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1.
The two chalcone-synthase forms from leaves ofSpinacia oleracea L. were purified to apparent homogeneity. Antibodies were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light.Abbreviations DEAE
diethylaminoethyl
- DTE
1,4-dithioerythritol
- EDTA
ethylenediaminetetraacetic acid
- HPLC
high-performance liquid chromatography
- IgG
immunoglobulin G
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Parts of the results were presented at the 14th International Botanical Congress at Berlin in July 1987 相似文献
2.
Phosphorylation of the plasma-membrane H+-ATPase of oat roots by a calcium-stimulated protein kinase
When plasma-membrane vesicles isolated from oat (Avena sativa L.) root cells were incubated with [-32P]ATP, the H+-ATPase was found to be phosphorylated at serine and threonine residues. Phosphotyrosine was not detected. Endogenous ATPase kinase activity was also observed in plasma-membrane vesicles isolated from potato (Solanum tuberosum L.) root cells as well as from yeast (Saccharomyces cerevisiae). Identity of the phosphorylated oat root Mr=100 000 polypeptide as the ATPase was confirmed using conventional glycerol density-gradient centrifugation to purify the native enzyme and by a new procedure for purifying the denatured polypeptide using reversephase high-performance liquid chromatography. Kinase-mediated phosphorylation of the oat root plasma-membrane H+-ATPase was stimulated by the addition of low concentrations of Ca2+ and by a decrease in pH, from 7.2 to 6.2. These results demonstrate that kinase-mediated phosphorylation of the H+-ATPase is a plausible mechanism for regulating activity. They further indicate that changes in the cytoplasmic [Ca2+] and pH are potentially important elements in modulating the kinase-mediated phosphorylation.Abbreviations EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- Mr
relative molecular mass
- RP-HPLC
reverse-phase high-performance liquid chromatography
- SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis 相似文献
3.
In flower extracts of defined genotypes of Matthiola incana, an enzyme was demonstrated which catalyzes the transfer of the glucosyl moiety of uridine 5-diphosphoglucose (UDPGlc) to the 5-hydroxyl group of pelargonidin and cyanidin 3-glycosides and acylated derivatives. The best substrate for 5-glucosylation is the 3-xylosylglucoside acylated with p-coumarate, followed by the 3-xylosylglucoside and by the acylated (p-coumarate) 3-glucoside. The 3-glucoside itself is a very poor substrate. Besides UDPGlc, thymine 5-diphosphoglucose is a suitable glucosyl-donor, but with a reduced reaction rate (42%). The anthocyanin 5-O-glucosyltransferase exhibits a pH optimum at 7.5 and is generally inhibited by divalent ions and by ethylenediaminetetraacetic acid and p-chloromercuribenzoate. Investigations on different genotypes showed that the 5-O-glucosyltransferase activity is clearly controlled by the gene l. In confirmation of earlier chemogenetic work, enzyme activity is only present in lines with the wild-type allele l+. The anthocyanin 5-O-glucosyltransferase activity is strictly correlated with the formation of 5-glucosylated anthocyanins during bud development.Abbreviations Cg
3,5-T-cyanidin 3-sambubioside-5-glucoside
- EDTA
ethylene diaminetetraacetic acid
- 5GT
UDP-glucose: anthocyanin 5-O-glucosyltransferase
- 3GT
UDP-glucose: anthocyanidin/flavonol 3-O-glucosyltransferase
- HPLC
high-performance liquid chromatography
- TLC
thin-layer chromatography
- UDPGlc
uridine 5-diphospho-glucose 相似文献
4.
Vishal V. Dawkar Umesh U. Jadhav Amar A. Telke Sanjay P. Govindwar 《Biotechnology and Bioprocess Engineering》2009,14(3):361-368
Peroxidase was purified by an ion exchange chromatography followed by gel filtration chromatography from dye degrading Bacillus sp. strain VUS. The optimum pH and temperature of the enzyme activity was 3.0 and 65°C, respectively. This enzyme showed
more activity with n-propanol than other substrates tested viz. xylidine, 3-(3,4-dihydroxy phenyl) Lalanine (L-DOPA), hydroxyquinone, ethanol, indole, and veratrole. Km value of the enzyme
was 0.076 mM towards n-propanol under standard assay conditions. Peroxidase was more active in presence of the metal ions like Li2+, Co2+, K2+, Zn2+, and Cu2+ where as it showed less activity in the presence of Ca2+ and Mn2+. Inhibitors like ethylenediamine tetraacetic acid (EDTA), glutamine, and phenylalanine inhibited the enzyme partially, while
sodium azide (NaN3) completely. The crude as well as the purified peroxidase was able to decolourize different industrial dyes. This enzyme
decolourized various textile dyes and enhanced percent decolourization in the presence of redox mediators. Aniline was the
most effective redox mediator than other mediators tested. Gas chromatography-Mass spectrometry (GC-MS) confirmed the formation
of 7-Acetylamino-4-hydroxy-naphthalene-2-sulphonic acid as the final product of Reactive Orange 16 indicating asymmetric cleavage
of the dye. 相似文献
5.
Yasuo Yajima Akio Enoki Mary B. Mayfield Michael H. Gold 《Archives of microbiology》1979,123(3):319-321
A soluble enzyme fraction from Phanerochaete chrysosporium catalyzed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone. The enzyme, partially purified by ammonium sulfate precipitation, required NADPH and molecular oxygen for activity. NADH was not effective. Optimal activity was displayed between pH 7.5–8.5. Neither EDTA, KCN, NaN3, nor o-phenanthroline (5 mM) were inhibitory. The enzyme was inducible with maximal activity displayed after incubation of previously grown cells with 0.1% vanillate for 30h.Abbreviations MHQ
Methoxy-p-hydroquinone
- GLC
gas liquid chromatography
- TMSi
trimethylsilane
- TLC
thin layer chromatography 相似文献
6.
Involvement of cinnamyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench 总被引:16,自引:0,他引:16
Christian Pillonel Marcel M. Mulder Jaap J. Boon Birgit Forster Andres Binder 《Planta》1991,185(4):538-544
The lignin structure and enzyme activities of normal and brown-midrib (BMR-6) mutant lines of Sorghum bicolor have been compared to identify the enzyme(s) involved in the reduction of the lignin content of the mutant. The results indicate that cinnamyl-alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase are depressed in the BMR-6 line, whereas the structural modifications correspond only to a reduction of CAD activity. Apparently, the change in the Sorghum lignin content, caused by depression of CAD activity, is accompanied by the incorporation of cinnamaldehydes into the core lignin.Abbreviations CAD
cinnamyl-alcohol dehydrogenase
- HPLC
high-performance liquid chromatography
- m/z
mass number
- OMT
caffeic acid O-methyltransferase 相似文献
7.
The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg2+ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom. 相似文献
8.
A plant lipid was isolated from zucchini (Cucurbita pepo L.) membranes and from soybean (Glycine max [L.] Merr) phospholipids by thinlayer chromatography and further purified by high-performance liquid chromatography. This plant lipid was chromatographically very similar to the platelet-activating factor, an ether phospho-lipid with hormone-like properties found in mammals. Both the plant lipid and the platelet-activating factor stimulated ATP-dependent H+ transport in isolated membrane vesicles from zucchini hypocotyls.Abbreviations HPLC
high-performance liquid chromatography
- PAF
platelet-activating factor 相似文献
9.
From isolated nuclei of suspension cultured cells of Nicotiana tabacum. DNA-dependent RNA polymerase II (E.C. 2.7.76) has been purified to homogeneity as evidenced by polyacrylamidegel electrophoresis under non-denaturing conditions. The purified enzyme had a specific activity of more than 15 nmol min-1·mg-1 with denatured calf thymus DNA as template. Sodium-dodecyl-sulfate gel electrophoresis and protein highperformance liquid chromatography revealed a subunit composition of four proteins with molecular weights of 165 000, 135 000, 35 000 and 25 000 and with a stoichiometry of 1:1:2:2. The RNA polymerase did not exhibit any detectable proteinkinase activity. The 25 000 subunit binds ADP in a molar ratio of 1:1; it could not be decided whether this subunit has an ATPase activity or is merely an acceptor of ADP.Abbreviations HPLC
high-performance liquid chromatography
- PMSF
phenylmethylsulfonyl fluoride
- SDS
sodium dodecyl sulfate
This contribution is dedicated to Professor Fritz Cramer on the occasion of his 60th birthday 相似文献
10.
Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots 总被引:2,自引:0,他引:2
Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA1 and GA4. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA9- as well as GA20-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod
– and fix
– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA
enzyme immunoassay
- GAn
gibberellin An
- GC-MS
gas chromatography-mass spectrometry
- HPLC
high-performance liquid chromatography
- IAA
indole-3-acetic acid
- Me
methyl ester
- RIA
radioimmunoassay
- TLC
thin-layer chromatography 相似文献
11.
A gibberellin (GA) C-20 hydroxylase that catalyses the conversion of GA53 to GA44 was purified from developing pea embryos by ammonium-sulfate precipitation, gel filtration and anion-exchange column chromatography. The purification was about 270-fold and 15% of the enzymic activity was recovered. The relative molecular mass was 44000 by Sephadex G-200 gel filtration. The apparent Michaelis constant was 0.7 M and the isoelectric point was 5.6–5.9. The enzymic activity was optimal at pH 7.0 2-Oxoglutarate and ascorbate were required for activity. Low concentrations of Fe2+ stimulated the reaction, but externally added Fe2+ was not essential, even in the most purified preparation. Catalase and bovine serum albumin also stimulated. Dithiothreitol preserved the activity during purification but was not needed during incubation. In fact, the simultaneous presence of dithiothreitol and Fe2+ in the incubation mixture was inhibitory to the purified enzyme. The cofactor requirements are typical for those of 2-oxoglutarate-dependent dioxygenases.When the incubation time was long enough, GA53 was converted to both GA44 and GA19. The proportions of these two products remained constant throughout the purification, but this does not necessarily mean that their formations is catalysed by a single enzyme. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the final preparation contained several proteins. Although the most prominent protein band was located within the range expected for the enzyme on the grounds of its molecular weight, this band did not represent the enzyme, since it separated from the GA C-20 hydroxylase activity on ultrathin-layer isoeletric focusing.Abbreviation BSA
bovine serum albumin
- DEAE
diethylaminoethyl
- DTT
dithiothreitol
- EDTA
ethylenediamine-tetraacetic acid
- GAn
gibberellin An
- HPLC
high-performance liquid chromatography
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
12.
Li Cui Ming Sheng Dong Xiao Hong Chen Mei Jiang Xin Lv Guijun Yan 《World journal of microbiology & biotechnology》2008,24(4):483-489
A novel fibrinolytic enzyme from Cordyceps militaris was purified and partially characterized for the first time, which was designated C. militaris fibrinolytic enzyme (CMase). This extracellular enzyme from C. militaris was isolated by ammonium sulphate fraction, and purified to electrophoretic homogeneity using gel filtration chromatography.
The apparent molecular mass of the purified enzyme was estimated to be 27.3 kDa by SDS-PAGE. The optimum pH and temperature
for the enzyme activity were pH 6.0 and 25 °C, respectively. In the presence of metal ions such as Mg2+ and Fe2+ ions the activity of the enzyme increased, whereas EDTA and Cu2+ ion inhibited the enzyme activity. Interestingly the N-terminal amino acid sequences of the enzyme is extremely similar to
those of the trypsin proteinases from insects, and has no significant homology with those of the fibrinolytic enzyme from
other medicinal mushroom. In conclusion, C. militaris produces a strong fibrinolytic enzyme CMase and may be considered as a new source for thrombolytic agents. 相似文献
13.
Summary An NADH-cytochromeb
5 reductase was purified from rat liver plasma membranes. Rat liver plasma membranes were prepared by aqueous two-phase partition. Peripheral proteins were removed by EDTA extraction and integral membrane proteins were solubilized with Triton X-100. The NADH-cytochromeb
5 reductase was purified by hydroxyapatite, anion exchange, and gel filtration chromatographies. The purified preparation was homogeneous and estimated to have an apparent molecular weight of 32 kDa on SDS-polyacrylamide gel electrophoresis. Two tryptic peptides of the purified enzyme had sequence homologies with rat, human, and bovine NADH-cytochromeb
5 reductases.Abbreviations CHAPS
3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate
- BCA
bicinchonicic acid
- EDTA
ethylenediamine tetraacetate acid disodium salt
- FeCN
ferricyanide
- HPLC
high-performance liquid chromatography
- NADH
nicotinamide adenine dinucleotide reduced form
- PMSF
-phenylmethylsulfonyl fluoride 相似文献
14.
Recently, some evidence for the occurence of a light-independent protochlorophyllide-reducing enzyme in greening barley plants has been presented. In the present work this problem was reinvestigated. -[14C] Aminolevulinic acid was fed to isolated barley shoots from plants which had been preilluminated for various lengths of time. Porphyrins which had been synthesized during the dark incubation were analyzed by high-performance liquid chromatography. There was no evidence for a light-independent synthesis of chlorophyll(ide). The 14C-labelled precursor was incorporated almost exclusively into protochlorophyllide. The reduction of labelled protochlorophyllide to chlorophyllide was strictly light-dependent. These results are not consistent with the existence of a light-independent protochlorophyllide-reductase in barley as proposed previously.Abbreviation HPLC
high-performance liquid chromatography 相似文献
15.
Reverse-phase high-performance liquid chromatography was used to analyse 14C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [14C]IAA, stelar segments had metabolised between 1–6% of the methanol-extractable radioactivity compared with 91–92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [14C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [14C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC
High-performance liquid chromatography
- IAA
Indole-3-acetic acid 相似文献
16.
Tobacco (N. tabacum cv. Xanthi) cell lines contained two forms of anthranilate synthase (AS; EC 4.1.3.27) which could be partially separated by gel-filtration chromatography. One form was resistant to feedback inihibition by 10 M tryptophan (trp) while the other form was almost completely inhibited by trp at the same concentration. Cell lines selected as resistant to 5-methyltryptophan (5MT) had more of the trp-resistant AS form. Only the trp-sensitive form was detected in plants regenerated from both normal and 5MT-resistant cell lines. Overexpression of the trp-resistant form in 5MT-resistant tobacco cells disappeared during plant regeneration but reappeared when callus was initiated from the leaves of these plants. The trp-sensitive form was localized in the particulate fraction and the trp-resistant form in the cytosol of tobacco cultured cell protoplasts. The trp-resistant form of AS from tobacco had an estimated MW of 200 000, determined by Sephacryl S-200 chromatography, compared to an estimated MW of 150 000 for the trp-sensitive form. The estimated molecular weights of AS from carrot and corn were 160 000 and 150 000, respectively. Analysis of AS activity from the diploid Nicotiana species Nicotiana otophora (chromosome number 2n=24) by high-performance liquid chromatography showed two activity peaks identical in elution time and trp inhibition characteristics to the activity from N. tabacum (chromosome No. 48). Thus the two enzyme forms found in tobacco did not appear to have originated individually from the progenitor species genomes which combined to make up the tobacco genome.Abbreviations AS
anthranilate synthase
- 2,4-D
2,4-dichlorophenoxyacetic acid
- HPLC
high-performance liquid chromatography
- 5MT
D1-5-methyltryptophan
- trp
L-tryptophan 相似文献
17.
A proteinase from the sarcocarp of melon (Cucumis Melo L. var. Prince) was purified by a three-step procedure involving batch-wise treatment with CM-cellulose fibers, column chromatography on CM-cellulose powder and gel filtration on Sephadex G-75. The final enzyme preparation was homogeneous on acrylamide gel electrophoresis. Its molecular weight was estimated by two different methods to be about 50,000. Anlayses indicated tha presence of 475 amino acid residues and at least 7 moles of hexose. The maximum activity was found in the alkaline pH region against casein as a substrate. The optimum temperature against casein was 70 degrees at pH 7.1. The enzyme was strongly inhibited by diisopropyl fluorophosphate, partly inhibited by HgCl2 and not inhibited by EDTA, p-chloromercuribenzoic acid, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor. The reduced and carboxymethylated insulin B-chain was cleaved at the peptide bonds of Asn3-Gln4, Cm-Cys7-Gly8, Glu13-Ala14, Leu15-Tyr16, Cm-Cys19-Gly20, Phe25-Tyr26, Pro28-Lys29, and Lys29-Ala30 by the enzyme. 相似文献
18.
E. Bräutigam F. Fiedler D. Woitzik H. T. Flammann J. Weckesser 《Archives of microbiology》1988,150(6):567-573
The capsule polysaccharide-protein-peptidoglycan complex (insoluble in boiling sodium dodecyl sulfate and hot phenol-water) from cell envelopes of Rhodobacter capsulatus St. Louis was characterized. Hydrofluoric, hydrochloric acid or alkaline hydrolysis solubilized the polysaccharide moiety, whereas the protein-peptidoglycan moiety remained insoluble. On treatment of the protein-peptidoglycan moiety with lysozyme, the protein with peptidoglycan-residues bound was solubilized. It showed a single, broad peptide band (M
r=about 17,000) on sodium dodecyl sulfate polyacrylamide gel-electrophoresis. The same protein was obtained by lysozyme digestion (without preceding hydrofluoric or hydrochloric acid treatment) of the protein-peptidoglycan complex of the phage-resistant mutant Rhodobacter capsulatus St. Louis RC1-, in which the capsule polysaccharide is present in a free form. A protein-peptidoglycan complex was isolated also from the capsulefree Rhodobacter capsulatus 37b4. Covalent binding between the protein and peptidoglycan moieties is likely for all three strains as is the lipoprotein nature of the protein moiety. The polysaccharide moiety of the complete complex from the wild-type Rhodobacter capsulatus St. Louis was at least partly removable from the complex in the presence of high salt concentrations or ethylene diamine tetraacetate. A specific amino acid pattern (with Ser, Gly, Glu, and Ala dominating) remained constantly associated with the capsule polysaccharide moiety independent of the separation procedure.Abbreviations A2pm
diaminopimelic acid
- Cetavlon
cetyltrimethyl-ammonium bromide
- EDTA
ethylene-diaminetetraacetate, disodium salt
- HF
hydrofluoric acid
- HPLC
high-performance liquid chromatography
- PAGL
polyacrylamide gel-electrophoresis
- SDS
sodium dodecyl sulfate
- TCA
trichloroacetic acid 相似文献
19.
Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus 总被引:1,自引:0,他引:1
Doddapaneni KK Tatineni R Vellanki RN Rachcha S Anabrolu N Narakuti V Mangamoori LN 《Microbiological research》2009,164(4):383-390
A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28 kDa, pH and temperature optima for this protease were 10 and 60 °C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu2+ ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents. 相似文献
20.
Molecular and functional properties of DNA topoisomerase I isolated from a hydrogen-oxidizing bacterium, Alcaligenes eutrophus H16, were investigated. Under native conditions the enzyme forms a monomer with a relative molar mass of 98.500. A rod-like shape of the molecule was derived from the calculated frictional coefficient. The isoelectric point of the enzyme was determined to be in the range of 7.6–8.0. The enzyme activity is strictly Mg2+ dependent with an optimum at 3 mM Mg2+. The pH optimum ranges within 7.5–9.0. A. eutrophus DNA topoisomerase I activity is inhibited by M13 ssDNA, high ionic strength, polyamines, heparin and by a number of intercalating drugs.Abbreviations DTT
dithiothreitol
- BSA
bovine serum albumin
- EDTA
ethylenediaminetetraacetic acid
- SDS
sodium dodecyl sulfate
- Tris
tris(hydroxymethyl)aminomethane
- PMSF
phenylmethanesulfonyl fluoride
- PAGE
polyacrylamide gel electrophoresis 相似文献