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1.
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 (M r) and structure, resistance to proteolysis) show similarity to phaseolin, the major 7S storage protein of Phaseolus 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 M r 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 M r 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
2.
The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat ( Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [ 35S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADH-cytochrome c reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles.Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with M r 21 000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of M r 23 000 and the mature monomer of M r 18000. When [ 3H]mannose was used in labeling studies, only the polypeptide of M r 23 000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of M r 20 000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745–6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide.Abbreviations ER
endoplasmic reticulum
- IgG
immunoglobulin G
- M r
relative molecular mass
- poly(A) +RNA
polyadenylated RNA
- SDS-PAGE
sodium dodecyl sulfatepolyacrylamide gel electrophoresis
- WGA
wheat-germ agglutinin 相似文献
3.
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 M r~50 000 (i.e., M r 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within M r~50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesis in vivo and in vitro which indicated that the vicilin subunits smaller than M r~50 000 arose by endoproteolytic cleavage of parent molecules within the M r~50 000 group. Cleavage in different M r 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 M r~50 000, with the possible exception of the M r34 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 M r~50 000 group. 相似文献
5.
The relative molecular mass (M r) of the native phytochrome monomer from etiolated Cucurbita pepo L., Pisum sativum L., Secale cereale L. and Zea mays L. seedlings has been determined using immunoblotting to visualize the chromoprotein in crude extracts subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single phytochrome band is observed for each plant species when the molecule is extracted under conditions previously demonstrated to inhibit the proteolysis of native Avena sativa L. phytochrome. A comparison among plant species indicates that the M r of native phytochrome is variable: Zea mays=127000; Secale=Avena=124000; Pisum=121000; Cucurbita=120000. The in-vitro phototransformation difference spectrum for native phytochrome from each species is similar to that observed in vivo in each case and is indistinguishable from that described for native Avena phytochrome. The difference minima between the red- and far-red-absorbing forms of the pigment (Pr-Pfr) are all at 730 nm and the spectral change ratios (A r/A fr) are near unity. When incubated in crude extracts, phytochrome from all four species is susceptible to Pr-specific limited proteolysis in a manner qualitatively similar to that observed for Avena phytochrome, albeit with slower rates and with the production of different M r degradation products. Further examination of the in-vitro proteolysis of Avena phytochrome by endogeneous proteases has identified several additional phytochrome degradation products and permitted construction of a peptide map of the molecule. The results indicate that both the 6000- and 4000-M r polypeptide segments cleaved by Pr-specific proteolysis are located at the NH 2-terminus of the chromoprotein and are adjacent to a 64000-M r polypeptide that contains the chromophore.Abbreviations and symbols A minimum
phototransformation difference spectrum (Pr-Pfr) minimum
- Ig
immunoglobulin
- Mops
3-(N-morpholino)propanesulfonic acid
- M r
relative molecular mass
- PAGE
polyacrylamide gel electrophoresis
- PMSF
phenylmethylsulfonyl fluoride
- SDS
sodium dodecyl sulfate 相似文献
6.
From a gene bank of the Acinetobacter calcoaceticus genome a plasmid was isolated that complements four different classes of PQQ - mutants. Subclones of this plasmid revealed that the four corresponding PQQ genes are located on a fragment of 5 kilobases. The nucleotide sequence of this 5 kb fragment was determined and by means of Tn5 insertion mutants the reading frames of the PQQ genes could be identified. Three of the PQQ genes code for proteins of M r 29700 (gene I), M r 10800 (gene II) and M r 43600 (gene III) respectively. In the DNA region where gene IV was mapped however the largest possible reading frame encodes for a polypeptide of only 24 amino acids. A possible role for this small polypeptide will be discussed. Finally we show that expression of the four PQQ genes in Acinetobacter lwoffi and Escherichia coli lead to the synthesis of the coenzyme in these organisms. 相似文献
7.
The major storage proteins, polypeptides of 31 and 47 kilodaltons (kDa), from the seeds of cocoa ( Theobroma cacao L.), have been identified and partially purified by preparative gel electrophoresis. The polypeptides were both N-terminally blocked, but some N-terminal amino-acid sequence was obtained from a cyanogen bromide peptide common to both polypeptides, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy-DNA (cDNA) clone from a library made from poly(A) + RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 566-amino-acid polypeptide of M r 65 612. The existence of a common precursor to the 31- and 47-kDa polypeptides of this size was confirmed by immunoprecipitation from total poly(A) +RNA translation products. The precursor has an N-terminal hydrophobic sequence which appears to be a typical signal sequence, with a predicted site of cleavage 20 amino acids after the start. This is followed by a very hydrophilic domain of 110 amino acids, which, by analogy with the cottonseed -globulin, is presumed to be cleaved off to leave a domain of approx. 47 kDa, very close to the observed size of the mature polypeptide. Like the hydrophilic domain of the cottonseed -globulin the cocoa hydrophilic domain is very rich in glutamine and charged residues (especially glutamate), and contains several Cys-X-X-X-Cys motifs. The cyanogen-bromide peptide common to the 47-kDa and 31-kDa polypeptides is very close to the proposed start of the mature domain, indicating that the 31-kDa polypeptide arises via further C-terminal processing. The polypeptide sequence is homologous to sequences of the vicilin class of storage proteins, previously found only in legumes and cotton. Most of these proteins have a mature polypeptide size of approx. 47 kDa, and are synthesised as precursors only slightly larger than this. Some, however, are larger polypeptides (e.g. -conglycinin from soybean is 72 kDa), usually due to an additional N-terminal domain. In cottonseed the situation appears to parallel that in cocoa in that the vicilin is synthesised as an approx. 70-kDa precursor and then processed to a 47-kDa (and in the case of cocoa also a 31-kDa) mature protein. In this context it is interesting that cotton is closer in evolutionary terms to cocoa than are the legumes, both cotton and cocoa being in the order Malvales.Abbreviations A
absorbance
- cDNA
copy DNA
- IgG
immunoglobulin G
- kb
kilobase pairs
- kDa
kilodaltons
- M r
relative molecular mass
- SDS-PAGE
sodium dodecyl sulphate-polyacylamide gel electrophoresis
The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies. 相似文献
8.
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 M r 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
- M r
relative molecular weight
- poly(A)
polyadenylated
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
9.
The effect of high nutrient levels of copper on the low-molecular-weight copper-proteins of leaves from plants of two cultivars of Pisum sativum L., with different sensitivity to copper, was investigated. Gel-filtration chromatography of leaf extracts from Cu-tolerant and Cu-sensitive plants grown with 1 M Cu(II), showed the presence of only two copper peaks (I and II), but growth of plants with 240 M Cu(II) induced two additional copper fractions (III and IV). Fractions II and III were purified by solvent extraction, gel-filtration and ion-exchange chromatography, and their molecular weights, subunit sizes, absorption spectra, metalprotein stoichiometry and amino-acid contents were determined. Fraction II was a polypeptide of M r 15000 composed of a single chain. The purification of fraction III produced a copper-containing fraction (III-1) of M r 3700, and a copper-protein (III-2) with an M r, by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, of 66000. The metal contents of fractions III-1 and III-2 were higher in Cu-tolerant than in Cu-sensitive plants. On the basis of amino-acid analyses, fraction III-1 appeared to be complexes of Cu(II)-poly- isoleucine and Cu(II)-poly-leucine. The results rule out the existence, in pea leaves, of any protein similar to either animal metallothioneins or to any of the low-molecularweight metal-binding proteins or peptides described in other plants and reported to be involved in metal tolerance. In the mechanism of copper tolerance at the leaf level, fractions III-1 (M r 3700), III-2 (M r 66000), and IV (M r 2000) appear to have a role, fraction IV being specifically induced in the tolerant cultivar by Cu(II). Fractions III-1 and III-2 could participate in a different mechanism, adaptive in character, involving an enhanced capacity to bind copper in Cu-tolerant plants.Abbreviations DEAE
diethylaminoethyl
- M r
relative molecular mass
- SDS
sodium dodecyl sulfate
- PAGE
polyacrylamide-gel electrophoresis
J.M. Palma was the recipient of a research fellowship from the Caja General de Ahorros y Monte de Piedad de Granada and CSIC. We are grateful to Dr. J. Moreno-Carretero, R + D Department, UNIASA, Granada, for conducting the amino-acid analyses. This work was supported by grant 603/275 from CAICYT-CSIC (Spain). 相似文献
10.
The major albumin, a polypeptide of 21 kilodaltons (kDa), from the seeds of cocoa ( Theobroma cacao L.), has been identified and partially purified by preparative gel electrophoresis. Some N-terminal sequence was obtained, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy DNA (cDNA) clone from a library made from poly(A) + RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 221-amino-acid polypeptide of M r 24003. The existence of a precursor to the 21-kDa polypeptide of this size was confirmed by immunoprecipitation from total poly(A) + RNA translation products. The polypeptide has a hydrophobic signal sequence of 26 amino acids before the mature start, and the mature polypeptide would have an M r of 21223. The protein sequence is homologous with sequences of the Kunitz protease and -amylase inhibitor family, and the protein probably functions to defend the seed's protein reserves from the digestive enzymes of invading pests. However because the protein comprises 25–30% of the total seed protein it may itself also function as a storage protein. Electron micrographs of immunogold-labelled embryo sections show that the protein is located in membrane-enclosed organelles.Abbreviations cDNA
copy DNA
- IgG
immunoglobulin G
- kb
kilobase pairs
- kDa
kilodaltons
- M r
relative molecular mass
- SDS-PAGE
sodium dodecyl sulphate-polyacylamide gel electrophoresis
The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies, and to Dr G. Cope, of the Biological Sciences Electron Microscopy Unit, Sheffield University, for taking the electron micrographs.To whom correspondence should be addressed. 相似文献
11.
In addition to the marked reduction in legumin synthesis and legumin mRNA levels reported earlier (Chandler, Higgins, Randall, Spencer 1983 Plant Physiol 71: 47-54), pulse labeling of S-deficient Pisum sativum L. seeds showed that a high relative level of total vicilin (vicilin plus convicilin) synthesis was maintained throughout the entire phase of protein accumulation, whereas in nondeficient seeds vicilin synthesis is largely confined to the first half of this phase. Fractionation of pulse-labeled proteins on Na-dodecylsulfate-polyacrylamide gels showed that the synthesis of the Mr 50,000 family of vicilin polypeptides was increased and greatly extended in S-deficient seeds whereas that of convicilin was slightly reduced. Other changes apparent from pulse-labeling experiments include a depression, to different degrees, in the synthesis of three major albumin polypeptides. The level of the mRNAs for seven major seed proteins was followed throughout development of control and sulfur-deficient seeds. In all cases, the changes in each mRNA closely reflected the pattern of synthesis of its corresponding polypeptide seen by pulse labeling. S-deficient seeds showed an elevated level of Mr 50,000 vicilin mRNA which remained high throughout seed formation, whereas legumin mRNA levels were greatly reduced at all stages of development. When S-deficient plants were given an adequate supply of sulfate midway through seed development, there was a shift toward the protein synthesis profile characteristic of healthy plants. The synthesis of legumin and two albumins rapidly increased and the synthesis of Mr 50,000 vicilin declined more slowly. Similar responses were seen in detached, S-deficient seeds supplied directly with adequate sulfate. 相似文献
12.
Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean ( Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (M r) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with M r 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-M r polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 M r. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [ 3H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, -mannosidase or -N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (M r=30000). The 4000 decrease in M r is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.Abbreviations Con A
concanavalin A
- Glc
glucose
- GlcNAc
N-acetylglucosamine
- IgG
immunoglobulin G
- Man
mannose
- M r
relative molecular mass
- SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis 相似文献
13.
Vicilin, a 7S globulin of Pisum sativum L. seed, accumulates in protein-storage vacuoles (protein bodies) of cotyledonary storage-parenchyma cells. The synthesis and proteolytic processing of various genetically engineered proteins within the leaf and seed of a heterologous (tobacco, Nicotiana tabacum L.) host was examined. A modified vicilin gene, in which the DNA sequence corresponding to the signal peptide was removed, resulted in a polypeptide of 50 kDa in the tobacco leaf and seed; none of the normal proteolytic cleavage products characteristic of expression of an unmodified vicilin gene were obtained. Likewise, no vacuolar accumulation of this mutant vicilin occurred in leaf protoplasts, which is also supportive of the predicted cytosolic localization for this protein. In-frame deletions were made within the region of the vicilin gene encoding the mature protein, to eliminate the N-terminal 28 and 121 amino acids and the C-terminal 69 residues, while maintaining an intact signal peptide. All of these mature deletion-mutant proteins were accumulated to only low levels in the host, but exhibited the predicted molecular weight and underwent some normal proteolytic processing in the seed. Mutant vicilin proteins having deletions in either the N-terminus (NT 121) or C-terminus (CT 69) were not found in appreciable amounts within the vacuolar fraction of transgenic tobacco leaf protoplasts, perhaps due to protein degradation in this compartment. Compared with the intact vicilin, oligomer assembly of the C-terminal deletion-mutant protein was disrupted in leaf cells, which may have further affected protein stability. The deletions of mature vicilin protein led to a much less dramatic reduction in protein accumulation in transgenic tobacco seed. Further, the same mutant proteins expressed within transgenic tobacco seed exhibited correct and highly specific proteolytic processing.Abbreviations CaMV
cauliflower mosaic virus
- M r
relative molecular mass
We gratefully acknowledge the technical assistance from Maria J. Still and help from M.R.I. Khan. Part of this research was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Operating and Equipment Grants to A.R.K. 相似文献
14.
A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5 and 3 regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number of integrated copies of the vicilin gene. Pea vicilin was confined to the seeds of transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of M
r50000 together with a range of smaller polypeptides. 相似文献
15.
The polypeptides of the subunits of 70S ribosomes isolated from rye ( Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (M r) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of M r 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L
polypeptide of large ribosomal subunit
- M r
relative molecular mass
- r-protein
ribosomal polypeptide
- S
polypeptide of small ribosomal subunit
- SDS
sodium dodecyl sulfate 相似文献
16.
The calmodulin-stimulated ATPase of maize ( Zea mays L.) coleoptiles has been purified by calcium-dependent binding to a calmodulin affinity column. In the presence of protease inhibitors (phenylmethylsulfonylfluoride and chymostatin) a polypeptide of relative molecular mass (M r) 140000 (±10000) is obtained on sodium-dodecylsulphate polyacrylamide gels. This polypeptide is recognised specifically by an affinity-purified polyclonal antibody to mammalian calmodulin-stimulated calcium-pumping ATPases and is of similar M r to the erythrocyte-membrane calcium pump (138000 M r).Abbreviations EGTA
ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid
- M r
apparent molecular mass
- SDS
sodium dodecyl sulphate 相似文献
17.
A method is described for the extraction of phytochrome from chlorophyllous shoots of Avena sativa L. Poly(ethyleneimine) and salt fractionation are used to reduce chlorophyll and to increase the phytochrome concentration sufficiently to permit spectral and immunochemical analyses. The phototransformation difference spectrum of this phytochrome is distinct from that of phytochrome from etiolated shoots in that the maximum in the red region of the difference spectrum is shifted about 15 nm to a shorter wavelength. Immunochemical probing of electroblotted proteins (Western blotting), using a method sensitive to 50 pg, demonstrates the presence of two polypeptides in green tissue that bind antiphytochrome antibodies: a predominant species with a relative molecular mass (M r) of 118000 and a lesser-abundant 124000-M r polypeptide. Under nondenaturing conditions all of the 124000-M r species is immunoprecipitable, but the 118000-M r species remains in the supernatant. Peptide mapping and immunochemical analysis with monoclonal antibodies show that the 118000-M r species has structural features that differ from etiolated-oat phytochrome. Mixing experiments show that these structural differences are intrinsic to the molecular species from these two tissues rather than being the result of post-homogenization modifications or interfering substances in the green-tissue extracts. Together the data indicate that the phytochrome that predominates in green-tissue has a polypeptide distinct from the well-characterized molecule from etiolated tissue.Abbreviations and symbols Ig
immunoglobulin
- M r
relative molecular mass
- Pfr, Pr
far-red-absorbing and red-absorbing forms of phytochrome respectively
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
-
max
R
,
max
FR
maxima of the phototransformation difference spectrum in the red and far-red region 相似文献
18.
A nuclear protein, present in carrot meristems and rapidly proliferating cultured cells of carrot ( Daucus carota L.) has been identified by the use of a monoclonal antibody (MAb 21D7). By combining the techniques of two-dimensional polyacrylamide gel analysis and blotting separated proteins onto nitrocellulose sheets, it was shown that the antibody detected a single polypeptide of apparent molecular mass ( M
r) of 45000 and an isoelectric focusing point (pI) of 6.7. This protein was found by subcellular fractionation and immunofluorescence to be highly concentrated in the nucleoli of somatic and zygotic embryos of a wide range of plants. It was not detectable in logarthmically growing cells of Escherichia coli, yeast, embryos of Drosophila melanogaster or cultured C3H mouse cells. These data indicate that this protein is a highly conserved non-histone protein associated with nuclei of rapidly dividing plant cells.Abbreviations
M
r
apparent molecular mass
- Da
dalton
- Ig
immunoglobulins
- MAb
monoclonal antibody
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- 2-D gel
two-dimensional gel electrophoresis
- 2,4-D
2,4-dichlorophenoxyacetic acid 相似文献
19.
The expression of members of two closely related abscisic acid (ABA)-responsive pea protein families, ABR17 and ABR18 (ABA-responsive 17200-M r and 18100-M r, 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
M r17200 ABA-responsive protein
- ABR18
M r-18100 ABA-responsive protein
- 2-D
two-dimensional
- DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea
- FW
fresh weight
- IgG
immunoglobulin G
- M r
apparent molecular mass
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis 相似文献
20.
Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with 125I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of 125I-labeled insulin to a polypeptide that gave an apparent Mr of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM unlabeled nerve growth factor. Using the same affinity labeling technique, 125I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an Mr 145 000 and an Mr 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation. 相似文献
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