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91.
The determination of the various endogenous cylokinins and their distribution among organs is important in understanding their role in growth and development in the intact plant. Cytokinins in young plants of Phaseolus vulgaris were purified by immunoaffinity chromatography and HPLC, and characterised by UV spectra. Zeatin nucleotide (zeatin riboside-5'-monophosphate) and isopentenyladenine nucleotide (isopentenyladenosne-5'-monopnosphate) were the most abundant cytokinins in all organs. Their identities were confirmed by GC-MS. The levels of zeatin, zeatin riboside, isopentenyladenine and isopentenyladenosine never exceeded 5% of the nucleotides, as assessed by a methodology that preserves cytokinin nucleotides. Three extraction methods were compared with qualitatively similar results, though differing in their suppression of nucleotidase activity. Cytokinin nucleotide levels were greater in the stems and petioles than in the roots and leaves on a per gram fresh weight basis, and were greater in the stems than in the other organs on a per plant basis. Levels of the zeatin and isopentenyladenine nucleotides were about equal in the stems and leaves, but in the petioles the zeatin nucleotide levels were about twice the level of isopentenyladenine nucleotide, while in the roots they were about half the isopentenyladenine nucleotide level. The importance of considering the cytokinin form is emphasised. 相似文献
92.
Osamu Shimomura 《Luminescence》1995,10(2):91-101
Bioluminescence of euphausiids takes place when a fluorescent tetrapyrrole F and a highly unstable protein P react in the presence of oxygen. A previous study on the euphausiid Meganyctiphanes norvegica indicated that F acts as a catalyst and P is consumed in the luminescence reaction, differing from the luminescence system of dinoflagellates in which a tetrapyrrole luciferin, nearly identical to F, is enzymatically oxidized in the presence of dinoflagellate luciferase. In the present study, P was extracted from Euphausia pacifica as well as from M. norvegica, then purified separately by affinity chromatography on a column of biliverdin–Sepharose 4B, completing the whole process in less than 5h. The samples of P obtained from both species had a molecular weight of 600,000, a purity of about 80%, and a specific activity 50–100 times greater than that previously found. The activity of P rapidly decreased in solutions, even at 0°C, and the inactivation of P derived from M. norvegica was more than four times faster than that derived from E. pacifica. The kinetics of the luminescence reaction was investigated with F and P whose concentrations were systematically varied. The reaction was characteristically slow and involved two different reaction rates; the turnover number at 0°C was 30/h for the initial 20 min and 20/h after the initial 1 h. The total light emitted in a 50-h period indicated that the bioluminescence quantum yield of F was about 0.6 at 0°C, and P recycled many times in the luminescence reaction. Thus, the present results conclusively show that F is a luciferin and P is a luciferase of an unusually slow-working type, contrary to early report. 相似文献
93.
Peter E. Molloy Barbara M. Graham Pauline M. Cupit Steven D. Grant Andrew J. R. Porter Charles Cunningham 《Molecular biotechnology》1995,4(3):239-245
This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity
or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain
their proper conformation. 相似文献
94.
The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO3
-H2S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c
3 from D. desulfuricans. The specific activity of mSiR was 103 nmol H2 min-1 mg-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO3
-H2S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations
SiR
sulfite reductase
-
mSiR
sulfite reductase purified from membranes
-
sSiR
sulfite reductase purified from the soluble fraction
Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c
3 hydrogenase, EC 1.12.2.1 相似文献
95.
Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.
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T. Kitlar F. Dring D. F. Diedrich R. Frank H. Wallmeier R. K. Kinne J. Deutscher 《Protein science : a publication of the Protein Society》1994,3(4):696-700
Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity. 相似文献
96.
By computer simulation of experimental dynamic gas chromatographic elution profiles, the rotational energy barrier ΔG= of racemic 2,2′-diisopropylbiphenyl has been determined as 114.6–115.0 kJ/mol (75–100°C). These data are in good agreement with a value that was determined previously by measuring the racemization kinetics of an enriched sample. This indicates that there is no measurable catalytic or inhibitory effect of the stationary phase. © 1994 Wiley-Liss, Inc. 相似文献
97.
A new covalently-bonded, silica-based stationary phase, using as the chiral selector the 1-(3-aminopropyl) derivative of (+)-(5R,8S,10R)-terguride, has been developed to resolve optically active isomers by HPLC. Good resolution of structurally related racemic ergot alkaloids were obtained using water-methanol mixtures as the eluent. Analysis of the influence of the type and concentration of the organic modifier, and the pH of the buffer in the mobile phase allowed the enantioseparation of these compounds to be optimized. Determination of the optical purity of a lisuride-containig drug is reported. © 1994 Wiley-Liss, Inc. 相似文献
98.
Lifibrol, a new drug for the treatment of hypercholesterolemia, contains a stereogenic center bearing a secondary alcohol group. A normal-phase achiral–chiral HPLC separation of the enantiomers of lifibrol and two of its metabolites was developed and validated for quantitation in dog plasma. A silica and a Chiralcel OD-H column were operated in series and all six enantiomeric components and internal standard were directly separated. An initial solid-phase extraction (phenyl) clean-up step and a column-switching step to eliminate late-eluting compounds were also utilized. The solid-phase extraction step was automated using a robotic system. Assay development, validation, and application of the method to a bioavailability study of the racemate and enantiomers of lifibrol in dogs are described. The lower limit of quantitation was 0.0125 μg/ml for each enantiomer of lifibrol using 200 μl of dog plasma with UV detection (255 nm). In dog plasma following oral or intravenous administration of the racemate, the (R)/(S) ratio of the enantiomers of lifibrol was greater than one and increased with time. Following administration of the individual enantiomers, chiral inversion of the (S)-enantiomer but not the (R)-enantiomer was observed. © 1994 Wiley-Liss, Inc. 相似文献
99.
Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography 总被引:2,自引:0,他引:2
A. Zamboni I. Giuntini D. Gianesello F. Maddalena F. Rognoni D. Herbst 《Cytotechnology》1994,16(2):79-87
The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using125I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP
-Fetoprotein
- BSA
bovine serum albumine
- FCS
Fetal calf serum
- HRP
horseradish peroxidase
- OPD
o-phenylenediamine dihydrochloride
- I.P.
isoelectric point
- IEF
isoelectric focussing
- PBS
Phosphate buffered saline 相似文献
100.
沈阳西部污灌水中有机污染物的分析刘海玲,张丽珊,姚家彪,于殿臣,朱岩,可夫,姜萍(中国科学院沈阳应用生态研究所,110015)AnalysisofOrganicPollutantsinIrrigatedsewageInWesternShenyang.... 相似文献