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1.
Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS1) and plastidic (GS2) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS1 and GS2, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS
glutamine synthetase
- GS1
cytosolic glutamine synthetase
- GS2
platidic glutamine synthetase
We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586. 相似文献
2.
Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants.Abbreviations GS Glutamine synthetase - UTR Untranslated region 相似文献
3.
During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase of chloroplastic glutamine synthetase (GS2) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS2. The synthesis of GS2 was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS2 protein content in green leaves is fivefold higher than in etiolated leaves.Abbreviations AbH
heavy chain of antibodies
- AbL
light chain of antibodies
- AP
acid phosphatase
- CH
cycloheximide
- G6PDH
glucose-6-phosphate dehydrogenase
- GS
glutamine synthetase
- GS1
cytosolic glutamine synthetase
- GS2
chloroplastic glutamine synthetase
- LC
lincomycin
- NAD-MDH
NAD malate dehydrogenase
- NADP-G3PDH
NADP glyceraldehyde-3-phosphate dehydrogenase 相似文献
4.
Ion-exchange chromatography has been used to separate the isoforms of glutamine synthetase (GS; EC 6.3.1.2) appearing in sunflower (Helianthus annuus L. cv. Peredovic) cotyledons during seedling growth under different light and nitrogen conditions. Both in dry and imbibed seeds, only a single form of GS (GSs) was detected. Upon seed germination, the GSs isoform was gradually replaced by cytosolic (GS1) and plastidic (GS2) isoforms. Light and nitrate decreased the levels of GS1. In contrast, the appearance of GS2 was greatly stimulated by light. Nitrate also had a positive effect, particularly in the light. Light and nitrate acted synergistically on the appearance of GS2. The GS2:GS1 ratio in cotyledons of 9-d-old seedlings ranged from about 2, in darkness and nitrate-deprivation conditions, to 16 under light and nitrate application. The possible physiological roles of the distinct GS isoforms appearing in the epigeal cotyledons of sunflower during germination, and their differential regulation by light and nitrate, are discussed.Abbreviations GS
glutamine synthetase
- GS1
cytosolic GS
- GS2
plastidic GS
- GSs
GS from seeds
This work was supported by a grant from Dirección General de Investigatión Científica y Técnica (PB90-0777) and Plan Andaluz de Investigación (3261), Spain. P.C. gratefully acknowledges receipt of a scholarship from Junta de Andalucía. The valuable technical assistance of Mrs. G. Alcalá is greatly appreciated. We are also grateful to Eurosemillas (Córdoba) for supplying us with sunflower seeds. 相似文献
5.
Cellular localization of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in vascular bundles of leaf blades of rice (Oryza sativa L.), at the stage at which leaf blades 6 (the lowest position) to 10 were fully expanded, was investigated immunocytologically with an affinity-purified anti-GS1 immunoglobulin G. Strong signals for GS1 protein were detected in companion cells of large vascular bundles when blades 6–8 were tested. Signals for GS1 were also observed in vascular-parenchyma cells of both large and small vascular bundles. The results further support our hypothesis that GS1 is important for the export of leaf nitrogen from senescing leaves. The signals in companion cells were less striking in the younger green leaves and were hardly detected in the non-green portion of the 11th blade. In the non-green blades, strong signals for GS1 protein were detected in sclerenchyma and xylemparenchyma cells. When total GS extracts prepared from the 6th,10th, and the non-green 11th blades were subjected to anion-exchange chromatography, the activity of GS1 was clearly separated from that of chloroplastic GS, indicating that GS1 proteins detected in the vascular tissues were able to synthesize glutamine. The function of GS1 detected in the developing leaves is discussed.Abbreviations Fd-GOGAT
ferredoxin-dependent glutamate synthase
- GS1
cytosolic glutamine synthetase
- GS2
plastidic glutamine synthetase
- IgG
immunoglobulin G 相似文献
6.
Work is described which suggests that glutamine synthetase (GS) could play an important and direct regulatory role in the control of NO3 assimilation by the alga. In both steady-state cells and ones disturbed physiologically by changes in light or nitrogen supply the assimilation of NO3 appears to be limited by the activity of GS. Moreover although in normal cells NH3 can completely inhibit NO3 uptake, promote the deactivation of nitrate reductase (NR) and repress the synthesis of NR and nitrite reductase (NIR), these controls are relaxed in cells in which GS is deactivated by treatment with L-methionine-DL-sulfoximine (MSO). It is proposed that the reversible deactivation of GS may play an important part in the regulation of NO3 assimilation although it is still not clear whether the enzyme itself or products of its metabolism are responsible.Abbreviations GS
glutamine synthetase
- GSs
glutamine synthetase, synthetase activity
- GSt
glutamine synthetase, transferase activity
- NR
nitrate reductase
- NIR
nitrite reductase
- GDH
glutamate dehydrogenase
- CHX
cycloheximide
- MSO
L-methionine-DL-sulfoximine
- FAD
flavine adenine dinucleotide 相似文献
7.
Purification and Characterization of Two Forms of Glutamine Synthetase from the Pedicel Region of Maize (Zea mays L.) Kernels 总被引:1,自引:1,他引:0
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Muhitch MJ 《Plant physiology》1989,91(3):868-875
Maize (Zea mays L.) kernel pedicels, including vascular tissues, pedicel parenchyma, placento-chalazal tissue, and the surrounding pericarp, contained two forms of glutamine synthetase (EC 6.3.1.2), separable by anion exchange chromatography under mildly acidic conditions. The earlier-eluting activity (GSp1), but not the later-eluting activity (GSp2), was chromatographically distinct from the maize leaf and root glutamine synthetases. The level of GSp1 activity changed in a developmentally dependent manner while GSp2 activity was constitutive. GSp1 and GSp2 exhibited distinct ratios of transferase to hydroxylamine-dependent synthetase activities (5 and 23, respectively), which did not change with kernel age. Purified pedicel glutamine synthetases had native relative molecular masses of 340,000, while the subunit relative molecular masses differed slightly at 38,900 and 40,500 for GSp1 and GSp2, respectively. Both GS forms required free Mg2+ with apparent Kms = 2.0 and 0.19 millimolar for GSp1 and GSp2, respectively. GSp1 had an apparent Km for glutamate of 35 millimolar and exhibited substrate inhibition at glutamate concentrations greater than 90 millimolar. In contrast, GSp2 exhibited simple Michaelis-Menten kinetics for glutamate with a Km value of 3.4 millimolar. Both isozymes exhibited positive cooperativity for ammonia, with S0.5 values of 100 and 45 micromolar, respectively. GSp1 appears to be a unique, kernel-specific form of plant glutamine synthetase. Possible functions for the pedicel GS isozymes in kernel nitrogen metabolism are discussed. 相似文献
8.
Jacqueline Freeman Antonio J. Marquez Roger M. Wallsgrove Ritva Saarelainen Brian G. Forde 《Plant molecular biology》1990,14(3):297-311
A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS2) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS1). A sequence identicalto an N-terminal sequence determined from a purified preparation of the mature GS2 polypeptide (NH2-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46–61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS2 subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS2-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS2 mRNA, and an anti-GS antiserum to assay for GS2 protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS2 mRNA; class II, which had normal or increased levels of GS2 mRNA but very little GS2 protein; and class III, which had significant amounts of GS2 protein but little or no GS2 activity. 相似文献
9.
M. Lara Julie V. Cullimore P. J. Lea B. J. Miflin A. W. B. Johnston J. W. Lamb 《Planta》1983,157(3):254-258
The activities of glutamine synthetase (GS), nitrogenase and leghaemoglobin were measured during nodule development in Phaseolus vulgaris infected with wild-type or two non-fixing (Fix-) mutants of Rhizobium phaseoli. The large increase in GS activity which was observed during nodulation with the wild-type rhizobial strain occurred concomitantly with the detection and increase in activity of nitrogenase and the amount of leghaemoglobin. Moreover, this increase in GS was found to be due entirely to the appearance of a novel form of the enzyme (GSn1) in the nodule. The activity of the form (GSn2) similar to the root enzyme (GSr) remained constant throughout the experiment. In nodules produced by infection with the two mutant strains of Rhizobium phaseoli (JL15 and JL19) only trace amounts of GSn1 and leghaemoglobin were detected.Abbreviations DEAE-Sephacel
diethylaminoethyl-Sephacel
- GS
glutamine synthetase 相似文献
10.
Jose Luis Ortega Olivia L. Wilson Champa Sengupta-Gopalan 《Molecular genetics and genomics : MGG》2012,287(11-12):881-893
11.
Glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design herbicidal α- and γ-substituted analogs of phosphinothricin (l-2-amino-4-(hydroxymethylphosphinyl)butanoic acid, PPT), a naturally occurring GS inhibitor and a potent herbicide. The substituted phosphinothricins inhibit cytosolic sorghum GS1 and chloroplastic GS2 competitively versusl-glutamate, with Ki values in the low micromolar range. At higher concentrations, these inhibitors inactivate glutamine synthetase, while dilution restores activity through enzyme-inhibitor dissociation. Herbicidal phosphinothricins exhibit low Ki values and slow enzyme turnover, as described by reactivation characteristics. Both the GS1 and GS2 isoforms of plant glutamine synthetase are similarly inhibited by the phosphinothricins, consistent with the broad-spectrum herbicidal activity observed for PPT itself as well as other active compounds in this series. 相似文献
12.
Summary We investigated the inducibility of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), and glutamine synthetase (GS; EC 6.3.1.2) isoforms in cotyledons of 7-day-old seedlings of sunflower (Helianthus annuus L.) in relation to light, nitrogen source (NO
3
–
, NO
2
–
or NH
4
+
), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO
2
–
or NH
4
+
the synthesis of NiR and chloroplastic GS (GS2) over the constitutive levels present in N-free-grown seedlings. In vivo inhibition of NR activity by tungsten application to seedlings and measurements of tissue NO
3
–
concentration indicate that NO
3
–
-dependent enzyme induction is elicited by NO
3
–
per se and not by a product of its assimilatory reduction, e.g., NO
2
–
or NH
4
+
. In the presence of NO
3
–
, light remarkably enhanced the appearance of NR, NiR, and GS2, while the activity of the cytosolic GS isoform (GS1) was adversely affected. Cycloheximide suppressed much more efficiently than chloramphenicol the light- and NO
3
–
-dependent increase of GS2 activity, indicating that sunflower chloroplastic GS is synthesized on cytoplasmic 80S ribosomes. When the plastids were damaged by photooxidation in cotyledons made carotenoid-free by application of norflurazon, the positive action of light and NO
3
–
on the appearance of NR, NiR, and GS2 isoform was greatly abolished. Therefore, it is suggested that intact chloroplasts are required for the inductive effect of light and NO
3
–
and/or for the accumulation of newly formed enzymes in the organelle.Abbreviations CAP
chloramphenicol
- CHX
cycloheximide
- GS
glutamine synthetase
- GS1
cytosolic GS
- GS2
plastidic (chloroplastic) GS
- NF
norflurazon
- NiR
nitrite reductase
- NR
nitrate reductase 相似文献
13.
Wacław L. Kretovich Zinaida G. Evstigneeva Alexander V. Pushkin Tina Z. Dzhokharidze 《Phytochemistry》1981,20(4):625-629
It has been shown that the leaves of pumpkin (Cucurbita pepo) contain two molecular forms of glutamine synthetase (GS), one occurring in the cytosol (GS1)and the other in the chloroplasts (GS2). The activities of both forms were greater when ammonium ion was infiltrated into the leaves and this was shown to be due to de novo synthesis. The two synthetases were purified by ammonium sulphate fractionation, ion exchange chromatography on DEAE-cellulose, selective adsorption on calcium phosphate gel, and preparative polyacrylamide gel electrophoresis. The MWs of GS1 and GS2, estimated by gel filtration on Sephacryl S-200, were 480 000 and 370 000 respectively. During polyacrylamide gel electrophoresis in the presence of SDS both GS1 and GS2 were dissociated into polypeptide chains with MWs of 58 000 and 50 000 respectively, suggesting that GS, 1 and GS2 are octamers consisting of identical monomers. The synthetases showed noticeable differences in their amino acid composition. In GS1 and GS2 the proportions of α- helical segments were 34 and 17 % respectively. In the presence of Mg2+, the pH optima for GS1 and GS2 were 7.25 and 7.75 respectively, and Km values toward l-glutamate were 13 and 46 mM respectively. From the experimental data it is inferred that GS1 and GS2 are isoenzymes. 相似文献
14.
The studies were performed on young triticale seedlings grown on a mineral medium containing 5 mM NO
3
−
as the nitrogen source, with the addition of 0.5 mM CdCl2. It was determined that cadmium ions accumulated mainly in the plant roots. Decreases in nitrate concentrations both in the
roots and shoots of seedlings, as well as decreases in soluble protein contents with simultaneous increases in endopeptidase
activity were also observed. Both in roots and shoots significant decreases in glutamic acid were noted. Toxic cadmium ion
accumulation in seedlings significantly modified activity of primary nitrogen assimilating enzymes, i.e. glutamine synthetase (GS, EC 6.3.1.2) and glutamate dehydrogenase (GDH, EC 1.4.1.2). There was a significant decrease in
GS activity both in roots and in shoots of the stressed plants, in comparison to plants grown without cadmium. In shoots of
the control plants and plants subjected to stress two GS isoforms were discovered: cytoplasmatic (GS1) and chloroplastic (GS2). Substantial decreases in total glutamine synthetase activity in green parts of seedlings, occurring under stress conditions,
result from dramatic decrease in GS2 activity (by 60 % in relation to the control plants); despite simultaneous increases in the cytoplasmatic isoform (GS1) activity by approx. 96 %.
Cadmium ions accumulating in roots and shoots of seedlings not only increased GDH activity, but also modified its coenzymatic
specificity. 相似文献
15.
Francisco Romero Francisco Javier Caballero Francisco Castillo José Manuel Roldán 《Archives of microbiology》1985,143(2):111-116
Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn2+-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS
glutamine synthetase
- MOPS
2-(N-morpholine) propane sulfonate
- MSX
l-methionine-d,l-sulfoximine 相似文献
16.
G. Chandler P. Ladley S.F. McNally M. Patel G.R. Stewart N. Sumar 《Journal of plant physiology》1985,121(1):13-21
Anion exchange chromatography and immunoprecipitation have been used to demonstrate the presence of two forms (GS1, and GS2) of glutamine synthetase in the leaves of nine species of Panicum representative of C3, C4 and C3-C4 intermediate-type photosynthesis. GS2 from the Panicum species, P. miliaceum and P. maximum was more thermostable than GS1, GS1, and GS2 from P. laxum were equally thermostable but GS2 from all the Panicum species examined was more sensitive to inhibition by N-ethylmaleimide than GS1. GS1, and GS2 were characterised as being cytoplasmic and chloroplastic isoforms respectively by their reaction with N-ethylmaleimide and by immunoprecipitation with antibodies raised against the cytosolic isoform in barley and the chloroplastic form in tobacco. C3 species were found to have higher activity of the chloroplastic isoform of glutamine synthetase than C4 species. C3-C4 intermediate species had total leaf glutamine synthetase activities similar to those in C3 species but were found to have a lower chloroplastic isoform content. The results are consistent with the reassimilation of photorespiratory ammonia by chloroplastic glutamine synthetase. 相似文献
17.
Oats Tolerant of Pseudomonas syringae pv. tabaci Contain Tabtoxinine-beta-Lactam-Insensitive Leaf Glutamine Synthetases
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Pseudomonas syringae pv. tabaci, a commonly recognized leaf pathogen of tobacco, can infest the rhizosphere of many plants, including oats. Normal oat plants do not survive this infestation as a consequence of the complete and irreversible inactivation of all of their glutamine synthetases by tabtoxinine-β-lactam (TβL), a toxin released by pv. tabaci. We have identified a population of oat (Avena sativa L. var Lodi) plants that are tolerant of pv. tabaci. The tolerant plants had no detectable TβL-detoxification mechanisms. Pathogen growth on these plant roots was not inhibited. These plants contain leaf glutamine synthetases (GS1 and GS2) that were less sensitive to inactivation by TβL in vitro; these GSs have normal Km values for glutamate and ATP when compared with those of GS in control plants. Root glutamine synthetase of the tolerant plants was inactivated in vivo during infestation by the pathogen or by TβL in vitro. When growing without pv. tabaci, the tolerant plants contained normal levels of glutamine synthetase in their roots and leaves and normal levels of protein, ammonia, glutamate, and glutamine in their leaves. However, when the tolerant plants' rhizosphere was infested with pv. tabaci, the plant leaves contained elevated levels of glutamine synthetase activity, protein, ammonia, glutamate, and glutamine. No changes in glutamate dehydrogenase activity were detected in leaves and roots of pathogen-infested tolerant plants. 相似文献
18.
Ray D. Blackwell Alan J. S. Murray Peter J. Lea Alan C. Kendall Nigel P. Hall Janice C. Turner Roger M. Wallsgrove 《Photosynthesis research》1988,16(1-2):155-176
Manipulation of the CO2 concentration of the atmosphere allows the selection of photorespiratory mutants from populations of seeds treated with powerful mutagens such as sodium azide. So far, barley lines deficient in activity of phosphoglycolate phosphatase, catalase, the glycine to serine conversion, glutamine synthetase, glutamate synthase, 2-oxoglutarate uptake and serine: glyoxylate aminotransferase have been isolated. In addition one line of pea lacking glutamate synthase activity and one barley line containing reduced levels of Rubisco are available. The characteristics of these mutations are described and compared with similar mutants isolated from populations of Arabidopsis. As yet, no mutant lacking glutamine synthetase activity has been isolated from Arabidopsis and possible reasons for this difference between barley and Arabidopsis are discussed. The value of these mutant plants in the elucidation of the mechanism of photorespiration and its relationships with CO2 fixation and amino acid metabolism are highlighted.Abbreviations GS
cytoplasmic glutamine synthetase
- GS2
chloroplastic glutamine synthetase
- PFR
Photon fluence rate
- Rubisco
Ribulose-1,5-bisphosphate carboxylase/oxygenase
- RuBP
Ribulose-1,5-bisphosphate
- SGAT
serine:glyoxylate aminotransferase 相似文献
19.
Escherichia coli glutamine synthetase (GS) preparations composed of 12 adenylylated subunits (GS12?) are almost completely precipitated by sheep Anti-AMP immunoglobulin G (IgG), whereas glutamine synthetase preparations containing 6 adenylylated subunits (GS6?) are only partially precipitated by the antibodies (R.J. Hohman, S.G. Rhee, and E.R. Stadtman, 1980, Proc. Nat. Acad. Sci. USA77, 7410–7414). By means of 125I-labeled anti-AMP antibodies and double immunoprecipitation techniques, in which rabbit antiserum to sheep IgG or anti-GS antibodies were used to precipitate soluble immune complexes, it was demonstrated that under optimal conditions, both the soluble and insoluble immune complexes obtained with either GS6? or GS12? contain 0.5 mol antibody/mol adenylylated subunit. In agreement with the lattice theory of immuno-precipitation, soluble immune complexes are formed in antibody excess. Scatchard plots of binding data indicate that under conditions of antibody excess, one antibody molecule is bound to each AMP moiety of GS12?, whereas GS6? binds a maximum of only 0.68 antibody molecule/adenylylated subunit. We propose that with some species of GS6?, the distribution of adenylylated subunits favors monogamous interactions of the bivalent antibody with two subunits within the same GS molecule and thereby leads to the formation of small, soluble, immune complexes. Other explanations are considered. Only 30% of the antibody population that recognizes unconjugated 5′-AMP binds to the AMP moiety of adenylylated GS. Anti-AMP antiserum can be fractionated on a GS12?-Sepharose matrix into two subpopulations of antibody with strikingly different immunoprecipitation characteristics. Conversely, species of GS with various states of adenylylation ranging from 0 to 8 were separated from a GS6? preparation by means of affinity chromatography on an anti-AMP antibody-Sepharose matrix. Under optimal conditions, antibodies purified by affinity chromatography precipitated a smaller fraction of a GS6? preparation than did unfractionated antiserum. Competence of the purified antibody was nearly restored to that of the unfractionated serum by the addition of an enhancement factor present in the IgG fraction of nonimmune serum. The enhancement factor was not required for complete precipitation of GS?12 by purified antibodies. Contrary to most antibody-antigen reactions, immunoprecipitation of GS6? with anti-AMP antibodies is greater at 30 °C than at 4 °C. 相似文献
20.
Studies on the glutamine synthetases (GS, EC 6.3.1.2) of green (GS2) and etiolated leaves (GSet) ofSinapis alba L. (cv. Steinacher) revealed striking similarities between the respective enzyme proteins. The enzymes showed corresponding
chromatographic properties, both on dimethylaminoethyl-Sephacel and on hydroxylapatite columns. The purified GS proteins were
also identical with regard to the molecular weight of their subunits. Isoelectrofocusing of pure GSet yielded two distinct polypeptide bands in the pH 5.6 region of the gels. This pattern corresponded to the two strong bands
of GS2. Two charge variants of GS polypeptides could be detected by Western-blot analysis of the soluble protein of green leaves
using antibodies against mustard GS2. In immunoprecipitation experiments, the holoenzymes of GS2 and GSet were recognized with identical affinities by this antiserum. We conclude that strong similarities exist between the proteins
of the GS enzymes in green and etiolated leaves of mustard. Most probably only one GS form, namely the plastidic enzyme, can
be found in the epigeal organs ofSinapis. The polypeptides of the GS2 subunits showed no differences in the hydrophobicity of the polypeptide chains. Neither glucosyl nor mannosyl residues could
be detected.
Dedicated to Professor Dr. H. Mohr on the occasion of his 60th birthday 相似文献