Storage proteins from Lathyrus sativus seeds

The proteins from Lathyrus sativus Linn. (chickling vetch or grass pea) seeds were investigated. Protein constitutes approximately 20% of the seed dry weight, >60% of which is composed by globulins and 30% by albumins. A single, 24 kDa polypeptide comprises more than half of the protein present in the albumin fraction. The globulins may be fractionated into three main components, which were named alpha-lathyrin (the major globulin), beta-lathyrin, and gamma-lathyrin. alpha-Lathyrin, with a sedimentation coefficient of approximately 18S, is composed of three main types of unglycosylated subunits (50-66 kDa), each of which produce, upon reduction, a heavy and a light polypeptide chain, by analogy with 11S. beta-Lathyrin, with a sedimentation coefficient of 13S, is composed by a relatively large number of subunits (8-66 kDa). Two major polypeptides are glycosylated and exhibit structural similarity with beta-conglutin from Lupinus albus. One of these possesses an internal disulfide bond. gamma-Lathyrin, with a sedimentation coefficient of approximately 5S, contains two interacting, unglycosylated polypeptides, with no disulfide bonds: the major 24 kDa albumin and the heavier (20 kDa) polypeptide chain of La. sativus lectin.     

Characterization of the proteins from Vigna unguiculata seeds

The proteins from Vigna unguiculata (L.) Walp. (cowpea) seeds were investigated. Globulins constitute over 51% of the total seed protein, with albumins composing approximately 45%. The globulins may be fractionated by native electrophoresis or anion exchange chromatography into three main components, which were termed (in decreasing order of anodic mobility) alpha-vignin, beta-vignin, and gamma-vignin. alpha-Vignin, with a sedimentation coefficient of 16.5S, is a major, nonglycosylated globulin, composed of a major 80 kDa subunit, which upon reduction, produces two polypeptides (20 and 60 kDa). beta-Vignin, with a sedimentation coefficient of 13S, is a major, glycosylated globulin, composed of two main polypeptides (55 and 60 kDa) with no disulfide bonds. Finally, gamma-vignin, a minor globulin, is composed by one main type of subunit (22 kDa), which upon reduction, is converted into a single, apparently heavier polypeptide chain (30 kDa) due to the presence of an internal disulfide bond. Immunological analyses revealed structural homology between beta-vignin and beta-conglutin (the vicilin from Lupinus seeds) but not between alpha- or gamma-vignins and their Lupinus counterparts. Haemagglutination activity toward trypsinized rabbit erythrocytes was found exclusively in the albumin fraction and was strongly inhibited by N-acetylglucosamine or chitin.     

Surface physicochemical properties of globulin-P amaranth protein

Globulin-P, the polymerized 11S amaranth globulin, is composed of 280 kDa unitary molecules (UM, 23%) and aggregates larger than 500 kDa (A, 70%). Antibodies against these proteins were prepared to study their surface characteristics and to assess their homology with other storage proteins. Results showed that globulin-P unitary molecules and aggregates had similar reactive surfaces. A polypeptide of 56 kDa was found to be the most reactive to the antibodies assayed, followed by the acidic polypeptides. Such results support previous information, according to which these polypeptides appeared to be the most exposed on the molecule surface. Globulin-P fraction presented cross-reactivity with the remaining amaranth protein fractions: 11S-globulin, glutelins, and albumins. Globulin-P and 11S-globulin showed similar reactive surfaces whereas glutelin and albumins presented a lower cross-reactivity. The reactivity of the glutelin fraction depended on its sequence. Globulin-P fraction presented cross-reactivity with quinoa globulins, and to a lesser extent with globulins of sunflower and rice. Moreover, the anti-Gp serum was unable to detect either conformational or sequence epitopes in globulins of soybean, wheat, buckwheat, rice, and rye.     

Characterization of an aspartic proteinase activity in buckwheat (Fagopyrum esculentum Moench) seeds

The pepstatin A sensitive acidic proteolytic activity of total protein extracts of buckwheat seeds has been analyzed in developing, mature, and germinating seeds by activity measurements as well as by electrophoretic and immunochemical techniques. Immunoblot analysis using cross-reactive antibodies raised against barley phytepsin suggested that specific proteolytic activity could be attributed to a 47 kDa heterodimeric polypeptide, composed of two subunits: 31 and 16 kDa polypeptides. The analysis of time course expression revealed that the 47 kDa heterodimer accumulated during seed maturation starting from 12 days after pollination and was also present at the beginning of germination. Milk-clotting activity of this proteinase was also indicated.     

Proteome profiling of seed storage proteins reveals the nutritional potential of Salicornia brachiata Roxb., an extreme halophyte

Salicornia brachiata is an extreme halophyte that grows in salty marshes and is considered to be a potential alternative crop for seawater agriculture. Salicornia seeds are rich in protein, and its tender shoots are eaten as salad greens. Seed storage proteins were fractionated by sequential extraction using different solvents, including distilled water for albumins, NaCl (1.0 M) for globulins, NaOH (0.1 N) for glutelins, and ethanol (70% v/v) for prolamins. Globulins accounted for 54.75% of the total seed storage proteins followed by albumins (34.30%) and glutelins (8.70%). The fractionated proteins were characterized using 2D-diagonal SDS-PAGE and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The globulin fraction, composed of seven intermolecular disulfide-linked polypeptide pairs of molecular mass 63.5, 62.5, 54.7, 53.0, 43.2, 38.5, and 35.1 kDa, encompassed a basic and an acidic subunit. Two-dimensional gels revealed approximately 32 spots, with isoelectric points and molecular masses ranging from 4.93 to 11.6 and from ～5.2 to ～109.4 kDa, respectively. Protein spots were identified by MALDI-TOF MS peptide mass fingerprint analysis and further classified. Homology analysis demonstrated that 19% of the proteins were involved in metabolism, 16% were involved in signaling, and 15% were regulatory proteins. Peptide mass fingerprint analysis confirmed the presence of inter- and intramolecular disulfide linkages in the globulin fraction. Sulfur-rich proteins are of high nutritional value, and disulfides make S. brachiata a potential source of dietary supplementation.     

Amaranth (Amaranthus hypochondriacus) vicilin subunit structure

The 7S-globulin fraction is a minor component of the amaranth storage proteins. The present work provides new information about this protein. The amaranth 7S-globulin or vicilin presented a sedimentation coefficient of 8.6 ± 0.6 S and was composed of main subunits of 66, 52, 38, and 16 kDa. On the basis of mass spectrometry (MS) analysis of tryptic fragments, the 52, 38, and 16 kDa subunits presented sequence homology with sesame vicilin, whereas the 66 kDa subunit showed sequence similarity with a putative vicilin. Several characteristics of the 66 kDa subunit were similar to members of the convicilin family. Results support the hypothesis that the 7S-globulin molecules are composed of subunits coming from at least two gene families with primary products of 66 and 52 kDa, respectively. According to the present information, amaranth vicilin may be classified into the vicilin group that includes pea, broad bean, and sesame vicilins, among others.     

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.     

Characterization of yam bean (Pachyrhizus erosus) proteins

Seed proteins from Mexican yam bean seeds (Pachyrhizus erosus L.) were sequentially extracted according to the Osborne classification. Albumins were the major fraction (52.1-31.0%), followed by globulins (30.7-27.5%). The minor protein fraction was prolamins (0.8%). Defatting with chloroform/methanol remarkably affected the distribution of protein solubility classes; albumins were the most affected fraction (4.3-17.5%). Electrophoretic patterns of albumins showed bands at 55, 40, 35, and 31 kDa. After reduction of the globulin fraction exhibited two triplets, one from 35 to 31 kDa and the second from 19 to 21 kDa, these could be compared to the acid and basic polypeptides of 11S-like proteins. Prolamins showed one band at 31 kDa, and glutelins after reduction showed three main bands at 52, 27, and 14 kDa. Trypsin inhibitors were assayed in saline extracts; the values found (1232-2608 IU/g of meal) were lower than those of other legumes. In general, yam bean seed proteins showed an excellent balance of all essential amino acids; albumins contain the highest amount of essential amino acids.     

Seed protein electrophoretic characterization of cowpea (Vigna unguiculata) germplasm from IITA gene bank

Summary The total seed protein, globulin and albumin fractions of 20 cowpea (V. unguiculata) accessions from IITA gene bank were investigated by SDS-polyacrylamide gel electrophoresis. Total seed protein extracts were prepared from the defatted meal by homogenisation and centrifugation in Tris-glycine buffer. The globulins and albumins were obtained from the total protein extract by exhaustive dialysis against sodium acetate buffer. Two main total seed protein electrophoretic patterns were observed with respect to the 39 and 20 kD subunits, which were present in six out of the twenty accessions analysed. While there was no correlation between seed colour and total seed protein banding pattern, the six insect-resistant cultivars were characterized by the presence of the 39 and 20 kD subunits. The globulins were the predominant class of the total seed proteins and consisted mainly of 64, 58, 56 and 14 kD subunits which make up CP1 and CP2, the major globulins. The albumins in all accessions were a heterogeneous protein fraction consisting of both high and low molecular weight subunits. It was suggested that the insect-resistant cultivars may be genetically related and that the 39 and 20 kD subunits may be involved in the insect resistance mechanism.     

Characterization of methanol-soluble and methanol-insoluble proteins from developing peanut seed

The 85% methanol-soluble proteins are known to specifically contribute to the production of flavor of roasted peanut. To determine the nature of the 85% methanol-soluble proteins, they were isolated from the peanut seed, and the 85% methanol-soluble (MS) and 85% methanol-insoluble (MIS) fractions were characterized using polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The results showed that the 85% MS fraction contained lower amounts (9-10%) of protein than the MIS fraction (15-33%). Protein content of the MIS fraction increased more significantly during seed maturation than it did in the MS fraction. Unlike the protein, free amino acids and soluble sugars levels of the MS fraction decreased significantly during seed maturation. The 85% MS fraction contained predominantly low molecular weight (<20 kDa) proteins/polypeptides, whereas the MIS fraction contained a mixture of polypeptides with molecular weight between 14 kDa and 90 kDa. SDS-PAGE showed no major changes in the polypeptide composition of the MS fraction during seed maturation. Capillary electrophoretic analysis revealed major qualitative and quantitative changes in the protein and polypeptide composition of the MS and MIS fractions during seed maturation. Fatty acid analysis of these fractions indicated that the MS fraction is lipoprotein in nature and rich in oleic and linoleic acids.     

Comparative proteomical analysis of zygotic embryo and endosperm from Coffea arabica seeds

During coffee seed development, proteins are predominantly deposited in cotyledons and in the endosperm. Reserve proteins of the 11S family are the most abundant globulins in coffee seeds, acting as a nitrogen source during roasting and guaranteeing flavor and aroma. The aim of the present study was to compare the protein profiles of endosperm and zygotic embryos of coffee seeds. Proteins were extracted from whole seed as well as from embryo and endosperm, separately. Total proteins were analyzed by two-dimensional electrophoresis (2-DE) followed by identification by mass spectrometry (MS). The most abundant spots observed in the gels of coffee seeds were excised, digested with trypsin, and identified by MS as subunits of the 11S globulin. Spots with identical pI and molecular masses were also observed in the protein profiles of coffee endosperm and embryo, indicating that 11S protein is also highly expressed in those tissues. Peptide sequence coverage of about 20% of the entire 11S globulin was obtained. Three other proteins were identified in the embryo and endosperm 2-DE profiles as a Cupin superfamily protein, an allergenic protein (Pru ar 1), exclusive to the endosperm 2D map, and a hypothetical protein, observed only in the zygotic embryo profile.     

Electrophoretic seed globulin patterns in some New World Lupinus species

Comparative SDS-PAGE analysis of seed globulins covered 74 accessions representing 19 New World Lupinus species, 18 North American lupins and one South American species: L. mutabilis. The species investigated showed major, well-defined polypeptide bands in the approximate M _r range of 40–75 kDa. In general, intraspecific variation blurred interspecific differences; L. mutabilis could not be distinguished from the North American lupins. Of the taxa investigated, only L. subcarnosus and L. succulentus displayed some species-specific features. The American species examined showed different seed globulin patterns from those reported earlier for the smooth-seeded and for the rough-seeded Old World lupins.     

Biochemical characterization of soluble proteins in pecan [Carya illinoinensis (Wangenh.) K. Koch

Pecans (cv. Desirable) contained approximately 10% protein on a dry weight basis. The minimum nitrogen solubility (5.9-7.5%) at 0.25-0.75 M trichloroacetic acid represented the nonprotein nitrogen. Among the solvents assessed for protein solubilization, 0.1 M NaOH was the most effective, while borate saline buffer (pH 8.45) was judged to be optimal for protein solubilization. The protein solubility was minimal in the pH range of 3-7 and significantly increased on either side of this pH range. Increasing the NaCl concentration from 0 to 4 M significantly improved ( approximately 8-fold increase) protein solubilization. Following Osborne protein fractionation, the alkali-soluble glutelin fraction (60.1%) accounted for a major portion of pecan proteins followed by globulin (31.5%), prolamin (3.4%), and albumin (1.5%), respectively. The majority of pecan polypeptides were in the molecular mass range of 12-66 kDa and in the pI range of 4.0-8.3. The pecan globulin fraction was characterized by the presence of several glycoprotein polypeptides. Lysine was the first limiting essential amino acid in the defatted flour, globulin, prolamin, and alkaline glutelin fractions. Leucine and tryptophan were the first limiting essential amino acids in albumin and acid glutelin fractions, respectively. Rabbit polyclonal antibodies detected a range of pecan polypeptides in the 12-60 kDa range, of which the globulin fraction contained the most reactive polypeptides.     

Molecular cloning of 11S globulin and 2S albumin, the two major seed storage proteins in sesame

Insoluble 11S globulin and soluble 2S albumin, conventionally termed alpha-globulin and beta-globulin, are the two major storage proteins and constitute 80-90% of total seed proteins in sesame. Two full-length cDNA clones were sequenced and deduced to encode sesame 11S globulin and 2S albumin precursors, respectively. Deduced amino acid composition reveals that 2S albumin, but not 11S globulin, is a sulfur-rich protein. Three abundant polypeptides of 50-60 kDa were resolved on SDS-PAGE when seed-purified 11S globulin was prepared in nonreducing conditions. Immunological analysis suggests that these three polypeptides are encoded by homologous genes. Immunodetection on the overexpressed protein of the 11S globulin clone in Escherichia coli indicates that this clone encodes the precursor protein of one of the three purified 11S globulin polypeptides.     

Calcium- and magnesium-dependent aggregation of legume seed storage proteins.

The solubility characteristics and sedimentation behavior of total or individual globulins from legume seeds [Lupinus albus L., Pisum sativum L., and Glycine max (L.) Merr.] were investigated. The typical insolubility of globulins detected during their extraction seems to be due to the presence of a low molecular weight factor(s) in the seed extract. The solubility of the purified globulins decreases with increasing concentrations of calcium and/or magnesium, but not of other cations, showing minimum values at concentrations that vary with the particular globulin considered. Ultracentrifugation analyses revealed that the Ca(2+)- and/or Mg(2+)-induced insolubilization of the globulins involves the formation of high-order aggregates of molecules of the same or of different globulins. These macromolecular structures are dissociated under conditions of high ionic strength, suggesting the involvement of electrostatic interactions in the aggregation process. The degree of association relies heavily on the amount of Ca(2+) and/or Mg(2+) available, on the presence of chelating agents for these divalent cations, and on the ionic strength of the surrounding medium. The possible physiological significance of the findings is discussed.     

Identification and variation of glutelin alpha polypeptides in the genus Oryza assessed by two-dimensional electrophoresis and step-by-step immunodetection

To obtain fundamental information for nutritional improvement of rice (Oryza sativa) seed proteins, the alpha polypeptides of the major storage protein glutelin varied over the genus Oryza were qualitatively and quantitatively characterized with unique methods. The polypeptides were maximally separated by two-dimensional electrophoresis (2D-PAGE) composed of nonequilibrium pH gradient gel electrophoresis (NEPHGE) and higher temperature SDS-PAGE. Then the subunit for each polypeptide spot was identified with the sequential immunodetection called a step-by-step detection method, making use of highly subunit-specific antibodies. The comparative analysis showed considerable variation in the accumulation level of A-type and B-type glutelin subunits and found unknown glutelin subunits that were unable to be identified with the antibodies used. Wild species accumulating a high amount of lysine-rich B-type glutelin subunits and unknown unique subunits were identified as they might play a crucial role in nutritional quality improvement of the cultivated rice.     

Characterization of Oat Endoproteinases that Hydrolyze Oat Globulins

During the germination of oats, the major seed storage proteins (globulins) are hydrolyzed by endoproteinases. We have used two methods to characterize these endoproteinases. A qualitative PAGE method that used oat globulins as gel‐incorporated substrates was used to determine which enzymes hydrolyzed the globulins. The proteolytic hydrolysis products were studied by hydrolyzing the globulins in vitro with the endoproteinases and analyzing the products by SDS‐PAGE. Class‐specific proteinase inhibitors were used to show that the globulin hydrolyzing enzymes were cysteine‐class proteinases. The proteinases were active at pH 3.8. Using the gel analysis method, a little activity was present at the beginning of seed germination, but the major activity only appeared on the sixth day of germination. Extracts from four‐day germinated oats contained cysteine proteinases that hydrolyzed the globulins in vitro to form a polypeptide of intermediate size (MW ≈34,500). Cysteine proteases from an eight‐day germinated sample totally hydrolyzed the globulins in <1 hr. Very little hydrolysis occurred at pH 6.2, the pH of germinated oats endosperm tissue. The fact that hydrolysis occurred quickly at pH 3.8 implies that there is probably pH compartmentalization within the endosperm, with some areas of the seed having a low pH value where the globulins can be degraded.     

Biochemical characterization of amandin,the major storage protein in almond (Prunus dulcis L.)

The almond major storage protein, amandin, was prepared by column chromatography (amandin-1), cryoprecipitation (amandin-2), and isoelectric precipitation (amandin-3) methods. Amandin is a legumin type protein characterized by a sedimentation value of 14S. Amandin is composed of two major types of polypeptides with estimated molecular weights of 42-46 and 20-22 kDa linked via disulfide bonds. Several additional minor polypeptides were also present in amandin. Amandin is a storage protein with an estimated molecular weight of 427,300 +/- 47,600 Da (n = 7) and a Stokes radius of 65.88 +/- 3.21 A (n = 7). Amandin is not a glycoprotein. Amandin-1, amandin-2, and amandin-3 are antigenically related and have similar biochemical properties. Amandin-3 is more negatively charged than either amandin-1 or amandin-2. Methionine is the first essential limiting amino acid in amandin followed by lysine and threonine.     

11S and 7S globulins of coconut (Cocos nucifera L.): purification and characterization

Total globulins extracted with 0.4 M NaCl in buffer from coconut endosperm separated into two peaks on gel filtration: peak I corresponding to 11S globulin or cocosin and peak II to 7S globulin with native molecular weights of 326 000 and 156 000, respectively. The percent composition of total globulins was estimated to be 11S, 86% and 7S, 14%. On SDS-PAGE, cocosin resolved into two closely migrating bands at approximately 34 000 (acidic polypeptide) and another set of 2 bands at 24 000 (basic polypeptide). Each set consisted of one darkly stained band and one lightly stained band. The 7S globulin consisted of three bands of 16 000, 22 000, and 24 000. Three isoforms of cocosin were identified after anion exchange chromatography. Cocosin, but not the 7S, was found to have disulfide bonds. Using periodic acid-Schiff's reagent, all of the bands of cocosin on SDS-PAGE were positive for carbohydrate. However, when con A-peroxidase was used, only the basic polypeptide stained positively for carbohydrate. For the 7S globulin, no carbohydrate group was detected using the PAS and con A-peroxidase tests. The 7S globulin was easily extracted with 0.10-0.15 M NaCl, whereas cocosin was extracted with 0.35 M NaCl. The N-terminal amino acid sequences of the 34 k band and 24 k band of cocosin were SVRSVNEFRXE and GLEETQ, respectively, and that of the 7S was EQEDPELQK.     

Purification and characterization of a mannan-binding lectin specifically expressed in corms of saffron plant (Crocus sativus L.)

Despite the economical interest of Crocus sativus, its biochemistry has been poorly studied. Herein, we have isolated a lectin present in saffron corm by gel-filtration, anion-exchange, and reversed-phase chromatography. One- and two-dimensional PAGE, MALDI-MS, and N-terminal amino acid sequence analyses indicated that the native protein forms noncovalently linked aggregates of about 80 kDa apparent molecular mass, mainly composed of two charged heterogeneous (pI's, 6.69-6.93) basic subunits of approximately 12 kDa. Their N-terminal sequences shared 25% similarity and were homologous to the N- and C-terminal domains of monocotyledonous mannose-binding lectins, respectively. An additional polypeptide of around 28 kDa apparent molecular mass was also detected, probably corresponding to a precursor processed into two mature subunits. In addition, the N-terminal domain subunit exhibited 56% similarity with curculin, a sweet protein with taste-modifying activity. The native lectin specifically interacts with a yeast mannan and is a major corm protein specifically expressed in this organ.