and differs qualitatively and quantitatively among individual subtypes and subunit combinations. Received: 12 October 1995/Revised: 17 January 1996     

Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.     

Polymorphism and Expression of cDNAs Encoding Glycinin Subunits

The nucleotide sequence of cDNA encoding the glycinin A₂B_1a subunit from var. Shirotsurunoko was determined and compared with that in the case of var. Bonminori. The comparison showed six nucleotide substitutions in the coding sequence, one of which results in one amino acid replacement, and three in the 3'-noncoding region. These differences indicate the occurrence of polymorphism of the glycinin A₂B_1a subunit gene between the cultivars. The present data together with the previous results indicating the polymorphism of the A_1aB_1b subunit gene [(Utsumi et al., J. Agric. Food Chem., 35, 210 (1987)] suggest that the polymorphism is a general property of glycinin subunit genes. The expression of cDNAs encoding the A₂B_1a and A_1aB_1b subunits was examined. The results obtained in both in vivo- and in vitro-expression experiments indicate that the resultant products were readily degraded.     

Yeast holoenzyme of protein kinase CK2 requires both β and β′ regulatory subunits for its activity

Protein kinase CK2 is a highly conserved Ser/Thr protein kinase that is ubiquitous among eucaryotic organisms and appears to play an important role in many cellular functions. This enzyme in yeast has a tetrameric structure composed of two catalytic (α and/or α′) subunits and two regulatory β and β′ subunits. Previously, we have reported isolation from yeast cells four active forms of CK2, composed of αα′ββ′, α₂ββ′, α′₂ββ′ and a free α′-catalytic subunit. Now, we report that in Saccharomyces cerevisiae CK2 holoenzyme regulatory β subunit cannot substitute other β′ subunit and only both of them can form fully active enzymatic unit. We have examined the subunit composition of tetrameric complexes of yeast CK2 by transformation of yeast strains containing single deletion of the β or β′ regulatory subunits with vectors carrying lacking CKB1 or CKB2 genes. CK2 holoenzyme activity was restored only in cases when both of them were present in the cell. Additional, co-immunoprecypitation experiments show that polyadenylation factor Fip1 interacts with catalytic α subunits of CK2 and interaction with beta subunits in the holoenzyme decreases CK2 activity towards this protein substrate. These data may help to elucidate the role of yeast protein kinase CK2β/β′ subunits in the regulation of holoenzyme assembly and phosphotransferase activity.     

The effect of pH on heat denaturation and gel forming properties of soy proteins

This study is focussed on the influence of pH on the gel forming properties of soy protein isolate and purified glycinin in relation to denaturation and aggregation. At pH 7.6 more fine-stranded gels were formed characterised by low G' values, and a smooth, slightly turbid appearance, whereas at pH 3.8 coarse gels were obtained with a high stiffness and a granulated, white appearance. Low G' values, as found at pH 7.6, correlate with a high solubility of glycinin and soy protein isolate (ca. 50%) after heating at low protein concentration. At pH 3.8 all protein precipitated upon heating, which correlates with relatively high G' values. The role of beta-conglycinin during gelation of SPI seems to be minor at pH 7.6, which is indicated by the fact that, in contrast to pH 3.8, notable gel formation did not start upon heat denaturation of beta-conglycinin. Furthermore, the mechanism of gel formation seems to be affected by pH, because at pH 7.6, in contrast to pH 3.8, the disulphide bridge between the acidic and the basic polypeptide of glycinin is broken upon heating.     

Determination of Optimal Protein Quantity Required to Identify Abundant and Less Abundant Soybean Seed Proteins by 2D-PAGE and MS

Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (75, 100, 125, 150, and 200 μg) by 2D-PAGE. Following 2D-PAGE and spot excision, proteins were identified by mass spectrometry analysis. The number of visible protein spots was increased with an increase in the amount of protein loaded. The intensity of highly abundant proteins [β-conglycinin β-homotrimer and glycinin G4 (A5A4B3) precursors] increased linearly between 75 and 125 μg, whereas the proglycinin G3 (A1ab1b) homotrimer showed linearity between 75 and 150 μg. The spot intensity of less abundant proteins, glycinin G2 (A2b1a) precursor and proglycinin G3 (A1ab1b) homotrimer, increased linearly with an increase in the amount of protein through 200 μg, whereas spot intensity of β-conglycinin β-homotrimer and the allergen Gly m bd 28K increased linearly until 150 μg and did not increase further at 200 μg. These results suggest that 150 μg protein was a suitable amount for the separation of abundant proteins, and 200 μg protein was suitable for the separation of less abundant proteins prepared from soybean seeds. Mention of trade name, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable.