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1.
A glycoprotein (GP57) was purified by ion-exchange and hydroxylapatite column chromatography from the 70%-ethanol precipitate of culture medium of non-embryogenic carrot cells (Daucus carota L.) grown with 2,4-dichlorophen-oxyacetic acid (2,4-D). Its apparent molecular mass (M
r) was estimated to be 57000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 50000 by gel filtration. GP57 contained 14% (w/w) carbohydrate; the M
r of the peptide portion was estimated to be 55000 after deglycosylation by trifluoromethanesulfonic acid. GP57 is composed of two polypeptides with the same Mr and with very similar amino-acid composition but different pI values, 8.8 and 9.5. Both are rich in aspartic acid, serine and threonine, and may possess N-linked oligosaccharide chains, including fucose and xylose. A monoclonal antibody (MAb) against the purified GP57 reacted with both the pI 8.8 and the 9.5 components, as well as the deglycosylated GP57. Immunoblotting with the MAb indicated that GP57 is synthesized in and released from cultured cells which have been supplied with auxin. In immunocytochemical studies, GP57 was found in the space between the embryo and the endosperm of dry seeds, and its content decreased during germination. GP57 was also found in the endodermis and epidermis of young roots, the periderm of mature taproots, and the epidermis of petioles and young leaves.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- GP57
M
r-57000 glycoprotein
- GP65
M
r-65000 glycoprotein
- MAb
monoclonal antibody
-
M
r
apparent molecular mass
- SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis
- TFMS
trifluoromethanesulfonic acid 相似文献
2.
Glycoproteins extracted from microsomes of in-vitro-cultured cells of Daucus carota L. cv. US-Harumakigosun were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by peroxidase-conjugated concanavalin A. The appearance of a glycoprotein with Mr 31 000 (GP 31) was correlated with the ability of cells to form somatic embryos. GP 31 appeared in embryogenic cells cultured in 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, but not in somatic embryos and non-embryogenic cells; it disappeared when the cultures were transferred to auxin-free medium. Another glycoprotein with Mr 32 000 (GP 32) was detected only in non-embryogenic cells, regardless of the presence or absence of 2,4-D. Both glycoproteins, GP 31 and GP 32, were associated with the rough endoplasmic reticulum and were extractable with 0.05% deoxycholate.Abbreviations Con A
concanavalin A
- 2,4-D
2,4-dichlorophenoxyacetic acid
- ER
endoplasmic reticulum
- GP 31, GP 32
a glycoprotein with an apparent molecular mass of 31 or 32 kdalton
- kDa
kilodalton
- MS
Murashige and Skoog
- Mr
apparent molecular mass
- SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis 相似文献
3.
Auxin-controlled glycoprotein release into the medium of embryogenic carrot cells 总被引:5,自引:4,他引:5
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Glycoproteins released from carrot cells into culture media were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by staining with Coomassie brilliant blue or with the periodic-acid Schiff procedure. The appearance or disappearance of two glycoproteins of Mr 65,000 (GP65) and Mr 57,000 (GP57) was closely related to the formation of somatic embroys. GP65 was released specifically from embryogenic cells cultured in a medium without 2,4-dichlorophenoxyacetic acid, in which they can form somatic embryos. GP57 was released from the same embryogenic cells, if they were cultured in a medium with 2,4-dichlorophenoxyacetic acid, in which they cannot form somatic embryos. Nonembryogenic cells which cannot form somatic embryos, released only GP57. 相似文献
4.
Immunogold-localization and synthesis of an oil-body membrane protein in developing soybean seeds 总被引:5,自引:0,他引:5
Eliot M. Herman 《Planta》1987,172(3):336-345
The synthesis of a major oil-body membrane brotein was studied in maturing soybean (Glycine max (L.) Merr.) cotyledons. The membrane contained four abundant proteins with apparent molecular mass (Mr) of 34000, 24000, 18000 and 17000. The Mr=24000 protein (mP 24) was selected for more detailed analysis. The protein was purified to apparent homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isolated from the gel by electroelution or chemical hydrolysis of gel crosslinks. It was then used to elicit rabbit antibodies which were judged to be specific when assayed by SDS-PAGE-immunoblot procedures. The mP 24 was localized in immature soybean cotyledon cells by indirect immunogold procedures on thin sections of Lowicryl- and LR-White-embedded tissue. Indirect labeling with the primary antiserum followed by colloidal gold-protein A showed specific labeling of the oil-body membrane and an absence of label on the other subcellular organelles including the endoplasmic reticulum (ER). Parallel tissue samples were studied by conventional transmission electron microscopy. Although segments of the ER were observed to be closely juxtaposed to the oil bodies, continuity between the two organelles was not observed. The synthesis of mP 24 was studied by in-vitro translation and in-vivo labeling with [3H]leucine followed by indirect immunoaffinity isolation of the labeled products. The SDS-PAGE fluorography results indicated that the primary translation product and the in-vivo synthesized protein have the same Mr, and this is also the same Mr as the protein in the mature membrane.Abbreviations and symbols DATD
N N'-diallyltartardiamide
- EM
electron microscopy/scopic
- ER
endoplasmic reticulum
- IgG
immunoglobulin G
- Mr
apparent molecular mass
- PBS
phosphate-buffered saline
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TBS
Trisbuffered saline 相似文献
5.
Spatiotemporal patterns of expression of the cell-surface arabinogalactan-protein epitope defined by monoclonal antibody JIM4 (J.P. Knox et al., 1989, Development 106, 47–56) have been characterized by indirect immunofluorescence during the process of somatic embryogenesis in Daucus carota L. The JIM 4 epitope (J4e) occurred on cells established in culture from hypocotyl explants which appeared to derive, at least in part, from the epidermal cells of the hypocotyl. Cultures maintained in the presence of 2,4-dichlorophenoxyacetic acid developed proembryogenic masses of which only infrequent cells at the surface expressed J4e. Sub-culture at a low cell density and withdrawl of the synthetic auxin resulted in an increase in J4e expression in most surface cells and most abundantly in surface layers of cells at the future shoot end of developing embryos. The transition to heart-shaped embryos occurred concurrently with the expression of J4e by groups of cells beneath the developing cotyledons, at the junction of the future root and shoot. At this stage, J4e was also expressed by a single well-defined layer of cells at the surface of the embryos. Advancement to the mature torpedo stage was accompanied by the expression of the epitope on cells forming two regions of the future stele and of cells associated with the cotyledonary provascular tissue characteristic of the carrot seedling. At this stage there was substantially less expression of the marker antigen by epidermal cells, although infrequent expression by isolated cells of the epidermis was maintained. The correlation of J4e expression with the development and distinction of plant tissue patterns during somatic embryogenesis indicates a role for plasma-membrane arabinogalactan proteins in these processes.Abbreviations AGP
arabinogalactan protein
- 2,4-D
2,4-di-chlorophenoxyacetic acid
- J4e
JIM 4 epitope
- PEM
proembryogenic mass
We thank Andrew Davis for photographic assistance and Roger Pennell for useful discussions. 相似文献
6.
Marcel A. J. Toonen Ed D. L. Schmidt Theo Hendriks Harrie A. Verhoeven Ab van Kammen Sacco C. de Vries 《Planta》1996,200(2):167-173
Certain single cells in carrot (Daucus carota L.) suspension cultures react with the monoclonal antibody JIM8, and it has been proposed that these cells represent a transitional stage in somatic embryo formation. Shortly after isolation of the single cells by sieving, up to 80% of the cells react with JIM8. Within 4 d, JIM8 labelling becomes restricted to 1% of the single cells. To obtain evidence for the proposed correlation between expression of the JIM8 cell wall epitope and somatic embryo formation the developmental fate of carrot single cells labelled with JIM8 was determined by cell tracking. The results, obtained by recording 43 000 cells, show that only few JIM8-labelled cells give rise to embryos, and most somatic embryos develop from cells devoid of the JIM8 cell wall epitope. We therefore conclude that the presence of the JIM8 cell wall epitope does not coincide with the ability of single suspension cells to form embryos.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- AGP
arabino galactan protein
- B5-0
Gamborg's B5 medium
- B5-0.2
Gamborg's B5 medium supplemented with 0.2 M 2,4-D
- FITC
fluoresceïn isothiocyanate
- PBS
phosphate-buffered saline 相似文献
7.
Tomohiro Kiyosue Jiro Nakayama Shinobu Satoh Akira Isogai Akinori Suzuki Hiroshi Kamada Hiroshi Harada 《Planta》1992,186(3):337-342
ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA
abscisic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- LEA protein
late-embryogenesis-abundant protein
To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government. 相似文献
8.
Nuclei were isolated from somatic embryos of carrot (Daucus carota L.) using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified membrane-depleted nuclei were digested with DNase I in combination with RNase A, followed by extraction with 1 M NaCl. The DNA residue in the final insoluble fraction was less than 4% of that in isolated nuclei, and most of the residual nuclei retained their sphericity. Electron microscopy revealed that the nuclear matrix was composed of a distinct peripheral layer, an internal matrix structure and some fibrils; residual nucleoli were observed when exogeneous RNase was not incorporated. The proteins extracted from the nuclei and nuclear subfractions were compared by gel electrophoresis, which showed that the residual fraction contained many minor proteins. To identify proteins showing specific localization at the nuclear periphery, we prepared monoclonal antibodies (MAbs) against an ion-exchange chromatography fraction extracted from carrot nuclear matrices. Immunofluorescence microscopy with one of the MAbs, CML-1, showed exclusive staining of the nuclear periphery. The MAb recognized several spots showing microheterogeneity, with a narrow range of pI and molecular mass upon immunoblotting. A complete set of these spots was shown to be conserved in nuclear matrices. On the other hand, MAb CML-13 appeared to react with the nuclear interior as well as the periphery, recognizing a 96-kDa polypeptide of the nuclear matrix. These proteins were thus demonstrated to lie at the nuclear periphery, and to constitute the nuclear matrices in carrot. The 96-kDa polypeptide is suggested to be similar to the 92-kDa nuclear protein reported by Beven et al. in carrot (Beven et al., 1991, J. Cell Sci. 98, 293–302).Abbreviations DEAE
diethylaminoethyl
- MAb
monoclonal antibody
- NEPHGE
nonequilibrium pH gradient electrophoresis
We wish to thank Ms. Akiko Itoh for excellent technical assistance. This work was supported by a Grant-in-Aid (05640738) from the Ministry of Education of Japan. 相似文献
9.
The expression of members of two closely related abscisic acid (ABA)-responsive pea protein families, ABR17 and ABR18 (ABA-responsive 17200-Mr and 18100-Mr, respectively), is developmentally, tissueand stress-specifically regulated. Two-dimensional polyacrylamide gel electrophoresis revealed a number of ABR polypeptides on fluorographs of immunoprecipitated translation products of mRNAs, depending on the tissue, stage of development or type of stress. High endogenous ABA, or added ABA, enhanced the accumulation of translatable mRNA for specific ABR members under certain conditions, but high endogenous ABA was not a pre-requisite for accumulation of translatable ABR mRNA. The accumulation of ABR polypeptides was examined by Western blot analysis of acetate-buffer-extracted proteins. In fully expanded, young unstressed leaves, the ABR17 polypeptides (ABR18 polypeptides not detectable) accumulated to markedly higher levels in the epidermis than in the mesophyll. Dehydration stress caused an increased (ABR17) and detectable (ABR18) polypeptide accumulation which occurred predominantly in the epidermis. Detached leaves were used further to characterise factors affecting ABR polypeptide accumulation. An enhanced (ABR17) and detectable (ABR18) polypeptide accumulation occurred in the presence of ABA (10–4 M) but ABR18-polypeptide accumulation required light. The accumulation of both ABR polypeptides was stimulated in the presence of metabolisable and non-metabolisable carbohydrate sources but not in water or glutamine, indicating an osmotic rather than metabolic response. This carbohydrate-stimulated accumulation was markedly enhanced by light but unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis, indicating other photoreceptive processes besides photosynthesis were involved. The function of the ABR proteins remains unknown but their accumulation in aging tissues indicates a role in senescence. The results clearly demonstrate highly complex interactions between different environmental and developmental signals leading to the expression of these stressrelated proteins. In light of these results, the induction of protein expression of the newly-termed intracellular pathogenesis-related proteins, to which the ABR proteins are closely related, is discussed.Abbreviations ABA
(±)cis, trans-abscisic acid
- ABR17
Mr17200 ABA-responsive protein
- ABR18
Mr-18100 ABA-responsive protein
- 2-D
two-dimensional
- DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea
- FW
fresh weight
- IgG
immunoglobulin G
- Mr
apparent molecular mass
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis 相似文献
10.
Two abscisic acid (ABA)-responsive seed proteins, ABR17 and ABR18 (ABA-responsive 17000-Mr and 18000-Mr, respectively), previously found to be induced in cultured embryos of pea (Pisum sativum L.) are major components synthesised during normal seed desiccation. The ABR17 and ABR18 proteins showed different patterns of accumulation. The ABR18 protein was abundant in the testa during early seed development but in desiccating seed it was synthesised in the embryo, indicating spacial as well as temporal regulation of expression. The ABR18 protein was undetectable soon after germination but reappeared after adding ABA. The ABR17 protein was not detected in the testa but appeared in the embryo just prior to maximum fresh weight. The ABR17 protein continued to be synthesised during germination and was also present in non-stressed leaves. A high level of endogenous ABA or added ABA increased levels of translatable ABR17 mRNA. The ABR17 and ABR18 proteins were further characterised so as to help determine their structure and function. Neither protein appeared to contain a signal peptide but both proteins appeared to be glycosylated. The proteins had similar amino-acid compositions and limited Nterminal analysis showed 56% sequence identity. Neither protein had any significant N-terminal sequence homology to any of the late embryogenesis-abundant (LEA) proteins or dehydrins. Both proteins, however, show striking homology with a pea disease-resistance-response protein and the major birch pollen allergen, indicating that the ABR17 and ABR18 proteins may be members of a distinct group of stress-induced proteins.Abbreviations ABA
(±) cis,trans-abscisic acid
- ABR17
Mr-17200 ABA-responsive protein
- ABR18
Mr-18 100 ABA-responsive protein
- FW
fresh weight
- IgG
immunoglobulin G
- LEA
late embryogenesis-abundant
- Mr
apparent molecularmass
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid
This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station. 相似文献
11.
The effect of cold treatment on gene expression in two different barley (Hordeum vulgare L.) cultivars has been studied. Cold stress induced a set of new mRNAs as determined by in-vitro translation of coleoptile RNA obtained from control and stressed seedlings. These mRNAs accumulated with different kinetics, and the cold-induced proteins could be grouped into five categories. The first category (a) is represented by a single protein with Mr of 75 kDa that reaches its highest level of expression after 6 h at 5°C. This polypeptide readily accumulates in the plant tissues and it can be detected when proteins separated by two-dimensional electrophoresis are stained with silver nitrate. The other polypeptides appear later during the 1- to 4-d stress period (protein groups b and c), increase (group d), or decrease during the period of treatment (group e). Only minor differences between the two cultivars with different cold-resistance capacities were found when the in-vitro translation products were compared. The results obtained demonstrate that several mRNAs are specifically expressed as a response to cold treatment in barley coleoptiles.Abbreviations 2-D
two-dimensional
- IEF
isoelectrofocusing
- Mr
relative molecular weight
- poly(A)
polyadenylated
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
12.
The extraction and partial purification of phytochrome from light-grownAtrichum undulatum P. Beauv., a chlorophyllous moss, is described. Polyethyleneimine and salt fractionation followed by hydroxyapatite and Affi-gel-blue chromatography were used to separate phytochrome from chlorophyll, and to purify the pigment. All steps were performed in the presence of Triton X-100 which improved the yield by a factor of about three. The protein has a molecular weight some-what larger than that ofAvena phytochrome (124 kDa), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. It cross-reacts with a monoclonal antibody against phytochrome from etiolated corn (Zea) and a polyclonal antibody against phytochrome from etiolated oat (Avena), and its photoreversibility is similar to that of phytochrome from greenAvena.Abbreviations EDTA
ethylenediaminetetraacetic acid
- FMN
flavinmononucleotide
- PMSF
phenylmethylsulfonylfluoride
- Pr(Pfr)
red(far-red)-absorbing form of phytochrome
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
13.
Sacco C. de Vries Hilbert Booij Peter Meyerink Gert Huisman H. Dayton Wilde Terry L. Thomas Ab van Kammen 《Planta》1988,176(2):196-204
Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [35S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [35S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- cDNA
complementary DNA
- PAGE
polyacrylamide gel electrophoresis
- PEM
proembryogenic mass 相似文献
14.
Alice J. Adler Charlotte D. Evans 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,761(3):217-222
A large retinol-binding protein, interphotoreceptor retinol-binding protein, is found only in the interphotoreceptor matrix of the eye, and may function in vitamin A transport for the visual cycle. Interphotoreceptor retinol-binding protein is the major glycoprotein of this matrix, and can be isolated rapidly by affinity-adsorption onto concanavalin A-Sepharose. The yield is approx. 0.25 mg per bovine eye. Its apparent Mr is 250 000 by gel-filtration chromatography, and 225 000 by native polyacrylamide-gradient gel electrophoresis; this protein band displays endogenous retinol fluorescence on such gels. As measured by SDS-polyacrylamide gel electrophoresis, the apparent Mr is 140 000. In the interphotoreceptor matrix most vitamin A-binding sites on this retinol-binding protein are unoccupied; however, addition of exogenous all-trans-retinol can saturate these sites. The apparent dissociation constant for retinol is 10−6 M, as measured by fluorimetric titration. 相似文献
15.
Richard M. Napier Michael A. Venis Michael A. Bolton Louise I. Richardson Goeffrey W. Butcher 《Planta》1988,176(4):519-526
Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.Abbreviations ABP
auxin-binding protein
- DEAE
diethylaminoethyl
- Ig
immunoglobulin
- kDa
kilodalton
- NAA
naphthalene-1-acetic acid
- Mr
relative molecular mass
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate 相似文献
16.
Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7–10 d following seed germination. We assayed cotyledon extracts for protease activity by using [3H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [3H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M
rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M
r=30 000). Optimum hydrolysis of [3H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [3H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.Abbreviations Da
dalton
- DTT
dithiothreitol
-
M
r
apparent molecular weight
- PAGE
polyacrylamide gel electrophoresis
- PHA
phytohemagglutinin
- SDS
sodium dodecyl sulfate 相似文献
17.
Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of Mr=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (Mr=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP
dithiobis(succinimidyl propionate)
- EDTA
ethylenediaminetetraacetic acid
- ER
endoplasmic reticulum
- kDa
kilodalton
- Mr
relative molecular mass
- PHA
phytohemagglutinin
- SDS
sodium dodecylsulfate
- PAGE
polyacrylamide gel electrophoresis 相似文献
18.
Summary Embryogenic callus was initiated from radicles of mature embryos removed from imbibed seeds (24 h). Embryogenic and other
nonembryogenic types of callus proliferated on a modified half-strength Murashige-Skoog medium (MS) basal medium (BM) supplemented
withmyo-inositol, casein hydrolysate (CH), L-glutamine (gln) and growth regulators kinetin (KN), N6-benzyladenine (BAP) each (20×10−6
M), 2,4-dichlorophenoxyacetic acid (2,4-D) (50×10−6
M) Embryogenic callus bearing suspensor-like cells in a mucilaginous gel matrix was isolated and maintained by subculture every
10 to 12 days on BM with KN, BAP each (2×10−6
M) and 2,4-D (5×10−6
M). Somatic embryos developed spontaneously from the callus on this medium at 23±1° C. Closer examination revealed that numerous
polyembryonic clusters, comprised of elongated cells (suspensors) and small dense cells with large nuclei (somatic embryos),
occurred in the viscous gel. When this enriched embryonal-suspensor mass was subcultured to low 2,4-D (1×10−6
M), globular embryos developed by 40 to 60 days. Upon transfer to a liquid medium without growth regulators, the embryos elongated
and developed cotyledons and shoots with needles. Plantlet development was completed by 30 days in a basal medium without
CH, gln and growth regulators. The total culture time was 150 days. Approximately 40±10 embryos were formed from 500 mg of
initial callus. Somatic embryogenesis became aberrant if embryos remained attached to the callus mass and were not subcultured
within 10 to 12 days according to the described protocol. Somatic embryos were encapsulated in an alginate gel and stored
at 4° C for nearly two months without visible adverse effects on viability.
Editor's Statement This paper presents advances in the in vitro regeneration of a commercially useful plant species from stored
seeds. In addition, data is presented on short-term storage of the plantlets, and long-term proliferation of the embryonal
mass in vitro. 相似文献
19.
When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the
total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial
characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to
homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins.
Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular
masses (Mrs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous
on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products)
in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins
appear to be produced late in seed development but are capable of being induced early in development by culturing embryos
in vitro and are markedly enhanced by ABA. 相似文献
20.
A partially purified preparation of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) from tomato (Lycopersicon esculentum (Mill.) fruit tissue was used to generate monoclonal antibodies (MAb) specific for the two different MAbs yielded a 50-kDa polypeptide as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An enzyme-linked immunosorbent assay (ELISA) capable of detecting <1 ng of antigen was developed. The ELISA system was used to demonstrate that two of the MAbs recognized different epitopes on the ACC-synthase protein. Wound-induced increases in ACC-synthase activity in tomato fruit tissue were correlated with changes in ELISA-detectable protein. In-vivo labeling of wounded tissue with [35S]methionine followed by extraction and immunopurification in the presence of various protease inhibitors yielded one major radioactive band of 50 kDa molecular mass. Pulse labeling with [35S]methionine at various times after wounding indicated that the wound-induced increase in ACC-synthase activity involved de-novo synthesis of a rapidly turning over 50-kDa polypeptide.Abbreviations ACC
1-aminocyclopropane-1-carboxylic acid
- ELISA
enzyme-linked immunosorbent assay
- MAb
monoclonal antibody
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献