Vicilin Subunits Research Articles

Narbonin, a 2 S globulin from Vicia narbonensis L. : Crystallization and preliminary crystallographic data

A seed globulin from Vicia narbonensis L. has been crystallized by vapour diffusion induced pH-shift. Crystals are suitable for high-resolution X-ray structural analysis and diffract to better than 1.5 A. Narbonin crystallizes in the monoclinic space group P21 with alpha = 46.9 A, b = 75.5 A, c = 50.9 A, alpha = gamma = 90 degrees, beta = 120.5 degrees. The protein consists of one polypeptide chain that does not coincide with the subunits of legumin or vicilin after SDS/polyacrylamide gel electrophoresis and has a relative molecular mass of about 33,000.

The problem of refolding and reassembling recombinant vicilin subunits

The reassociation of a storage protein (pea (Pisum sativum L) vicilin) expressed in yeast (Saccharomyces cerevisiae MC16 (ade 2–1, leu 2–3, lys 2–1, his 4–712FS, SUF 2)) was investigated. The corresponding protein in French, bean (Phaseolus vulgaris L), termed phaseolin, was used as a model to produce a methodology for the refolding of the recombinant protein. Phaseolin was successfully renatured from its dissociated state in 5 M guanidinium chloride. Application of this rationale to yeast-derived vicilin proved unsuccessful. The nature of the yeast vicilin and its failure to refold and reassemble correctly are discussed together with the further experimental approaches required.

Inheritance and mapping of vicilin storage protein genes in Pisum Sativum L

Gel electrophoresis techniques have been used to investigate genetically controlled variation in the major subunits (Mr 50,000) of vicilin, a storage protein of Pisum sativum L. The F1 protein band patterns were shown to be additive with respect to those of the parental lines and to be identical in reciprocal crosses. Genetic analysis of the F2 plants indicated that the 50,000 Mr vicilin subunit band pattern is controlled by a pair of co-dominant genes at single locus. The F2 data were used to locate this major vicilin gene locus (Vc-1) to chromosome 7, closely linked to the r locus (for round and wrinkled seed surface).

Sequence interrelationships of the subunits of vicilin from pea seeds

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than Mr∼50 000 (i.e., Mr 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within Mr∼50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than Mr∼50 000 arose by endoproteolytic cleavage of parent molecules within the Mr∼50 000 group. Cleavage in different Mr 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than Mr∼50 000, with the possible exception of the Mr34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the Mr∼50 000 group.

The post-translational proteolysis of the subunits of vicilin from pea (Pisum sativum L.)

Tryptic-peptide profiles and amino acid sequencing of purified pea (Pisum sativum L.) vicilin subunits were used to show that their sequences were interrelated. Comparison with the nucleotide sequence of a cloned vicilin complementary DNA (mRNA) showed that all vicilin subunits could be derived from 50 000-Mr precursors containing up to two sites for post-translational proteolytic cleavage, and allowed these subunits to be located relative to the precursor.

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.

The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.).

A third storage protein, distinct from legumin and vicilin, has been purified from the seeds of pea (Pisum sativum L.). This protein has been named 'convicilin' and is present in protein bodies isolated from pea seeds. Convicilin has a subunit mol.wt. of 71 000 and a mol.wt. in its native form of 290 000. Convicilin is antigenically dissimilar to legumin, but gives a reaction of identity with vicilin when tested against antibodies raised against both proteins. However, convicilin contains no vicilin subunits and may be clearly separated from vicilin by non-dissociating techniques. Unlike vicilin, convicilin does not interact with concanavalin A, and contains insignificant amounts of carbohydrates. Limited heterogeneity, as shown by isoelectric focusing, N-terminal analysis, and CNBr cleavage, is present in convicilin isolated from a single pea variety; genetic variation of the protein between pea lines has also been observed.

Pulse-labeling Studies on Protein Synthesis in Developing Pea Seeds and Evidence of a Precursor Form of Legumin Small Subunit

Intact cotyledons were taken from pea seeds at various stages during seed development and pulse-labeled with (14)C-amino acids. Salt-soluble proteins then were extracted and fractionated on Na dodecyl sulfate-polyacrylamide gels. Storage proteins in these extracts were identified by their binding to immunoaffinity columns. The labeling studies showed that the synthesis of storage protein polypeptides accounts for a major part of total protein synthesis of developing cotyledons between 10 and 22 days after flowering. The distribution of the incorporated radioactivity between individual storage protein polypeptides varied with stage of development. For example, the synthesis of the 50 kilodalton complex of vicilin subunits dominated the early stages of protein accumulation but was a negligible proportion of the total incorporation in the later stages. On the other hand, the 75 kilodalton vicilin subunit was synthesized throughout this entire period. The major small subunit of legumin (20 kilodaltons) was not detected by either Coomassie blue staining or by 2-hour labeling during this period. It was found to arise during the desiccation phase of seed maturation from a long-lived precursor with a relative electrophoretic mobility equivalent to 19 kilodaltons.

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.

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.

Studies on Seed Globulins from Legumes VII. Narbonin, a 2S Globulin from Vicia narbonensis L.

Narbonin ,isolated by zone precipitation and purified by gel chromatography and crystallization, has a molecular weight of 33500 ± 1500 D according to ultracentrifugation. Isoelectric focusing in polyacrylamide gels reveals an isoelectric point of p H 5.17 ± 0.10. The absorption coefficient at 281 nm is A 1% 1 cm = 11.1. It consists of only one polypeptide chain that does not coincide with the subunits of legumin and vicilin after SDS gel electrophoresis. No serological similarities have been observed between narbonin and other seed globulins.

The structure of vicilin of Vicia faba

Four sub-units of vicilin were separated by sodium dodecyl sulphate gel electrophoresis and their molecular weights determined by calibration. The amino acid composition of vicilin was determined by ion-exchange chromatography and by other methods for half-cystine and tryptophan. Tryptic peptide maps and N-terminal analysis following cyanogen bromide cleavage, were used to deduce a chemical molecular weight of vicilin of 100–130 × 10 3. These structural results on vicilin are discussed in relationship to its biological role.

Analysis of protein complex and isoflavones content of the soybean and chickpea seed in the connection of food direction breeding

Aim. The goal of research was to study the features of protein complex and isoflavones content in the soybean and checkpea seed for development the characterictics of legume selection of food direction. Methods. 7S and 11S globu-lins were separated by method, which was developed in the Laboratory of Plant Biochemistry (Patent # 42181). Protein content was determined by Kjeldahl method. Isoflavones content were determined by spectrophotometric method. Results. The features of protein complex and isoflavones content in the soybean and checkpea seed of genotypes of Ukrainian and foreign plant breeding were researched. It was shown that the studied genotypes of soybean, chickpea differ widely in the content of total protein, 7S and 11S globulins and isoflavones. The polymorphism between varieties on the electrophoresis spectrums of component composition of 7S and 11S globulins of soybean was established. The features of 7S and 11S globulins content, its ratio and component composition of genotypes of different phylogenesis origin, F3.F8 hybrids of soybean and their paternal forms were identified. It was established that soybean and checkpea genotypes of different phylogenesis origin were characterized by different content in the component composition of storage proteins of α, α', β subunits of β-conglycinin, А3, А5, А, B components of glycinin, 2S albumin, vicilin subunit (50 kDa), legumin subunit (20 kDa). These subunits and components have negative and positive influence on the man health. Conclusions. The got results open fundamentally new approach of estimation of legumes on a quality of seed and can be used for authetication of legume varieties of the food direction. Keywords: Legume, 11S globulin, 7S globulin, isoflavones.