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91.
用快速琼脂糖层析纯化磷酸甘油酸激酶及磷酸甘油醛脱氢酶 总被引:1,自引:0,他引:1
研究了用快速琼脂糖离子交换层析(DEAE—Fast Flow sepharose)结台PEG 4000/Reppal PES双水相体系从黄豆中分离纯化磷酸甘油酸激酶(PGK)及磷酸甘油醛脱氢酶(GAPDH)。控制床层高度(10~20cm),径向放大具有压降低的优点.设计多点进料取代传统的中心管进料,解决了径向流场不均匀的问题。GAPr)H的总收率及纯化倍数分别为58%和144,PGK的总收率及纯化倍数分别为41%和44。工艺成本为2.92美元/ku GPADH,具有一定的实用价值。 相似文献
92.
Gernot Falkner Ferdinand Wagner John Victor Small Renate Falkner 《Journal of phycology》1995,31(5):745-753
The blue-green alga Anacystis nidulans Drouet (Synechococcus leopoliensis Raciborski) cultivated under phosphate-limited conditions adopts a threshold value in the nanomolar range below which uptake ceases. In this study, we investigated the influence of phosphate pulses on the regulation of uptake behavior during reestablishment of the threshold value. Short-term pulses had only a slight effect on uptake kinetics and, hence, on the threshold value, even if the population had been exposed several times to elevated concentrations above the steady-state level in the growth medium. The threshold value was also practically insensitive to the amount of phosphate stored during these short-term fluctuations. Long-term phosphate pulses resulted in a transition to a metastable state that was characterized by a severalfold higher threshold value. This transition, apparently an adaptation to the transiently elevated phosphate concentrations, was further studied by following the influx of 32 P-phosphate at constant external concentrations and was shown to be complete after a period of 10–20 min. After adaptation to pulses, the uptake behavior followed a linear flow-force relation over a wide range of external concentrations. This behavior was explained by the simultaneous operation of at least two uptake systems with different, but coordinated kinetic parameters. This linear flow-force relation facilitated a direct determination of the threshold value from uptake measurements. For applicability in the field the force-flow relation can be a diagnostic tool to assay for fluctuating phosphate and to establish threshold values below the normal measurable range . 相似文献
93.
Ulrich Nowitzki Ralf Wyrich Peter Westhoff Katrin Henze Claus Schnarrenberger William Martin 《Plant molecular biology》1995,29(6):1279-1291
Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E
d-ribulose-5-phosphate 3-epimerase
- RPI
ribose-5-phosphate isomerase
- TKL
transketolase
- PRK
phosphoribulokinase
- GAPDH
glyceraldehyde-3-phosphate dehydrogenase
- FBP
fructose-1,6-bisphophatase
- FBP
fructose 1,6-bisphosphate
- G6PDH
glucose-6-phosphate dehydrogenase
- 6PGDH
6-phosphogluconate dehydrogenase
- OPPP
oxidative pentose phosphate pathway
- Rubisco
ribulose-1,5-bisphosphate carboxylase/oxygenase
- FBA
fructose-1,6-bisphophate aldolase
- IPTG
isopropyl -d-thiogalactoside
- GST
glutathione S-tranferase
- PBS
phosphate-buffered saline
- TPI
triosephosphate isomerase 相似文献
94.
95.
D'Souza S.E. Altekar W. D'Souza S.F. 《World journal of microbiology & biotechnology》1997,13(5):561-564
Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) of Haloferax mediterranei was immobilized by treating the cell extract in the presence of 10% BSA, with the cross-linking reagent, 0.5% glutaraldehyde for 15min, with the retention of 60% of its original activity. The immobilized preparation exhibited a shift in the temperature optimum from 55°C to 65°C. The enzyme showed enhanced stability towards inactivation by radiation and storage (0–5°C) on immobilization. Immobilization also made the enzyme less halophilic, reducing its denaturation on prolonged storage in a non-salt medium, as well as exhibiting optimal activity at a lower KCl concentration (0.5m) as compared to the soluble enzyme (1–2m). 相似文献
96.
K. A. Buss C. Ingram-Smith J. G. Ferry D. A. Sanders M. S. Hasson 《Protein science : a publication of the Protein Society》1997,6(12):2659-2662
The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family. 相似文献
97.
Naruo Nikoh Naoyuki Iwabe Kei-ichi Kuma Mutsuhito Ohno Tsutomu Sugiyama Yoko Watanabe Kinya Yasui Zhang Shi-cui Katsuji Hori Yoshiro Shimura Takashi Miyata 《Journal of molecular evolution》1997,45(1):97-106
Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately
constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined
based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially
unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and
even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for
inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata
and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and sequenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the
freshwater sponge with known sequences revealed that the Parazoa–Eumetazoa split occurred about 940 million years ago (Ma)
as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates
and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes
and protostomes. 相似文献
98.
A index for monitoring the trophic status of rivers based on diatom composition (‚trophic diatom index’, TDI) has been developed,
in response to the National Rivers Authority (England & Wales)'s needs under the terms of the Urban Wastewater Treatment Directive
of the European Community. The index is based on a suite of 86 taxa selected both for their indicator value and ease of identification.
When tested on a dataset from 70 sites free of significant organic pollution, this index was more highly correlated with aqueous
P concentrations than previous diatom indices. However, where there was heavy organic pollution, it was difficult to separate
the effects of eutrophication from other effects. For this reason, the value of TDI is supplemented by an indication of the
proportion of the sample that is composed of taxa tolerant to organic pollution.
The index was tested on the R. Browney, N-E. England, above and below a major sewage discharge. TDI values indicated that
the effect of inorganic nutrients on the river downstream of the discharge was slight as the river was already nutrient-rich,
but there was a large increase in the proportion of organic pollution-tolerant taxa. This indicates that the river was already
so eutrophic upstream of the discharge that tertiary treatment to remove P would not be effective unless other aspects of
the discharge were also improved. 相似文献
99.
A photomicrobial sensor consisting of immobilized Chlorella vulgaris and an oxygen electrode has been developed for selective determination of phosphate. When 40 mM phosphate was added to the sensor system, the photocurrent increased to a maximum under light irradiation with a response time of 1 min. The current increased with increasing phosphate concentration in the range 8–70 mM. Selectivity of the sensor was satisfactory. Good agreement was obtained between the phosphate concentrations in lake water determined by the photomicrobial sensor and by conventional colorimetry (correlation coefficient 0.96). 相似文献
100.
A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D 总被引:1,自引:0,他引:1
We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells. 相似文献