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.     

Characterization of the Major Protease Involved in the Soybean beta-Conglycinin Storage Protein Mobilization

Protease C1, the protease responsible for the initial degradation of the α′ and α subunits of the soybean β-conglycinin storage protein (Glycine max [L.] Merrill), has been purified. The enzyme was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of 70,000 and a pH optimum of 3.5 to 4.5. Susceptibility to protease inhibitors indicates that protease C1 is a serine protease. Study of the proteolytic intermediates generated suggests that the cleavage of the α′ and α subunits of β-conglycinin by protease C1 results in intermediates that are 1 or 2 kilodaltons smaller than the native α′ and α subunits. Following that, a succession of intermediates exhibiting molecular masses of 70.0 and 58.0 kilodaltons, then 63.0, 61.0, 55.0, and 53.5 kilodaltons, are observed. A 50.0- and a 48.0- kilodalton intermediate are the final products of protease C1 action. Comparison of these intermediates with the prominent anti-β-conglycinin cross-reacting bands that increase during the first few days of germination and early growth show that protease C1 plays an important physiological role, but not an exclusive one, in the living plant.     

Developmental Regulation of beta-Conglycinin in Soybean Axes and Cotyledons

Analysis of the expression of genes encoding the β-conglycinin seed storage proteins in soybean has been used to extend our understanding of developmental gene expression in plants. The α, α′, and β subunits of β-conglycinin are encoded by a multigene family which is organ-specific in its expression. In this study we report the differentially programmed accumulation of the α, α′, and β subunits of β-conglycinin. Multiple isomeric forms of each subunit are present in the dry seed, but the timing of their accumulation is unique for each subunit. The previously reported variation in amount of α′ and α subunits in axis and cotyledons is also reflected in the amount of subunit specific mRNA which is present in each tissue. The β subunit, previously undetected in soybean axes, is found to be synthesized but rapidly degraded. These differences in β-conglycinin protein accumulation may be reflected by the morphological differences observed in protein bodies between these two tissues.     

Purification and reconstitution of the proton-pumping ATPase of fungal and plant plasma membranes

The 11S storage protein (glycinin) of soybean [Glycine max (L.) Merr., cv. Raiden] was studied by polyacrylamide gel electrophoresis and amino acid sequence analysis. It contained the following subunits composed of acidic (A) and basic (B) polypeptides: A1aB2, A1bB1b, A2B1a, and A3B4. However, it lacked polypeptides A4, A5, and B3 which are present in many other cultivars. A new acidic polypeptide called A6 was present in a low amount and was characterized by amino acid sequence analysis. It was homologous to A4, although of a smaller apparent molecular weight. Since Raiden has an average protein content of about 40% and its glycinin fraction can be purified as a 350,000 D complex which is typical of other cultivars, the results imply polymorphism with respect to glycinin subunit composition. Because there is a wide variation in the methionine content of the various subunits, these findings suggest the possibility of genetically manipulating the nutritional quality of soybean seed protein by altering glycinin subunit composition.     

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.     

Biochemical characterization of rice glutelin

The two major subunits of rice glutelin, the acidic (α) and basic (β) polypeptides were purified by chromatofocusing and cation exchange chromatography, respectively. The molecular weight range of the α polypeptides was 28.5 to 30.8 kilodaltons and the molecular weight range of the β polypeptides was 20.6 to 21.6 kilodaltons. Electrofocusing in polyacrylamide gels showed that the isoelectric points of the α and β polypeptides were 6.5 to 7.5 and 9.4 to 10.3, respectively. At least 12 polypeptides of the α-group and nine polypeptides of the β-group could be separated by electrofocusing. The amino acid compositions of whole glutelin, and the purified α and β subunits were analyzed. The α subunit contained more glutamic acid/glutamine, serine, and glycine, and less alanine, lysine, aspartic acid/asparagine, and isoleucine than the β subunit. A comparison of the amino acid composition of rice glutelin subunits with those of the 11S proteins from eight other plant species indicated that there is more similarity between the β subunits than the α subunits of several diverse plant species.     

Role of endoplasmic reticulum in biosynthesis of oat globulin precursors

Oat (Avena sativa L.) groats were labeled with radioactive leucine and salt-soluble proteins were extracted and analyzed. Polyacrylamide gel electrophoresis followed by fluorography indicated two radioactive polypeptides with molecular weight 58 to 62 kilodaltons which were similar in size to unreduced globulin α-β dimers. The role of endoplasmic reticulum in the synthesis of these globulin polypeptides was investigated by in vivo and in vitro protein synthesis studies. Labeled tissue was fractionated by centrifugation and rough endoplasmic reticulum was isolated. Two polypeptides which had molecular weights of 58 to 62 kilodaltons and were immunoprecipitable with antiglobulin immunoglobulin G were found to be transiently associated with the endoplasmic reticulum. Rough endoplasmic reticulum, as well as membrane-bound polysomes, directed the in vitro synthesis of two polypeptides with molecular weight 58 to 62 kilodaltons corresponding in size to unreduced α-β dimers and could be immunoprecipitated with antiglobulin immunoglobulin G. The translation products of free polysomes did not show this. In pulse-labeling, globulin polypeptides with molecular weight 58 to 62 kilodaltons, as well as the α + β subunits, were labeled in protein bodies.

The data suggest that oat globulin polypeptides are synthesized as higher molecular weight precursors on ER-associated polysomes. These precursors are probably transported into protein bodies and cleaved into smaller α and β subunits.

     

Phycobilisome Structure of Porphyridium cruentum: POLYPEPTIDE COMPOSITION

Purified phycobilisomes of Porphyridium cruentum were solubilized in sodium dodecyl sulfate and resolved by sodium dodecyl sulfate-acrylamide gel electrophoresis into nine colored and nine colorless polypeptides. The colored polypeptides accounted for about 84% of the total stainable protein, and the colorless polypeptides accounted for the remaining 16%. Five of the colored polypeptides ranging in molecular weight from 13,300 to 19,500 were identified as the α and β subunits of allophycocyanin, R-phycocyanin, and phycoerythrin. Three others (29,000-30,500) were orange and are probably related to the γ subunit of phycoerythrin. Another colored polypeptide had a molecular weight of 95,000 and the characteristics of long wavelength-emitting allophycocyanin. Sequential dissociation of phycobilisomes, and analysis of the polypeptides in each fraction, revealed the association of a 32,500 molecular weight colorless polypeptide with a phycoerythrin fraction. The remaining eight colorless polypeptides were in the core fraction of the phycobilisome, which also was enriched in allophycocyanin. In addition, the core fraction was enriched in a colored 95,000 dalton polypeptide. Inasmuch as a polypeptide with the same molecular weight is found in thylakoid membranes (free of phycobilisomes), it is suggested that this polypeptide is involved in anchoring phycobilisomes to thylakoid membranes.     

Purification and characterization of mRNA from soybean seeds. Identification of glycinin and beta-conglycinin precursors

Poly(A)-rich RNA was isolated from developing soybean seeds (Glycine max (L.) Merr.) and fractionated on linear log sucrose gradients. Two major fractions sedimenting at 18 S and 20 S were separated and then purified by further sucrose gradient fractionation. Both fractions were active as messengers when added to a rabbit reticulocyte lysate protein synthesis system. The 18 S fraction caused proteins migrating primarily to the 60,000-dalton region of a sodium dodecyl sulfate gel to be produced, while translation of the 20 S fraction preferentially directed the synthesis of polypeptides similar in size to the alpha and alpha' subunits of beta-conglycinin. Evidence that many of the 60,000-dalton polypeptides were related to glycinin and the high molecular weight 20 S translation products were related to beta-conglycinin was obtained by immunoprecipitation using monospecific antibodies against glycinin and beta-conglycinin, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated products revealed that the glycinin precursor region contained at least three different size components and that the family of glycinin precursors had larger apparent molecular weight (58,000-63,000) than the disulfide-linked complexes between acidic and basic glycinin subunits (57,000). Unlike the disulfide-linked glycinin complexes which were cleaved by disulfide reduction, glycinin precursors were insensitive to reducing agents. The alpha and alpha' subunits synthesized in vitro also had slightly larger apparent molecular weights than purified alpha and alpha' standards.     

Initiation of the degradation of the soybean kunitz and bowman-birk trypsin inhibitors by a cysteine protease

Protease K1 activity initiates the degradation of the Kunitz soybean trypsin inhibitor (KSTI) during germination and early seedling growth. This enzyme was purified nearly 1300-fold from the cotyledons of 4-day-old soybean (Glycine max [L.] Merrill) seedlings. Protease K1 is a cysteine protease with a molecular weight of approximately 29,000. It cleaves the native form of KSTI, Ti^a, to Ti^a_m, the same modified form observed in vivo. In addition to attacking KSTI, protease K1 is also active toward the major Bowman-Birk soybean trypsin inhibitor, as well as the α, α′, and β subunits of soybean β-conglycinin. The properties and temporal variation of protease K1 during germination indicate that it is responsible for initiating the degradation of both KSTI and Bowman-Birk soybean trypsin inhibitor in the soybean cotyledon.     

The amino acid sequence of the A2B1a subunit of glycinin

The amino acid sequences of the acidic and basic components of the A2B1a subunit of glycinin, the major seed reserve protein of the soybean (Glycine max L. Merr.), were determined. They contain 278 and 180 amino acids, respectively, and have molecular weights of 31,600 +/- 100 and 19,900 +/- 100. The molecular weight of the acidic component is considerably less than that estimated by sodium dodecyl sulfate-gel electrophoresis (37,000). Sequence heterogeneity was detected at several positions scattered throughout the primary structures of both components, indicating that the preparation sequenced was composed of several nearly identical polypeptides. These data, in conjunction with a recently determined nucleotide sequence of the 3'-terminal two-thirds of the analogous glycinin subunit gene, illustrate the complexity of the gene family responsible for synthesis of glycinin subunits.     

An analysis of the subunit structure of the crystalloid protein complex from castor bean endosperm

Chromatographic and electrophoretic studies have shown that the subunits of the crystalloid protein, isolated from mature castor bean (Ricinus communis L. cv Hale) seed endosperm protein bodies, are heterogeneous with molecular weights in the range 49 to 53.5 kilodaltons (kD), and are quantitatively in unequal amounts. Each subunit comprises an αβ polypeptide pair which are reduced by 2-mercaptoethanol in two subgroups with molecular weights in the 29 to 34 kD and 20.5 to 23.5 kD ranges. Subunits and corresponding polypeptide pairs are also seen to be heterogeneous in pI following isoelectric focusing. In general, large polypeptides are acidic (pI 4.8-6.2) and small polypeptides basic (pI 7.4-9.4), although overlap of some isoelectric isomers does occur, notably in polypeptides derived from subunits which are quantitatively present in smaller amounts.     

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.     

Effect of Soy Protein Subunit Composition on the Rheological Properties of Soymilk during Acidification

The effect of soy protein subunit composition on the acid-induced aggregation of soymilk was investigated by preparing soymilk from different soybean lines lacking specific glycinin and β-conglycinin subunits. Acid gelation was induced by glucono-δ-lactone (GDL) and analysis was done using diffusing wave spectroscopy and rheology. Aggregation occurred near pH 5.8 and the increase in radius corresponded to an increase in the elastic modulus measured by small deformation rheology. Diffusing wave spectroscopy was also employed to follow acid gelation, and data indicated that particle interactions start to occur at a higher pH than the pH of onset of gelation (corresponding to the start of the rapid increase in elastic modulus). The protein subunit composition significantly affected the development of structure during acidification. The onset of aggregation occurred at a higher pH for soymilk samples containing group IIb (the acidic subunit A₃) of glycinin, than for samples prepared from Harovinton (a commercial variety containing all subunits) or from genotypes null in glycinin. The gels made from lines containing group I (A₁, A₂) and group IIb (A₃) of glycinin resulted in stiffer acid gels compared to the lines containing only β-conglycinin. These results confirmed that the ratio of glycinin/β-conglycinin has a significant effect on gel structure, with an increase in glycinin causing an increase in gel stiffness. The type of glycinin subunits also affected the aggregation behavior of soymilk.     

Equal Expression of the Maternal and Paternal Alleles for the Polypeptide Subunits of the Major Storage Protein of the Bean Phaseolus vulgaris L

Discontinuous sodium dodecyl sulfate slab gel electrophoresis of G1 globulin from several strains of Phaseolus vulgaris L. seed permitted clear resolution of the constituent polypeptides. Three strains (Tendergreen, Canadian Wonder, and BBL 240) had subunits of molecular weight 53,000, 47,000 and 43,000 while two strains (Seafarer and PI 229,815) had 50,500, 47,000 and 43,000 molecular weight subunits. F₁ seed from the cross BBL 240 × PI 229,815 showed four polypeptides on dissociation of the G1 protein; however, the amount of each of the 53,000 and 50,500 subunits was half that of the 47,000 subunit. This is interpreted as evidence that both the maternal and paternal loci for these polypeptides are transcribed and translated with similar efficiency. All of the polypeptides were found to have associated sugar residues.     

Soybean β-Conglycinin Induces Inflammation and Oxidation and Causes Dysfunction of Intestinal Digestion and Absorption in Fish

β-conglycinin has been identified as one of the major feed allergens. However, studies of β-conglycinin on fish are scarce. This study investigated the effects of β-conglycinin on the growth, digestive and absorptive ability, inflammatory response, oxidative status and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and their enterocytes in vitro. The results indicated that the specific growth rate (SGR), feed intake, and feed efficiency were reduced by β-conglycinin. In addition, activities of trypsin, chymotrypsin, lipase, creatine kinase, Na⁺,K⁺-ATPase and alkaline phosphatase in the intestine showed similar tendencies. The protein content of the hepatopancreas and intestines, and the weight and length of the intestines were all reduced by β-conglycinin. β-conglycinin increased lipid and protein oxidation in the detected tissues and cells. However, β-conglycinin decreased superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in the intestine and enterocytes. Similar antioxidant activity in the hepatopancreas was observed, except for GST. The expression of target of rapamycin (TOR) gene was reduced by β-conglycinin. Furthermore, mRNA levels of interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β) genes were increased by β-conglycinin. However, β-conglycinin increased CuZnSOD, MnSOD, CAT, and GPx1b gene expression. In conclusion, this study indicates that β-conglycinin induces inflammation and oxidation, and causes dysfunction of intestinal digestion and absorption in fish, and finally reduces fish growth. The results of this study provide some information to the mechanism of β-conglycinin-induced negative effects.     

Microheterogeneity of globulin-1 storage protein from French bean with isoelectrofocusing

The major storage protein fraction, globulin-1 protein, of French bean (Phaseolus vulgaris L.) was analyzed by two-dimensional electrophoresis. The protein pattern suggested a more complex system for globulin-1 protein than the model of three polypeptides, α, β, and γ, differing in molecular weight. Isoelectrofocusing analyses of the individual proteins showed that each exhibited charge microheterogeneity over a similar pH range. Isoelectrofocusing banding patterns may help to understand the relationships between the globulin-1 polypeptide subunits.     

Expression of Two Different Glutamine Synthetase Polypeptides during Root Development in Phaseolus vulgaris L

Immunoprecipitation and two-dimensional gel electrophoresis analysis of the glutamine synthetase (GS) polypeptides (α and β) during Phaseolus vulgaris root development shows that the α polypeptide is the main component of the enzyme in the embryo and in up to 5 day old roots. From 5 days on, the β polypeptide becomes the root predominant GS monomer. The α/β ratio of the in vitro translated GS polypeptides from the total polysomal RNA isolated at different root ages correlates with the α/β ratio observed in the root extracts. These results suggest that the two root GS polypeptides are encoded by different mRNA species in Phaseolus vulgaris.     

Separation and properties of β-galactosidase, β-glucosidase, β-glucuronidase and n-acetyl-β-glucosaminidase from rat kidney

1. The activities of β-galactosidase, β-glucosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7·0 indicated slow- and fast-moving components of rat-kidney β-galactosidase. 3. The fast-moving component is also associated with the total β-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the β-glucosidase component is a small acidic molecule, of molecular weight approx. 40000–50000, with optimum pH5·5–6·0 for β-galactosidase and β-glucosidase activities. 5. The major β-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3·7.