Vicilin Fraction Research Articles

Characterization of pea vicilin. 2. Consequences of compositional heterogeneity on heat-induced gelation behavior.

The gelling characteristics of two vicilin fractions from pea (Pisum sativum L.) were compared over a range of pH and salt conditions after preliminary results showed that despite having equal opportunity to unfold, and expose hydrophobic residues, they had different minimum gelling concentrations (at pH 7.6). Furthermore, at this pH one fraction formed turbid gels and the other formed transparent gels. The fraction that formed transparent gels contained a substantial amount of the 70 kDa alpha-subunits of vicilin, and thus it was hypothesized that the highly charged N-terminal extension region on these 70 kDa subunits hinders gelation of this vicilin fraction at pH 7.6 and I = 0.2 due to repulsion of the net negative charge. The experiments designed to test this hypothesis are presented and discussed in this paper and prove that the hypothesis was true, which offers the possibility to control or modify the gelation behavior of vicilin on the basis of information of its subunit composition.

Characterization of pea vicilin. 1. Denoting convicilin as the alpha-subunit of the Pisum vicilin family.

Vicilin, a major globulin protein of pea that has been described as "extremely heterogeneous in terms of its polypeptide composition", was extracted from pea flour under alkaline conditions and subsequently fractionated by salt under acid conditions. This procedure induced the separation of vicilin into two fractions, which, after purification, were called vicilin 1 degrees and vicilin 2 degrees. Vicilin 2 degrees was seen on SDS-PAGE to contain the third globulin protein of pea, convicilin (a band at approximately 70 kDa). Vicilin fractions were thus characterized using gel electrophoresis, differential scanning calorimetry, circular dichroism, and pH-dependent solubility in order to determine whether the convicilin should in fact be considered as a third separate globulin protein of pea. On the basis of the results obtained it was concluded that this distinct polypeptide of the Pisum vicilin gene family should be further denoted as a subunit of the salt extractable protein vicilin. The definition of vicilin heterogeneity should therefore be extended to acknowledge the possible oligomeric inclusion of the 70 kDa polypeptide that is here denoted as the alpha-subunit.

Vicilin-class globulins and their degradation during cocoa fermentation

Cocoa beans were fermented for 144 h using shallow wooden boxes at ambient temperature. Two major polypeptides were found to consist of the storage protein and an albumin fraction. The storage protein comprises two vicilin fractions with molecular weights of 47.1 and 39.2 kDa, and the albumin fraction has a molecular weight of 21.1 kDa. The degradation of vicilin fractions during the course of fermentation was visually detected by sodium docecyl sulphate-polyacrylamide gel electrophoresis. The albumin fraction was found to be the most resistant to proteolysis during fermentation. At the end of fermentation, the 39.2 kDa polypeptide was completely degraded but the 47.1 kDa polypeptide was still present at low intensity. The protein concentrations of 47.1 and 39.2 kDa polypeptides decreased from 1.74 to 0.03 μg and from 0.93 to 0.02 μg, respectively. The protein concentration of 46 and 46.5 kDa polypeptides increased from 0.06 to 0.34 μg and from 0.03 to 0.23 μg, respectively. This could be due to the result of the degradation products of the 47.1 kDa polypeptide.

Seed protein content and composition of near-isogenic and induced mutant pea lines

AbstractTotal protein content and amounts of albumin, legumin and vicilin have been determined for pea seeds from lines near-isogenic except for genes at therugosusloci,randrb(RR/RbRb; rr/RbRb; RR/rbrb; rr/rbrb). Seeds with the wildtype, round-seeded phenotype (RR/RbRb) had less protein on a total seed dry-weight basis than any of the wrinkled-seeded lines and this protein had a lower proportion of albumin. The lines which had recessive alleles at bothrandrbloci had the highest proportion of protein and the highest proportion of albumin. The roundseeded peas possessed nearly two-fold more legumin than the double recessive line, with proportions for the two single recessive lines falling in between these extremes. Vicilin levels were similarfor all four near-isogenic lines. SDS-PAGE analysis of the isolated albumin, legumin and vicilin fractions revealed no significant differences between the four lines. Differential scanning calorimetry of protein extracts showed that all the wrinkled-seeded near-isolines possessed legumin fractions with diminished thermal stability relative to that from the roundseeded, wild-type line.Chemically-induced mutants were also analysed for protein content and composition. These mutants have previously been shown to display great variation in starch and lipid levels. Total protein varied from 20.3% to 37.9%; however, relative proportions of albumin, legumin and vicilin were similar in all mutant lines. SDS-PAGE analysis identified two mutant pea lines which possessed a legumin A-chain of 65 000 Mras well as the typical 45 000 Mrform. Differential scanning calorimetry of protein extracts indicated that the legumin in all mutants had lower enthalpies of denaturation than the legumin in the round-seeded parent.The mutant pea lines possess exceptional variation with respect to starch, lipid and protein which raises opportunities for their use in the food and animal feedstuff industries.

Cloning, expression, and crystallization of jack bean (Canavalia ensiformis) canavalin.

Canavalin is the major storage protein of the jack bean (Canavalia ensiformis) and belongs to the classical vicilin fraction. A full-length cDNA for canavalin was generated by the polymerase chain reaction. The nucleotide sequence coding for canavalin and the corresponding amino acid sequence were determined and shown to be homologous with those of other seed storage proteins. The amino acid sequence contained an internal sequence duplication corresponding to the structural redundancy in the monomer demonstrated by crystallographic analysis. The coding region of the canavalin cDNA was inserted into a T7 RNA polymerase expression vector and used to transform Escherichia coli. A recombinant protein with a molecular mass of 47 kilodaltons was expressed and purified to 95% homogeneity. The protein exhibited the same physical, immunological, and biochemical properties as native jack bean canavalin. Recombinant canavalin, following treatment with trypsin, was crystallized in two forms. Crystals of a rhombohedral habit grew to 1 mm in the longest dimension and diffracted to beyond 3-A resolution. Three-dimensional diffraction data demonstrated crystals of the recombinant protein to be isomorphous with crystals of the natural plant protein, thereby confirming the identity of their structures.

Characterization of Oat Vicilin-Like Polypeptides

The 7S and 3S globulin fractions were extracted and characterized from Avena sativa L. seeds which showed similar solubility characteristics and holoprotein size to those of the vicilin fraction in legumes. These holoproteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectrofocussing, and two-dimensional electrophoresis. Among the components of the oat reserve globulins, the 7S fraction was capable of binding to concanavalin A-Sepharose, thus indicating it to be a glycoprotein. This pattern of the glycosylation of the reserve proteins in oat resembles that observed in certain legume seeds such as Pisum sativum and Glycine max. The results support the notion that the protein components of cereal and legume globulins may be homologous.

Control of storage-protein synthesis during seed development in pea (Pisum sativum L.).

The tissue-specific syntheses of seed storage proteins in the cotyledons of developing pea (Pisum sativum L.) seeds have been demonstrated by estimates of their qualitative and quantitative accumulation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and rocket immunoelectrophoresis respectively. Vicilin-fraction proteins initially accumulated faster than legumin, but whereas legumin was accumulated throughout development, different components of the vicilin fraction had their predominant periods of synthesis at different stages of development. The translation products in vitro of polysomes isolated from cotyledons at different stages of development reflected the synthesis in vivo of storage-protein polypeptides at corresponding times. The levels of storage-protein mRNA species during development were estimated by 'Northern' hybridization using cloned complementary-DNA probes. This technique showed that the levels of legumin and vicilin (47000-Mr precursors) mRNA species increased and decreased in agreement with estimated rates of synthesis of the respective polypeptides. The relative amounts of these messages, estimated by kinetic hybridization were also consistent. Legumin mRNA was present in leaf poly(A)+ RNA at less than one-thousandth of the level in cotyledon poly(A)+ (polyadenylated) RNA, demonstrating tissue-specific expression. Evidence is presented that storage-protein mRNA species are relatively long-lived, and it is suggested that storage-protein synthesis is regulated primarily at the transcriptional level.

Characterisation and subunit structures of the vicilin storage proteins of pea (Pisum sativum L.).

Investigations of the vicilin fraction of the storage proteins of pea (Pisum sativum L.) have shown that its major components are a number of protein species of Mr 170 000. Convicilin (Mr 280 000, composed of 71 000-Mr subunits) is a separable component of this fraction. The vicilin proteins are composed principally of approximately equal to 50 000-Mr polypeptides, but also contain a number of smaller polypeptides. The sub-unit polypeptide composition of vicilin changes during seed development quantitatively and qualitatively. Vicilin sub-units have been shown to be synthesised as polypeptides of Mr approximately equal to 50 000 by means of pulse-labelling experiments in vivo, and synthesis of vicilin in vitro directed by mRNA, polysomes and microsomes extracted from pea cotyledons in cell-free translation systems. Polypeptides then undergo two distinct types of proteolytic modification: (a) co-translational removal of a small polypeptide (Mr less than 1000); (b) 'nicking' of polypeptide chains in assembled vicilin molecules, which occurs more than 4 h after their initial synthesis. The basic structure of the vicilin molecule is thus a multimer, possibly a trimer, of approximately equal to 50 000-Mr subunits. The heterogeneity of the initially synthesised 50 000-Mr subunits accounts not only for the several different 50 000-Mr polypeptides found in vicilin, but also for the range of minor polypeptides, since the 'nicking' points will differ among subunits. It also accounts for the observed partial separation of vicilin into different molecular species, since different subunit combinations will give rise to molecules with different properties. Vicilin is also glycosylated and this is a source of further variation.

Comparative Studies on Protein Bodies and Storage Proteins of Pisum sativum L. and Vicia faba L.

Summary Protein bodies of Vicia and Pisum seeds were isolated in aqueous and nonaqueous media. Besides a similar ultrastructure, there are very similar globulin patterns in both species, although quantitative differences occur. Serological relationships suggest partial identities between the vicilins of Vicia and Pisum , and between undefined proteins in the vicilin fraction. Using antisera against Vicia globulins, legumins of both species seem closely related, as do the “pseudolegumins”. Phytohemagglutinins (PHA’s), present in protein bodies, are identical in Vicia and Pisum . Using Concanavalin A we observed different degrees of glycosylation in the main fractions of seed storage globulins of selected legumes. The globulins of Vicia and Pisum showed only a diffuse precipitin line, in which individual components could not be recognized, whereas we observed strong reactions with globulins of Glycine and Phaselous , respectively. Predominant albumins typical of our Pisum material were found in protein bodies. There is, however, no information about possible enzymic activity or a presumptive storage protein function.

Characterisation of the storage protein subunits synthesised in vitro by polyribosomes and RNA from developing pea (Pisum sativum L.)

Polypeptide material has been immunoprecipitated by antivicilin antibodies from translation products of polyribosomes and poly(A)-rich RNA isolated from developing seeds of Pisum sativum in the wheat germ and reticulocyte lysate cell-free synthesis systems. Analysis of this material by SDS-PAGE shows it to consist of three bands, of molecular weights 70,000, 50,000 and 47,000. The in vitro vicilin polypeptides of 70,000 and 50,000 mol. wt. have been shown to be very similar to the 70,000 and 50,000 mol. wt. subunits of vicilin by specific immunoprecipitation, and behaviour on treatment with cyanogen bromide and trypsin. The 50,000 mol. wt. in vitro vicilin polypeptide contains no significant extra sequence compared to the 50,000 mol. wt. vicilin subunit. The 47,000 mol. wt. in vitro vicilin polypeptide has no corresponding subunit in vicilin from mature seeds, but a 47,000 mol. wt. subunit is present in vicilin isolated from developing seeds. Comparison of translation products from polysomes isolated from seeds at middle and late stages of development shows that synthesis of the 50,000 and 47,000 mol. wt., but not 70,000 mol. wt. polypeptides is very much reduced at late stages of development. These results are discussed with reference to the nature of the vicilin fraction.

The Carbohydrate of Storage Glycoproteins From Seeds of Pisum sativum: Characterization and Distribution on Component Polypeptides

Storage proteins were extracted from isolated protein bodies from mature seeds of Pisum sativum and separated into one legumin and four vicilin fractions. A comparative study was made of the glycosylation of each fraction and all were found to contain covalently attached carbohydrate at a level of between 0.9 and 1.4% of the protein weight. The carbohydrate composition of the legumin fraction was different from that of the vicilin fractions: the major sugar in legumin was glucose (Glc) with some mannose (Man) and glucosamine (GlcN) while, for all vicilin fractions, Man was the major sugar wlth GlcN, Glc and galactose (Gal) in lesser amounts. There were minor differences in carbohydrate composition between vicilin fractions. Each of the vicilin fractions was further separated by concanavalin A (Con A)-Sepharose 4B chromatography into two fractions. The protein which bound to Con A always contained more carbohydrate than the unbound fraction and, in one vicilin fraction, the unbound protein was not glycosylated. No legumin bound to Con A. Both the 20 and 40 kdalton polypeptides of legumin are glycosylated while the 14 kdalton polypeptide was the major site of glycosylation for all vicilin fractions. One vicilin fraction also has a similar amount of carbohydrate associated with a 50 kdalton polypeptide. Some other polypeptides in the vicilins were glycosylated but to a much lesser extent. These findings are compared to those for other legume storage proteins and their significance in storage protein biosynthesis is discussed.

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.)

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.

Cotyledonary Storage Proteins in Pisum Sativum. II. Hereditary Variation in Components of the Legumin and Vicilin Fractions

Gel electrophoresis has been used to investigate genetically controlled variation in storage-protein constituents forming five series of bands (LA-LE) derived from legumin fractions, and three series of bands (VA-VC) from vicilin fractions, of pea seeds. In each variant system, the phenotypes of the storage-protein polypeptides from F1 seeds were additive with respect to the band patterns of the parental lines, and identical in reciprocal crosses. Neither dominance nor formation of new interaction products was observed. Variation in the three systems involving vicilin polypeptides and two of those involving legumin components was found to be based on allelic alternatives at single loci designated Vicilin A (Vca), Vicilin B (Vcb), Vicilin C (Vcc), Legumin A (Lga) and Legumin C (Lgc). For each of these variant systems, the gene products involved and the basis of the phenotypic variation have been discussed. Variants of the VC band complex, in which mobility of two bands both composed of 12 and 14 kdalton polypeptides is altered, appear likely to correspond to vicilin variants described previously. Type lines are specified for each of the variant phenotypes analysed, and for the genes designated.

Cotyledonary Storage Proteins in Pisum sativum. III. Patterns of Accumulation During Development

An antiserum raised against proteins from isolated protein bodies of mature pea seeds recognized seven antigenically distinct protein families in cotyledon extracts analysed by crossed immunoelectrophoresis. This antiserum has been used to follow the sequence of appearance and pattern of accumulation of these proteins in peas grown under controlled environmental conditions. One antigen present in mature cotyledons was shown to be a haemagglutinin, one corresponded to legumin, and four distinct, non-crossreacting antigenic species were related to the vicilin series of proteins. Both the number of components detected and the order of their appearance during development were similar in three genotypes examined, but the quantitative representation of legumin relative to other components was genotype-specific. Quantitative differences were observed in the polypeptide composition of the vicilin components responsible for separate antigenic specificities, but qualitative differences were not excluded. During development, similar conspicuous quantitative changes in polypeptide composition, accompanied by a change in mean electrophoretic mobility, occurred amongst the molecules carrying the predominant antigenic determinant related to vicilin (peak 4); again, qualitative differences might also distinguish these components. The antigen responsible for peak 6 contained two major polypeptides distinct from the components of the legumin and vicilin fractions. Peak 6 represented the first fully immunocompetent species to appear during development, but later became quantitatively less significant as the other antigenic species accumuIated. Legumin and the antigen forming peak 6 were first detected immunologically in an apparently incomplete form. These putative precursors disappeared during subsequent development, so that only the corresponding stable precipitin peaks were seen in the immunoelectrophoretic profiles of extracts from mature cotyledons.

Cotyledonary Storage Proteins in Pisum sativum. I. Molecular Heterogeneity

The two storage-protein fractions of pea seeds, legumin and vicilin, have each been resolved electrophoretically on a cellulose acetate gel matrix into multiple molecular species distinguished by electrophoretic mobility and by quantitative or qualitative differences in subunit composition or both. Electrophoretograms of these apparent holoproteins from a range of lines and cultivars were found to be genotype-specific, generally showing three strong bands, together with up to three additional minor bands, assignable to a vicilin series of components. A further three or four bands could be assigned to a legumin series, although the slowest of these showed apparent admixture of certain polypeptides typical of the vicilin fractions. Each putative holoprotein band in both the legumin and vicilin series behaved additively in the electrophoretograms of F1 offspring from reciprocal crosses between lines showing distinct patterns. For comparison with the proteins distinguished electrophoretically on cellulose acetate gels, storage proteins from lyophilized protein bodies were fractionated on the basis of differential solubility at various ionic strengths and pH values. The single legumin and three vicilin fractions obtained by this method showed sedimentation velocities typical of the respective holoproteins. No overlap in the polypeptide composition of legumin with that of vicilin fractions was observed. The components represented in the four fractions accounted for all the major polypeptides in total storage-protein extracts and in the bands eluted from cellulose acetate gels. The distinctive polypeptide pattern and electrophoretic mobility of vicilin fraction 4 identified this protein as a contaminant of slow legumin bands in cellulose acetate gels, and as an additional vicilin species not recognized directly from the electrophoretic analysis.