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91.
alpha-chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid-liquid and the solid state-liquid transfer; (2)the back-transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back-extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back-transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer. 相似文献
92.
Extraction of whole lobes of normal rat liver with dimethyl sulphoxide (DMSO) under N2 gives extracts which contain 5—10 μmol/l·O?2 (50-100 nmol·O?2 per 10 ml extract per 4 g liver; 1.25-2.50 nmol·O?2 per millilitre per gram liver). Evidence for ·O?2 in the extracts is given by: (1) electron spin resonance signals (ESR), (2) differential pulse polarography (DPP), (3) chemiluminescence (CL), and (4) nitroblue tetrazolium reduction (NBT). All tests yield results identical with those obtained with authentic ·O?2. Extraction of ·O?2 is enhanced by tetrabutyl ammonium ion, and is maximal at 1-3 min. These results raise the possibility that substantial amounts of ·O?2 are normally sequestered in protective membranous sites in vivo. 相似文献
93.
94.
A method of enzyme release and aqueous two-phase extraction is described for the separation of penicillin acylase from Escherichia coli cells. Butyl acetate, 12% (v/v), treatment combined with freeze-thawing gives up to 70% enzyme release. For polyethylene glycol (PEG) + phosphate two-phase extraction systems the enzyme purity and yield were rather low. Modified PEG, including PEG-ampicillin, PEG-aniline, PEG-phosphate, and PEG-trimethylamine, were synthesized and used in aqueous two-phase systems; PEG-trimethylamine is the most satisfactory. A system containing 12% (w/w) PEG4000, 8% (w/w) of which is PEG-trimethylamine, with 0.7M potasium phosphate at pH 7.2, resulted in the enzyme selective partition being greatly enhanced by charge directed effects. Possible mechanisms for the separation process are discussed. (c) 1992 John Wiley & Sons, Inc. 相似文献
95.
Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation 总被引:3,自引:0,他引:3
Aqueous two-phase extraction incorporated affinity precipitation was examined as a technique for protein purification. An enteric coating polymer, Eudragit S100, was employed as a ligand carrier. Eudragit was specifically partitioned to the top phase in the aqueous two-phase systems. For application of this method to purification of recombinant protein A using human IgG coupled to Eudragit in an aqueous two-phase system, 80% of protein A added was recovered with 81% purity. The purity was enhanced 26-fold by thid method. The IgG-Eudragit could be used repeatedly for the purification process. This seperation method should be applicable to industrial-scale purification as a new purification procedure combining the advantages and compensating for the disadvantages of the aqueous two-phase method and affinity precipitation method. (c) 1992 John Wiley & Sons, Inc. 相似文献
96.
The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots 总被引:6,自引:0,他引:6
Anna M. Rychter Michele Chauveau Jean-Loup Bomsel Claude Lance 《Physiologia plantarum》1992,84(1):80-86
Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a Pi -deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—Pi , roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+Pi ) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -Pi mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—Pi roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -Pi roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher. 相似文献
97.
J. De Las Rivas B. Crystall P. J. Booth J. R. Durrant S. Özer G. Porter D. R. Klug J. Barber 《Photosynthesis research》1992,34(3):419-431
A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680+Pheo-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47
chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively
- D1 and D2 polypeptides
PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively
- HPLC
high performance liquid chromatography
- PS II
Photosystem two
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
- P680
primary electron donor of PS II
- Pheo
phenophytin a
- SPC
single photon counting
- PBQ
phenyl-p-benzoquinone
- DPC
1,5-diphenylcarbazide
AFRC Photosynthesis Research Group, Department of Biochemistry 相似文献
98.
A rapid procedure for the construction of PCR cDNA libraries from small amounts of plant tissue 总被引:6,自引:0,他引:6
Ian Jepson John Bray Gareth Jenkins Wolfgang Schuch Keith Edwards 《Plant Molecular Biology Reporter》1991,9(2):131-138
We describe a general method for the preparation of λZAP II cDNA libraries from very small amounts (<50 mg) of plant tissue.
We have achieved this by combining an efficient method for RNA extraction with a modified PCR protocol for the synthesis and
amplification of cDNA. Using this protocol we have found it possible to generate cDNA libraries containing more than 106 clones from as little as 1 μg of total RNA. 相似文献
99.
A rapid and efficient method for the extraction of total DNA from the sweet potato and its related species 总被引:2,自引:2,他引:0
Secondary metabolites, latex/mucilagenous secretions, polysaccharides, and proteins interfere with the extraction of high-quality,
restrictable total cellular DNA from sweet potato [Ipomoea batatas (L.) Lamk.] and related species. A method for the DNA extraction is described which overcomes these problems. 相似文献
100.
Although great strides have been made in understanding the genetics of Duchenne muscular dystrophy (DMD), uncertainty still remains as to the metabolic changes which are associated with the disease. We have used the recently discovered animal model of DMD, the mdx mouse, to study aspects of high energy phosphate metabolism and metabiolic control indices in dystrophic muscle. This model of DMD has the dual advantage of having a genetic defect which is homologous to that in human DMD, and it lacks the fatty infiltration and ncecrosis which makes biochemical analysis of DMD so difficult. We have used nuclear magnetic resonance sperctroscopy (NMR) to monitor developmental changes in high energy phosphates and pH. No differences were observed between young (< 40–50 days old) control and mdx mice. The pH increase and alterations in phosphate ratios (i.e., decline in PCr/ATP) observed in adult mdx vs. control mice are quantilatively similar to those observed in humans. Biochemical analysis showed a small decline in ATP and PCr content and a decline in some indices of energy status in adult mdx mice. As young mdx mice appeared to be normal, the lack of dystrophin does not correlate with metabolic changes. The changes which were observed were small enough that alterations in fibre composition could be the major contributory factor. 相似文献