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1.
Intact pea (Pisum sativum L.) cotyledons were incubated with [14C]glucosamine at several stages of seed development and the resultant radioactive proteins were analysed by gel electrophoresis combined with immunoaffinity chromatography and sucrose gradient fractionation. Glucosamine was incorporated into at least five vicilin polypeptides (approx. molecular weight 70,000; 50,000, two components; 14,000, two components). No incorporation was detected into the subunits of legumin. Tunicamycin at 50 g/ml largely inhibited glucosamine incorporation but had little effect on the incorporation of 14C-labelled amino acids into cotyledon proteins, including vicilin. The assembly of vicilin polypeptides into full-sized protein oligomers (7–9 S) was also unaffected by tunicamycin. Chromatography on concanavalin A confirmed that glycosylation of cotyledon proteins was inhibited by tunicamycin. It is concluded that glycosylation of most cotyledonary proteins involves lipid-linked sugar intermediates, but that glycosylation itself is not an essential step in the synthesis of vicilin polypeptides nor in their assembly into oligomers.Abbreviations IgG
immunoglobulin G
- M Wt
approximate molecular weight based on electrophoretic mobility relative to that of protein standards
- SDS-PAGE
Na-dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
2.
C. Horstmann B. Schlesier A. Otto S. Kostka K. Müntz 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1993,86(7):867-874
Legumin, which amounts to approximately 55% of the seed protein in field beans (Vicia faba L. var. minor), is a representative of the 12S storage globulin family. The 12S storage globulins are hexameric holoprotein molecules composed of different types of polymorphic subunits encoded by a multigene family. Type-A legumin subunits contain methionine whereas type-B are methionine-free subunits. Sequencing of two different type A-specific cDNAs, as well as an FPLC/HPLC-based improvement of subunit fractionation and peptide mapping with subsequent partial amino-acid sequencing, permit the assignment of some of the polymorphic legumin subunits to members of the multigene family. Two different type A subunits (A1 and A2) correspond to the two different cDNA clones pVfLa129 (A2) and 165 (A1), but microheterogeneity in the amino-acid sequences indicates that polymorphic variants of both representatives of this type may exist. Two groups of published type B-specific gene sequences (LeB7, and LeB2, LeB4, LeB6, respectively) are represented by two polymorphic subunit fractions (B3I, B3II, and B4I, B4II). A seventh clone, LeB3, encodes one of the large legumin subunits that is only a minor component of the legumin seed protein complex. 相似文献
3.
C. De Pace V. Delre G. T. Scarascia Mugnozza F. Maggini R. Cremonini M. Frediani P. G. Cionini 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1991,83(1):17-23
Summary Experiments were carried out on Vicia faba major involving (1) determination of the pattern of legumin accumulation during seed development, (2) protein purification from mature cotyledons, (3) the characterization of legumin mRNA, and (4) the chromosomal localization of the genes coding for legumins. In developing cotyledons the synthesis of legumin begins 28 days after petal desiccation (DAPD), and 4 days after initiation of vicilin synthesis. The two subunits (A and A) of legumin A appear 2 days earlier than those (B and B) of legumin B. While the accumulation of vicilin peaks on the 30th DAPD, that of legumin continues during further seed development, and the synthesis of legumin mRNA peaks on the 37th DAPD. Northern blot hybridizations using two DNA plasmids containing cDNA inserts with sequence homology to the A- and B-type legumin genes, respectively, indicated that legumin mRNAs extracted from cotyledons 36 DAPD band below the 18S RNA band. In addition, a faint band below that of the 25S RNA band can be observed in legumin mRNAs extracted from cotyledons at an earlier developmental stage (30 DAPD). By means of polyacrylamide gel electrophoresis in the presence or absence of SDS and 2-mercaptoethanol, two fractions could be eluted after zonal isoelectric precipitation of the globulins from mature seeds: one fraction contains mainly vicilin, the other, legumin. In situ hybridization showed that legumin genes are arranged in two clusters: the genes coding for legumin A are located in the longer arm of the one between the two shortest subtelocentric chromosome pairs whose centromere is in a less terminal position; those coding for legumin B are located in the non-satellited arm of the longer submetacentric pair. 相似文献
4.
The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation. 相似文献
5.
Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamin/glutamic acid-rich region encoded by the C-terminal part of the chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342–350) and limited own data. 相似文献
6.
Since there is some question as to whether or not legumin is glycosylated, this storage protein was isolated by various procedures from developing cotyledons of Pisum sativum L. supplied with [14C]-labeled glucosamine and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Legumin isolated by the classical method of Danielsson [(1949) Biochem. J. 44, 387–400] a procedure in which globulins extracted with a buffered salt solution are precipitated with ammonium sulfate (70% saturation) and legumin separated from vicilin by isoelectric precipitation, was labeled. The glucosamine incorporated into legumin was associated with low-molecular-weight polypeptides. In contrast, legumin isolated by the method of Casey [(1979) Biochem. J. 177, 509–520], a procedure where legumin is prepared by zonal isoelectric precipitation from globulins precipitated with 40–70% ammonium sulfate, was not labeled. However, the globulin fraction precipitated with 40% ammonium sulfate was labeled and the radioactive glucosamine was associated with low-molecular-weight polypeptides. Legumin isolated from protein bodies [Thomson et al. (1978) Aust. J. Plant Physiol. 5, 263–279] was not extensively labeled. However, the saltinsoluble fraction of protein body extracts was labeled and the radioactivity was associated with low-molecular-weight polypeptides. These results indicate that protein bodies contain a glycoprotein of low-molecular-weight that co-purifies with legumin isolated by the method of Danielsson but that is discarded when isolation methods developed more recently are used. 相似文献
7.
Isoelectric-focusing properties and carbohydrate content of pea (Pisum sativum) legumin 总被引:2,自引:2,他引:2
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. 相似文献
8.
An antiserum specific for the legumin and vicilin of Vicia faba was used to examine extracts of seeds of taxa of the Fabeae and Trifolieae for the presence of related storage proteins, Proteins related to legumin were found to be widely distributed, indicating considerable conservation of the genetic information for this protein. Only Pisum sativum contained a protein immunochemically identical with the vicilin of V. faba; the equivalent proteins of all other genera tested here were immunochemically different from vicilin. 相似文献
9.
We have characterized several Vicia faba genes encoding methionine residue-free group B subunits of the 11S or legumin storage proteins. The respective gene subfamily consists of 10 to 15 members, six of them having been studied by DNA sequence analysis. Four functional genes (LeB2, LeB4, LeB6, LeB7) are highly homologous in their coding region and 0.3 kb of their 3 flanking sequences. On the other hand, two pseudogenes (LeB1, LeB5) have accumulated a large number of mutations including an identical 0.7 kb internal deletion; they are both flanked by a repetitive element. Analysis of sequence changes show that transitions are nearly double as frequent as transversions. CpG is the most infrequent dinucleotide whereas TpA is significantly underrepresented in exon sequences. End points of deletions are correlated with short direct repeats and preferentially found in the two introns. Our studies indicate that the Vicia faba legumin B gene subfamily contains a group of expressed, highly homologous genes as well as more diverged pseudogenes. 相似文献
10.
11.
MATTA NARENDER; GATEHOUSE JOHN A.; BOULTER DONALD 《Journal of experimental botany》1981,32(1):183-197
Two-dimensional electrophoretic studies have shown a wide rangeof heterogeneity in the subunits of legumin isolated from seedsof Vicia faba L. As many as 10 disulphide-linked subunit pairsin the mol. wt. range 37 00079 000 have been observed.Each subunit pair separated on reduction by 2-mercaptoethanolinto a large acidic and a small basic subunit, each of whichwas shown to be heterogeneous in charge by isoelectric focusing.More heterogeneity was found in the large subunits (mol. wt.range 23 00058 000; pl range 4.66.1) than in thesmall subunits (mol. wt. range 21 00023 000; pl range8.28.5). Most legumin molecules seemed to be formed byrandom association of subunit pairs, although one subunit pairassociated only with itself to give a molecular type separableunder non-dissociating conditions. 相似文献
12.
Analyses of heterogeneity in legumin subunit patterns, legumin precursor polypeptides, and restriction fragments containing legumin genes have shown thatPisum (pea) genotypes vary in the extent of gene and polypeptide divergence but not in the degree of gene reiteration. Genotypes containing single and multiple M subunits had the same numbers of legumin genes. The potential value of this heterogeneity in genetical analyses is outlined.This work was supported by the Agricultural and Food Research Council via a grant-in-aid to the John Innes Institute. We acknowledge financial support from Agrigenetics Corporation, Boulder, Colorado, and from the CEC Biomolecular Engineering Programme, Contract GBI-4-113-UK. 相似文献
13.
Glutamine synthetase (GS) of Rhodopseudomonas sphaeroides is regulated by adenylylation and deadenylylation. The extent of adenylylation/deadenylylation of the enzyme in cell free extracts was influenced by inorganic phosphate (P
i), -ketoglutarate, ATP and other nucleotides. While P
i and -ketoglutarate stimulated deadenylylation, ATP and other nucleotides enhanced adenylylation of the GS. By using proper combinations of the effectors and incubation conditions, any desired adenylylation state of GS could be adjusted in vitro. The enzyme was purified to electrophoretic homogenity by three steps including affinity chromatography on 5-AMP-Sepharose. Adenylylated and deadenylylated enzyme showed different UV-spectra and isoelectric points. The native enzyme had a molecular weight of 600,000, deadenylylated subunits of 50,000±1,000. Electron microscopic investigations revealed a dodecameric arrangement of subunits in two hexameric planes. 相似文献
14.
Anne E. Simon Karen M. Tenbarge Steve R. Scofield Ruth R. Finkelstein Martha L. Crouch 《Plant molecular biology》1985,5(3):191-201
Summary The most abundant protein in seeds of Brassica napus (L.) is cruciferin, a legumin-like 12S storage protein. By in vitro translation of embryo RNA, and pulse-chase labelling of cultured embryos with 14C-leucine, we have shown that the 30 kd polypeptides and 20 kd polypeptides of cruciferin are synthesized as a family of 50 kd precursors which are cleaved post-translationally. One member of the cruciferin family was cloned from embryo cDNA and sequenced. The nucleotide sequence of the cruciferin cDNA clone, pC1, contains one long open reading frame, which originates in a hydrophobic signal peptide region. Therefore, the complete sequence of the cruciferin mRNA was obtained by primer extension of the cDNA. The predicted precursor polypeptide is 488 amino acids long, including the 22 amino acids of the putative signal sequence. The amino acid composition of cruciferin protein is very similar to the predicted composition of the precursor. Comparison with an amino acid sequence of legumin from peas, deduced from the nucleotide sequence of a genomic clone, shows that the polypeptide precedes the polypeptide on the precursor. Cruciferin and legumin share 40% homology in the regions which can be aligned. However, cruciferin contains a 38 amino acid region high in glutamine and glycine in the middle of the subunit, which is absent in legumin. Legumin has a highly charged region, 57 amino acids long, at the carboxyl-end of the subunit, which is not found in cruciferin. Both of these regions appear to have originated by reiteration of sequences. re]19850513 ac]19850715 相似文献
15.
A. D. Shutov I. A. Kakhovskaya H. Braun H. Bäumlein K. Müntz 《Journal of molecular evolution》1995,41(6):1057-1069
Legumin-like 11S and vicilin-like 7S globulins are the main storage proteins of most angiosperms and gymnosperms. The subunits of the hexameric legumin are synthesized as a precursor comprising a N-terminal acidic - and a C-terminal basic -chain. The trimeric vicilin molecule consists of subunits composed of two symmetrical N- and C-terminal structural domains.In a multiple alignment we have compared the N-terminal and C-terminal domains of 11 legumns and seven vicilins of several dicot, monocot, and gymnosperm species. The comparisons using all six possible pairwise combinations reveal that the N-terminal and C-terminal domains of both protein families are similar to each other. These results together with data on the distribution of variable and conserved regions, on the positions of susceptible sites for proteolytic attack, as well as on the published 7S protein tertiary structure suggest that both protein families share a common single-domain ancestor molecule and lead to the hypothesis that a triplication event has occurred during the evolution of a putative legumin/vicilin ancestor gene.Moreover, the comparison of the intron/exon pattern reveals that at least three out of five intron positions are precisely conserved between the genes of both protein families, further supporting the idea of a common evolutionary origin of recent legumin and vicilin encoding genes.
Correspondence to: H. Bäumlein 相似文献
16.
《Plant science》1986,46(3):159-167
The effects of abscisic acid (ABA), high osmotica, fluridone (an inhibitor of carotenoid biosynthesis), gibberellic acid (GA3) and an inhibitor of gibberellin biosynthesis, paclobutrazol (1-(4-chlorophenyl)-4,4-dimethyl-2-(1-24-triazol-1-yl)pentan-3-ol) on storage protein accumulation were studied in developing Vicia faba L. cotyledons cultured for 2 or 3 days in vitro. Extracts of these cotyledons were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. ABA stimulated the accumulation of vicilin and legumin polypeptides. GA3 did not noticeably stimulate the accumulation of any polypeptide. There was stimulation of vicilin and legumin polypeptide accumulation by high osmoticum (18% sucrose), which was further enhacedd by ABA and inhibited by fluridone. The fluridone inhibition was reversed by ABA addition.The data provides evidence that ABA modulates the synthesis of V. faba storage proteins. 相似文献
17.
Summary The deduced amino acid sequences of the -polypeptides ofPisum legumin from two loci on chromosome 1 were compared with one from a locus on chromosome 7. The chromosome 1-derived sequences were 80% identical, but each was only 50% homologous to the chromosome 7-derived sequence. Comparison of these sequences with those of homologous polypeptides from two other species of the Leguminoseae showed that the chromosome 1-derivedPisum sequences were more similar to legumin B than to legumin A fromVicia faba and were more closely related to group II than to group I glycinins fromGlycine max. The converse was true for the chromosome 7-derivedPisum sequences. This suggests that divergence of legumin-like sequences predated speciation in these three members of the Leguminosease.Among the threePisum sequence classes, a highly variable region was identified within the -polypeptide, just to the amino-terminal side of the processing site. This region varied considerably in length within the three classes ofPisum -polypeptide sequence, a variation which far exceeded that which has previously been described for other legumins and glycinins. The chromosome 7-derived, and one of the chromosome 1-derived -polypeptide sequences contained different tandem repeats in this region. 相似文献
18.
Seed storage proteins are thought to be accumulated exclusively in the cell-expansion phase of embryogenesis and metabolized during germination and seedling growth. Here we show by a sensitive immunohistological technique that the two Vicia faba L. storage proteins vicilin and legumin are accumulated in substantial amounts in the suspensor and coenocytic endosperm and to a lesser extent in the mid-globular embryo. Both proteins appear and disappear at precise stages specific for each tissue. In the endosperm the accumulation starts around 12 d after pollination (DAP). After a maximum attained at 14–15 DAP, storage proteins are degraded within about 4 d. Accumulation is restricted to that part of the endosperm which covers the embryo and displays the highest levels of endoploidy (maximum 96n). In all other parts of the endosperm, storage proteins do not appear to accumulate, although storage-protein-specific mRNA synthesis takes place. In the suspensor, storage proteins are already observed at 6 DAP and disappear very quickly at approximately 10 DAP. Low amounts of legumin and vicilin are also detectable in the mid-globular embryo, but disappear completely as the embryo enters the heart stage. We conclude that storage proteins of Vicia faba accumulated transiently during early seed development are used as nutritive reserves for the growing embryo.Abbreviation DAP
days after pollination
Dedicated to Prof. Rigomar Rieger in the occasion of his 65th birthdayThis research was supported by the Ministry of Science and Research, Land Sachsen-Anhalt, Germany. U.W. acknowledges additional support by the Fonds der Chemischen Industrie. 相似文献
19.
The major storage protein of jackbean (Canavalia ensiformis) has been purified by a protocol involving ammonium-sulphate precipitation, gel filtration and ion-exchange chromatography. The protein was shown by partial amino-acid-sequence data to be homologous to vicilin, a major storage protein of pea (Pisum sativum), and is thus a member of the family of legume 7S proteins exemplified by pea vicilin. This protein is thus referred to as jack-bean vicilin rather than canavalin or precanavalin as previously used. Other properties of the jack-bean vicilin (e.g. subunit relative molecular mass (Mr) and structure, resistance to proteolysis) show similarity to phaseolin, the major 7S storage protein ofPhaseolus vulgaris. Jack-bean vicilin contained no detectable -mannosidase activity, either as isolated from mature or germinating seeds, or after proteolytic treatment. -Mannosidase was also purified from jack beans, and was shown to have a subunit Mr of approx. 120,000; it was separated completely from jack-bean vicilin by a similar protocol to that used for purifying the latter. The -mannosidase was proteolytically cleaved after seed germination, but did not give polypeptides of the same Mr as jackbean vicilin. It was concluded that -mannosidase and jack-bean vicilin are not related proteins.Abbreviations DE
diethylaminoethyl
- M
relative molecular mass
- SDS
sodium dodecyl sulphate
- PAGE
polyacrylamide-gel electrophoresis 相似文献
20.
The effects of differemt S and methionine regimes on growthof developing Vicia faba cotyledons in vitro were studied. Basalmedium (containing adequate S) supplemented with 05 mM methioninemarginally increased d. wt and uncombined amino acid accumulationbut adding 15 mM methionine inhibited both growth andprotein accumulation. Sulphur deficiency reduced both d. wtand protein accumulation but incresed accumulation of uncombinedamino acids. Adding 1 mM methionine to the S-deficient mediumrestored growth, normal protein and uncombined amino acid acnunulation.High sulphate medium (7.5 mM ) decreased d. wt, protein anduncombined amino acid accumulation. High sulphate medium or basal medium+methionine (05 mM) changedthe proportions of the seed proteins; legumin increased butvicilin decreased. Sulphur deficiency caused a relative increasein vicilin but a decrease in legumin. The different S and methionineregimes markedly changed the composition of the uncombined aminoacids, especially those derived from aspartic acid but not thecomposition of the protein fraction, except during S deficiency. The data presented indicates a flexibility in the storage proteincomposition of developing cotyledons grown in vitro, with theS and methionine status having a regulatory effect. Vicia faba L., field bean, cotyledon, growth, in vitro culture, uncombined amino acids, protein composition, legumin, vicilin, methionine, sulphur 相似文献