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1.
Purification and characterization of mRNA from soybean seeds. Identification of glycinin and beta-conglycinin precursors 总被引:15,自引:0,他引:15
Poly(A)-rich RNA was isolated from developing soybean seeds (Glycine max (L.) Merr.) and fractionated on linear log sucrose gradients. Two major fractions sedimenting at 18 S and 20 S were separated and then purified by further sucrose gradient fractionation. Both fractions were active as messengers when added to a rabbit reticulocyte lysate protein synthesis system. The 18 S fraction caused proteins migrating primarily to the 60,000-dalton region of a sodium dodecyl sulfate gel to be produced, while translation of the 20 S fraction preferentially directed the synthesis of polypeptides similar in size to the alpha and alpha' subunits of beta-conglycinin. Evidence that many of the 60,000-dalton polypeptides were related to glycinin and the high molecular weight 20 S translation products were related to beta-conglycinin was obtained by immunoprecipitation using monospecific antibodies against glycinin and beta-conglycinin, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated products revealed that the glycinin precursor region contained at least three different size components and that the family of glycinin precursors had larger apparent molecular weight (58,000-63,000) than the disulfide-linked complexes between acidic and basic glycinin subunits (57,000). Unlike the disulfide-linked glycinin complexes which were cleaved by disulfide reduction, glycinin precursors were insensitive to reducing agents. The alpha and alpha' subunits synthesized in vitro also had slightly larger apparent molecular weights than purified alpha and alpha' standards. 相似文献
2.
Identification and characterization of a monofunctional glycosyltransferase from Staphylococcus aureus
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Wang QM Peery RB Johnson RB Alborn WE Yeh WK Skatrud PL 《Journal of bacteriology》2001,183(16):4779-4785
A gene (mgt) encoding a monofunctional glycosyltransferase (MGT) from Staphylococcus aureus has been identified. This first reported gram-positive MGT shared significant homology with several MGTs from gram-negative bacteria and the N-terminal glycosyltransferase domain of class A high-molecular-mass penicillin-binding proteins from different species. S. aureus MGT contained an N-terminal hydrophobic domain perhaps involved with membrane association. It was expressed in Escherichia coli cells as a truncated protein lacking the hydrophobic domain and purified to homogeneity. Analysis by circular dichroism revealed that secondary structural elements of purified truncated S. aureus MGT were consistent with predicted structural elements, indicating that the protein might exhibit the expected folding. In addition, purified S. aureus MGT catalyzed incorporation of UDP-N-acetylglucosamine into peptidoglycan, proving that it was enzymatically active. MGT activity was inhibited by moenomycin A, and the reaction product was sensitive to lysozyme treatment. Moreover, a protein matching the calculated molecular weight of S. aureus MGT was identified from an S. aureus cell lysate using antibodies developed against purified MGT. Taken together, our results suggest that this enzyme is natively present in S. aureus cells and that it may play a role in bacterial cell wall biosynthesis. 相似文献
3.
Dea Hwan Kim Seong Kyong Kim Jeong-Ho Kim Bong-Gyu Kim Joong-Hoon Ahn 《Plant Physiology and Biochemistry》2009,47(11-12):991-997
In this study, a flavonoid malonyltransferase (OsMaT-2) was cloned from Oryza sativa, and the recombinant protein OsMaT-2 was purified via affinity chromatography. OsMaT-2 utilized a variety of flavonoid glucosides, including flavanone glucosides, flavone glucosides, flavonol glucosides, and isoflavone glucosides as substrates, but did not utilize anthocyanin. As an acyl donor, OsMaT-2 utilized only malonyl-CoA. Based on reactions with various quercetin 3-O-sugars, we identified the probable position of malonylation as the 6″-hydroxyl group of the sugar. This is the first report, to the best of our knowledge, of the cloning of a flavonoid malonyltransferase from O. sativa. 相似文献
4.
《Phytochemistry》1986,25(5):1067-1071
Acetyl-CoA carboxylase from two lines of soybean (Glycine max) seeds has been purified to apparent homogeneity. The procedure included affinity chromatography of the enzyme on avidin-monomer-Sepharose 4B. The enzyme from both lines showed a single band on polyacrylamide gel electrophoresis. On sodium dodecyl sulphatepolyacrylamide gel electrophoresis, the enzyme from experimental line 9686 showed a single protein band having the M, 240 000. The enzyme from the commercial line Wayne, however, showed three protein bands having the M, s 240 000, 65 000 and 58 000, respectively. High concentrations of the enzyme were required for stability as well as the presence of dithiothreitol, glycerol and Triton X-100. The enzyme was active over a wide pH range, with an optimum at 8.2 for 9686 and 7.5 for Wayne. The enzyme from both 9686 and Wayne showed absolute specificity for acetyl-CoA as a substrate and this could not be replaced by propionyl-CoA, butyryl-CoA, hexanoyl-CoA or S-methylerotonyl-CoA. At the optimum pH the apparent Km values for the substrates were: bicarbonate, 1.13 mM; acetyl-CoA, 0.32 mM; ATP, 0.46 mM for the Wayne carboxylase and bicarbonate, 1.56 mM; acetyl-CoA, 0.17 mM; ATP, 0.14 mM for the 9686 enzyme. Citrate, at higher concentrations, was strongly inhibitory. Both ADP and AMP inhibited the enzyme from 9686 and Wayne. The enzyme from both 9686 and Wayne did not appear to be highly regulated by cellular metabolites. 相似文献
5.
A new protein has been isolated from the reserve proteins of the seeds of soybean (Glycine max) which is particularly deficient in methionine and cysteine. The protein dissociated in sodium dodecyl sulfate into a single polypeptide, Mr 48,000. The amino acid composition, N-terminal leucine and mobility on gel electrophoresis of this polypeptide all were indistinguishable from the β-subunit of β-conglycinin. In its nondissociated form, the protein behaved as a trimer of Mr, 137,000 ± 4000. Its sedimentation coefficient at ionic strength 0.5 was 7.5 S and it possessed antigenic determinants in common with β-conglycinin. This protein therefore has the properties of a new isomer of β-conglycinin—a homogeneous trimer of β subunits. 相似文献
6.
A set of Ds-element enhancer trap lines of Arabidopsis thaliana was generated and screened for expression patterns leading to the identification of a line that showed root-specific expression of the bacterial uidA reporter gene encoding beta-glucuronidase (GUS). The insertion of the Ds element was found to be immediately downstream to a glycosyltransferase gene At1g73160. Analysis of At1g73160 expression showed that it is highly root-specific. Isolation and characterization of the upstream region of the At1g73160 gene led to the definition of a 218 bp fragment that is sufficient to confer root-specific expression. Sequence analysis revealed that several regulatory elements were implicated in expression in root tissue. The promoter identified and characterized in this study has the potential to be applied in crop biotechnology for directing the root-specific expression of transgenes. 相似文献
7.
Purification and characterization of phytase from cotyledons of germinating soybean seeds 总被引:12,自引:0,他引:12
Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively. 相似文献
8.
Purification and characterization of acid phosphatase from cotyledons of germinating soybean seeds 总被引:6,自引:0,他引:6
Soybean acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was completely separated from phytase (EC 3.1.3.8) isolated from cotyledons of germinating seeds and purified to homogeneity. A four-step purification regimen consisting of ammonium sulfate fractionation, and ion-exchange, affinity, and chromatofocusing gel chromatographies was employed to achieve a homogeneous preparation. Acid phosphatase activity appeared as a major band of the three forms of acid phosphatase identified on native gels. The purified enzyme had a molecular weight of 53,000 when electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular weight of 53,000 from its mobility in a Fracto-gel TSK HW-50F gel permeation column. The molar extinction coefficient of the enzyme at 278 nm was estimated to be 4.2 X 10(4) M-1 cm-1. The isoelectric point of the protein, as revealed by chromatofocusing, was about 6.7. The optimal pH for activity, like other plant acid phosphatases, was 5.0. While the enzyme failed to accommodate phytate as a substrate, the enzyme did exhibit a broad substrate selectivity. The affinity of the enzyme for p-nitrophenyl phosphate was high (Km = 70 microM), and activity was competitively inhibited by orthophosphate (Ki = 280 microM). The estimated catalytic turnover number (Kcat) of the enzyme for p-nitrophenyl phosphate was about 430 per second. Although the purified enzyme was stable at 0 degrees C and exhibited maximum catalytic activity at 60 degrees C, thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 68 degrees C for 10 min. 相似文献
9.
Gupta Om Prakash Dahuja Anil Sachdev Archana Kumari Sweta Jain Pradeep Kumar Vinutha T. Praveen Shelly 《Molecular biology reports》2019,46(4):3713-3730
Molecular Biology Reports - Despite the significant importance of soybean isoflavone, the regulatory mechanism of miRNAs during its biosynthesis is highly unexplored. In the present work, nine... 相似文献
10.
Purification of allantoinase from soybean seeds and production and characterization of anti-allantoinase antibodies. 总被引:2,自引:1,他引:1
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Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, a reaction important in both biogenesis and degradation of ureides. Ureide production in cotyledons of germinating soybean (Glycine max L.) seeds has not been studied extensively but may be important in mobilizing nitrogen reserves. Allantoinase was purified approximately 2500-fold from a crude extract of soybean seeds by differential centrifugation, heat treatment, ammonium sulfate fractionation, ethanol fractionation, and fast protein liquid chromatography (Pharmacia) with Mono-Q and Superose columns. The purified enzyme had a subunit size of 30 kD. Polyclonal antibodies produced against the purified protein titrated allantoinase activity in a crude extract of seed proteins. Antibodies recognized the 30-kD band in western blot analysis of crude seed extracts, indicating that they were specific for allantoinase. 相似文献
11.
Isolation and characterization of an N-acetylgalactosamine specific lectin from Salvia sclarea seeds 总被引:1,自引:0,他引:1
Crude extracts from Salvia sclarea seeds were known to contain a lectin which specifically agglutinates Tn erythrocytes (Bird, G. W. G., and Wingham, G. (1974) Vox Sang. 26, 163-166). We have purified the lectin to homogeneity by ion-exchange chromatography and affinity chromatography. The agglutinin was found to be a glycoprotein of Mr = 50,000, composed of two identical subunits of Mr = 35,000 linked together by disulfide bonds. The purified lectin agglutinates specifically Tn erythrocytes and, at higher concentrations, also Cad erythrocytes. Native A, B, or O red blood cells are not agglutinated by the lectin and, even after treatment with sialidase or papain, these cells are not recognized. Tn red cells present 1.45 X 10(6) accessible sites to the lectin which binds to these erythrocytes with an association constant of 1.8 X 10(6) M-1. On Cad red cells, 1.73 X 10(6) sites are accessible to the lectin which binds with an association constant of 1.0 X 10(6) M-1. The carbohydrate specificity of the S. sclarea lectin has been determined in detail, using well defined monosaccharide, oligosaccharide, and glycopeptide structures. The lectin was found to be specific for terminal N-acetylgalactosamine (GalNAc) residues. It binds preferentially alpha GalNAc determinants either linked to Ser or Thr (as in Tn structures) or linked in 1-3 to a beta GalNAc or to an unsubstituted beta Gal. Although more weakly, the lectin binds beta GalNAc residues linked in 1-4 to a beta Gal (as in Cad structures). It does not recognize beta GalNAc determinants linked in 1-3 to a Gal (as in globoside) or the alpha GalNAc residues of blood group A structures. 相似文献
12.
Purification and characterization of a specific nucleoside diphosphatase from soybean root nodules 总被引:2,自引:2,他引:0
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A specific nucleoside diphosphatase was purified from the plant portion of soybean (Glycine max L.) root nodules. This enzyme is highly specific for nucleotide diphosphates; it is unable to hydrolyze nucleotide tri- and monophosphates or a variety of other phosphorylated compounds. It will, however, hydrolyze any nucleotide disphosphate tested. The pH optimum of the enzyme is about 7.5; it requires a divalent cation for activity; and it is neither inhibited nor activated by any of the metabolites tested. It appears that in vivo this enzyme would be very active, but its function is not clear. 相似文献
13.
Both in mammals and plants, excess lysine (Lys) is catabolized via saccharopine into alpha-amino adipic semialdehyde and glutamate by two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. To study the control of metabolite flux via this bifunctional enzyme, we have purified it from developing soybean (Glycine max) seeds. LKR activity of the bifunctional LKR/SDH possessed relatively high K(m) for its substrates, Lys and alpha-ketoglutarate, suggesting that this activity may serve as a rate-limiting step in Lys catabolism. Despite their linkage, the LKR and SDH enzymes possessed significantly different pH optima, suggesting that SDH activity of the bifunctional enzyme may also be rate-limiting in vivo. We have previously shown that Arabidopsis plants contain both a bifunctional LKR/SDH and a monofunctional SDH enzymes (G. Tang, D. Miron, J.X. Zhu-Shimoni, G. Galili [1997] Plant Cell 9: 1-13). In the present study, we found no evidence for the presence of such a monofunctional SDH enzyme in soybean seeds. These results may provide a plausible regulatory explanation as to why various plant species accumulate different catabolic products of Lys. 相似文献
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16.
Microsatellites, or simple sequence repeats (SSRs), are very useful molecular markers for a number of plant species. We used a new publicly available module (TROLL) to extract microsatellites from the public database of soybean expressed sequence tag (EST) sequences. A total of 12,833 sequences containing di- to penta-type SSRs were identified from 200,516 non-redundant soybean ESTs. On average, one SSR was found per 7.25?kb of EST sequences, with the tri-nucleotide motifs being the most abundant. Primer sequences flanking the SSR motifs were successfully designed for 9,638 soybean ESTs using the software primer3.0 and only 59 pairs of them were found in earlier studies. We synthesized 124 pairs of the primers to determine the polymorphism and heterozygosity among eight genotypes of soybean cultivars, which represented a wide range of the cultivated soybean cultivars. PCR amplification products with anticipated SSRs were obtained with 81 pairs of primers; 36 PCR products appeared to be homozygous and the remaining 45 PCR products appeared to be heterozygous and displayed polymorphism among the eight cultivars. We further analysed the EST sequences containing 45 polymorphic EST-SSR markers using the programs BLASTN and BLASTX. Sequence alignment showed that 29 ESTs have homologous sequences and 15 ESTs could be classified into a Uni-gene cluster with comparatively convincing protein products. Among these 15 ESTs belonging to a Uni-gene cluster, 9 SSRs were located in 3'-UTR, 4 SSRs were located in the intron region and 2 SSRs were located in the CDS region. None of these SSRs was located in the 5'-UTR. These novel SSRs identified in the ESTs of soybean provide useful information for gene mapping and cloning in future studies. 相似文献
17.
18.
Santosh Kumar Ramachandra Jadhav Krunal Arvind Patel Bhushan Bhalchandra Dholakia Bashir Mohammad Khan 《Bioinformation》2012,8(19):943-949
Medicinal plants are extensively utilized in traditional and herbal medicines, both in India and around the world due to the
presence of diverse low molecular weight natural products such as flavonoids, alkaloids, terpenoids and sterols. Flavonoids which
have health benefits for humans are the large class of phenylpropanoid-derived secondary metabolites and are mostly glycosylated
by UDP-glycosyltransferases (UGTs). Although large numbers of different UGTs are known from higher plants, very few protein
structures have been reported till now. In the present study, the three-dimensional model of flavonoid specific glycosyltransferases
(WsFGT) from Withania somnifera was constructed based on the crystal structure of plant UGTs. The resulted model was assessed
by various tools and the final refined model revealed GT-B type fold. Further, to understand the sugar donors and acceptors
interactions with the active site of WsFGT, docking studies were performed. The amino acids from conserved PSPG box were
interacted with sugar donor while His18, Asp110, Trp352 and Asn353 were important for catalytic function. This structural and
docking information will be useful to understand the glycosylation mechanism of flavonoid glucosides.
Abbreviations
DOPE - Discrete Optimized Potential Energy, PDB - Protein Data Bank, PSPG - Plant Secondary Product Glycosyltransferase, RMSD - Root Mean Squared Deviation, UDP - Uridine diphosphate, UGT - UDP-glycosyltransferases. 相似文献19.
Satouchi K Kodama Y Murakami K Tanaka T Iwamoto H Ishimoto M 《Bioscience, biotechnology, and biochemistry》2002,66(10):2154-2160
A lipase-inhibiting protein was isolated from lipoxygenase (LOX)-deficient soybean seeds. The molecular mass of the protein was 56.0-kDa and the N-terminal amino acid was blocked. The protein was identified by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. The masses of the lysyl endopeptidase-digested peptides of the 56.0-kDa inhibiting protein were almost identical to the calculated masses of the theoretically predicted lysyl endopeptidase-treated peptides of beta-amylase from soybean seed. In a previous paper (Biosci. Biotechnol. Biochem., 62, 1498-1503, 1998), we reported that LOX-1, an isozyme of soybean seed LOX, inhibited hydrolysis of soybean oil by pancreatic lipase. Purified beta-amylase also inhibited lipase activity, although the magnitude of inhibition was weaker than that by LOX-1. Thus, there are at least two lipase-inhibiting proteins, one is a LOX and the other is a beta-amylase, in soybean seed. 相似文献
20.
Kinetic characterization of the monofunctional glycosyltransferase from Staphylococcus aureus
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The glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs (MGTs) belong to the GT51 family in the sequence-based classification of GTs. They both possess five conserved motifs and use lipid II precursor (undecaprenyl-pyrophosphate-N-acetylglucosaminyl-N-acetylmuramoyl- pentapeptide) to synthesize the glycan chain of the bacterial wall peptidoglycan. MGTs appear to be dispensable for growth of some bacteria in vitro. However, new evidence shows that they may be essential for the infection process and development of pathogenic bacteria in their hosts. Only a small number of class A PBPs have been characterized so far, and no kinetic data are available on MGTs. In this study, we present the principal enzymatic properties of the Staphylococcus aureus MGT. The enzyme catalyzes glycan chain polymerization with an efficiency of approximately 5,800 M(-1) s(-1) and has a pH optimum of 7.5, and its activity requires metal ions with a maximum observed in the presence of Mn2+. The properties of S. aureus MGT are distinct from those of S. aureus PBP2 and Escherichia coli MGT, but they are similar to those of E. coli PBP1b. We examined the role of the conserved Glu100 of S. aureus MGT (equivalent to the proposed catalytic Glu233 of E. coli PBP1b) by site-directed mutagenesis. The Glu100Gln mutation results in a drastic loss of GT activity. This shows that Glu100 is also critical for catalysis in S. aureus MGT and confirms that the conserved glutamate of the first motif EDXXFXX(H/N)X(G/A) is likely the key catalytic residue in the GT51 active site. 相似文献