Homology between legumin-like polypeptides from cereals and pea.
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ABSTRACT:
SUBMITTER: Robert LS
PROVIDER: S-EPMC1144785 | biostudies-other | 1985 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The purification and characterization of a minor legumin species from Pisum sativum is described. Electrophoretic data indicate that it corresponds to a legumin subunit pair previously designated L1. The beta-polypeptides of the minor legumin have a phenylalanine N-terminus. This is the first time that an amino acid other than glycine has been reported as the N-terminus of the basic polypeptides from legumin-like proteins from any plant species. Sequence analyses of the isolated alpha- and beta-polypeptides of the minor legumin show that it does not correspond to any of the three legumin gene families that have previously been defined on the basis of DNA hybridizations and genetic analyses.
| S-EPMC1148942 | biostudies-other
Project description:A genomic clone from pea (Pisum sativum L.) contains all of one gene encoding a 'minor' (B-type) legumin polypeptide, and most of a second very similar gene. The two genes, designated LegJ and LegK, are arranged in tandem, separated by approx. 6 kb. A complete sequence of gene LegJ and its flanking sequences is given, with as much of the sequence of gene LegK as is present on the genomic clone. Hybridization of 3' flanking sequence probes to seed mRNA, and sequence comparisons with cDNA species, suggested that gene LegJ, and probably gene LegK, was expressed. The partial amino acid sequences of 'minor' legumin alpha- and beta-polypeptides were used to confirm the identity of these genes. The transciption start in gene LegJ was mapped. The 5' flanking sequence of gene LegJ contains a sequence conserved in legumin genes from pea and other species, which is likely to have functional significance in control of gene expression. Sequence comparisons with legumin genes and cDNA species from Vicia faba and soya bean show that separation of legumin genes into A- and B-type subfamilies occurred before separation of the Viciae and Glycinae tribes.
| S-EPMC1148808 | biostudies-other
Project description:Legumin from pea (Pisum sativum) is a molecule made up of six pairs of subunits, each pair consisting of an ;acidic' subunit (mol.wt. about 40000) and a ;basic' subunit (mol.wt. about 20000) linked by one or more disulphide bonds. The heterogeneity of legumin has been investigated by isoelectric focusing; undissociated legumin could not be focused satisfactorily, but legumin subunits could be analysed under dissociating conditions. 8m-Urea was not found to be a satisfactory medium for isoelectric focusing of legumin, as the ;basic' subunits showed a shift in pI with time of incubation in urea. A new dissociating medium for isoelectric focusing, namely 50% (v/v) formamide, was used for analysis of legumin, which gave pI values of 5.0-5.3 for the ;acidic' subunits, and 8.3-8.7 for the ;basic' subunits. Both types of subunits were shown to be heterogeneous in charge and molecular weight by two-dimensional analysis employing isoelectric focusing in the first dimension and sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the second. The ;basic' and ;acidic' subunits of legumin were separated on the preparative scale by ion-exchange chromatography in 50% formamide. Carbohydrate attached to the protein was investigated as a possible cause of the heterogeneity of legumin subunits. However, both a fluorescent-labelling technique and a sensitive radioactive-labelling technique failed to show any carbohydrate bound to legumin subunits, and it was concluded that legumin is not a glycoprotein.
| S-EPMC1161378 | biostudies-other
Project description:In isolates from different pea cultivars, the legumin-to-vicilin (L:V) ratio is known to vary from 66:33 to 10:90 (w/w). In this study, the effect of variations in the L:V ratio on the pea protein emulsifying properties (emulsion droplet size (d3,2) vs protein concentration (Cp)) at pH 7.0 was investigated using a purified pea legumin (PLFsol) and pea vicilin fraction (PVFsol). Despite a different Γmax,theo, the interfacial properties at the oil-water interface and the emulsifying properties were similar for PLFsol and PVFsol. Hence, the L:V ratio did not affect the pea protein emulsifying properties. Further, PLFsol and PVFsol were less efficient than whey protein isolate (WPIsol) in stabilizing the emulsion droplets against coalescence. This was explained by their larger radius and thus slower diffusion. For this reason, the difference in diffusion rate was added as a parameter to the surface coverage model. With this addition, the surface coverage model described the d3,2 versus Cp of the pea protein samples well.
| S-EPMC10375591 | biostudies-literature
Project description:The potential of immunoaffinity chromatography as a means of purifying legumin from a wide range of Pisum (pea) types was assessed. The method required small amounts of highly purified legumin from a single Pisum type, and this was obtained by salting out with (NH4)2SO4 followed by zonal isoelectric precipitation, ion-exchange chromatography on DEAE-cellulose and sucrose-density-gradient centrifugation. Some physiocochemical properties of purified legumin were determined, a number of which (Strokes radius, subunit molecular weights, subunit N-terminal residues and subunit molar ratios) have not previously been reported for Pisum legumin. Examination of Pisum legumin by two-dimensional gel isoelectric focusing/electrophoresis indicated the existence of extensive subunit heterogeneity, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate showed apparent variation in the nature of this heterogeneity from one Pisum variety to another. Despite this variation, immunoaffinity chromatography on immobilized anti-legumin (which was prepared by affinity chromatography on the immubolized purified legumin from the single Pisum type) was shown to be a generally applicable method for the purification of undegraded legumin from a range of pisum types, including two primate lines.
| S-EPMC1186401 | biostudies-other
Project description:The production of superoxide radicals (O2(-).) and the activities of ferricyanide reductase and cytochrome c reductase were investigated in peroxisomal membranes from pea (Pisum sativum L.) leaves using NADH and NADPH as electron donors. The generation of O2(-). by peroxisomal membranes was also assayed in native polyacrylamide gels using an in situ staining method with NitroBlue Tetrazolium (NBT). When peroxisomal membranes were assayed under native conditions using NADH or NADPH as inducer, two different O2(-).-dependent Formazan Blue bands were detected. Analysis by SDS/PAGE of these bands demonstrated that the NADH-induced NBT reduction band contained several polypeptides (PMP32, PMP61, PMP56 and PMP18, where PMP is peroxisomal membrane polypeptide and the number indicates molecular mass in kDa), while the NADPH-induced band was due exclusively to PMP29. PMP32 and PMP29 were purified by preparative SDS/PAGE and electroelution. Reconstituted PMP29 showed cytochrome c reductase activity and O2(-). production, and used NADPH specifically as electron donor. PMP32, however, had ferricyanide reductase and cytochrome c reductase activities, and was also able to generate O2(-). with NADH as electron donor, whereas NADPH was not effective as an inducer. The reductase activities of, and O2(-). production by, PMP32 were inhibited by quinacrine. Polyclonal antibodies against cucumber monodehydroascorbate reductase (MDHAR) recognized PMP32, and this polypeptide is likely to correspond to the MDHAR reported previously in pea leaf peroxisomal membranes.
| S-EPMC1220006 | biostudies-other
Project description:Mixed stand (MS) cropping of pea with small-grain cereals can produce more productive and environment-friendly grain crops relative to pure stand (PS) crops but may require selection to alleviate the pea competitive disadvantage. This study aimed to assess the pea variation for competitive ability and its associated traits and the efficiency of four phenotypic or genomic selection strategies. A set of 138 semi-leafless, semi-dwarf pea lines belonging to six recombinant inbred line populations and six parent lines were genotyped using genotyping-by-sequencing and grown in PS and in MS simultaneously with one barley and one bread wheat cultivar in two autumn-sown trials in Northern Italy. Cereal companions were selected in a preliminary study that highlighted the paucity of cultivars with sufficient earliness for association. Pea was severely outcompeted in both years albeit with variation for pea proportion ranging from nearly complete suppression (<3%) to values approaching a balanced mixture. Greater pea proportion in MS was associated with greater total yield of the mixture (r ≥ 0.46). The genetic correlation for pea yield across MS and PS conditions slightly exceeded 0.40 in both years. Later onset of flowering and taller plant height at flowering onset displayed a definite correlation with pea yield in MS (r ≥ 0.46) but not in PS, whereas tolerance to ascochyta blight exhibited the opposite pattern. Comparisons of phenotypic selection strategies within or across populations based on predicted or actual yield gains for independent years indicated an efficiency of 52-64% for indirect selection based on pea yield in PS relative to pea yield selection in MS. The efficiency of an indirect selection index including onset of flowering, plant height, and grain yield in PS was comparable to that of pea yield selection in MS. A genome-wide association study based on 5,909 SNP markers revealed the substantial diversity of genomic areas associated with pea yield in MS and PS. Genomic selection for pea yield in MS displayed an efficiency close to that of phenotypic selection for pea yield in MS, and nearly two-fold greater efficiency when also taking into account its shorter selection cycle and smaller evaluation cost.
| S-EPMC8495324 | biostudies-literature
Project description:We have determined the complete nucleotide sequence of RNA1 of the tobravirus pea early browning virus [PEBV] from an overlapping series of cDNA clones. The 7073 nucleotide sequence contains four open reading frames [ORFs]. The 5' proximal ORF encodes a 141K polypeptide, and readthrough of the opal [UGA] termination codon of this ORF would lead to the synthesis of a second, 201K polypeptide. Both of these polypeptides have extensive amino acid homology with the putative replicase proteins of tobacco rattle virus [TRV] and tobacco mosaic virus [TMV]. The third ORF encodes a 30K polypeptide which has homology with the TRV 29K and TMV 30K putative cell-to-cell spread proteins. The fourth, 3' proximal ORF encodes a 12K polypeptide which has extensive homology with the TRV 16K protein whose function is unknown. Examination of the amino acid sequences of the 12K and 16K gene products reveals in each the presence of two multiple-cysteine/histidine motifs, a finding which suggests that these proteins might have zinc and/or nucleic acid-binding properties.
| S-EPMC317594 | biostudies-other
Project description:An RNA polymerase activity has been purified from pea (Pisum sativum) chloroplast extracts with a distinct transcriptional specificity for a chloroplast messenger gene. This activity (ms-RNA pol) differs from the pea RNA polymerase preparation reported by Sun, Shapiro, Wu & Tewari [(1986) Plant Mol. Biol. 6, 429-439], which specifically transcribes only the rRNA gene (rb-RNA pol). The specificity of transcription has been assessed by the synthesis in vitro of discrete transcripts of predicted sizes using cloned promoter regions of the chloroplast psbA and 16 S rRNA genes. The ms-RNA pol preparation, with polypeptides ranging in apparent molecular mass from 22 to 180 kDa, correctly initiates transcription from recombinant plasmids containing the psbA promoter and does not support 16 S rRNA promoter-directed transcription. The two activities differ also in their response to Mn2+ ions. To investigate whether the two transcriptional activities share common functional polypeptides, monoclonal antibodies were developed against the rb-RNA pol preparation. Three clones were selected on the basis of their ability to inhibit transcription in vitro of the 16 S rRNA gene by rb-RNA pol. The antibodies from these clones independently recognized three polypeptides with molecular masses of 27, 90 and 95 kDa on immunoblots. Antibodies cross-reacting with the 90 kDa polypeptide completely eliminated the specific retardation of an end-labelled 16 S rRNA promoter fragment in a mobility-shift assay, whereas the antibodies against the 95 kDa polypeptide resulted in the formation of a ternary complex (enzyme-DNA-antibody). The antibodies cross-reacting with the 27 kDa polypeptide, however, did not alter the mobility of the retarded DNA-enzyme complex on the gel. These antibodies also inhibited transcription in vitro of the psbA gene by ms-RNA pol and recognized polypeptides of identical molecular masses in the ms-RNA pol. These results show that the three polypeptides are functional components of the chloroplast transcriptional complex and appear to be involved in the transcription of both rRNA and mRNA genes. Transcriptional specificity is probably conferred by ancillary transcription factor(s) which remain to be identified.
| S-EPMC1132980 | biostudies-other
Project description:Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may provide guidance for a future thermodynamically informed classification of protein homology.
| S-EPMC2845653 | biostudies-literature