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The presented pH-dyn assay serves as a versatile tool for screening enzymatically catalyzed reactions consuming or producing acids. The method is based on material balances of substrates and products. Ion balances relate concentrations of acids and bases to pH. pH-changes caused by the enzymatically catalyzed reaction in a well-defined buffer system are recorded by light-absorption measurements of a pH-indicator. Kinetic parameters are estimated by fitting the modeled pH changes to the experimentally observed ones. The enzymatically catalyzed hydrolysis of 4-nitrophenol is used as a model system. A pH indicator, bromothymol blue, is used to monitor the reaction progress. The reaction is monitored until the limiting substrate is completely consumed. This allows the estimation of the parameters of the Michaelis-Menten kinetics, K(M) and k(cat), in a single run. The results agree well with conventional spectrophotometric experiments and values reported in literature. Around pH 7, environmental CO(2) influences pH. Carbon dioxide influence was included in the model. Thus it was possible to estimate initial CO(2) concentrations as a model parameter, and therefore automatic correction for the CO(2) disturbances was achieved. This was important to detect low conversions at low buffer concentrations. 相似文献
6.
Quantitative estimates of gibberellin A9 in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A9, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.Abbreviations FW
fresh weight
- GA9
gibberellin A9
- GA9–Me
methylated GA9
- GC-MS
gas chromatography-mass spectrometry
- HPLC
high performance liquid chromatography
- MID
multiple-ion detection
- RIA
radioimmunoassay 相似文献
7.
George Harauz Lisa Borland Gunter F. Bahr Elmar Zeitler Marin van Heel 《Chromosoma》1987,95(5):366-374
A complete human metaphase chromosome has been reconstructed from a series of electron microscopical projections obtained by tilting the specimen stage at 3 degree intervals from –60 to +60 degrees. The reconstructed structure is about 3.0 m long, 1.6 m wide, and 0.8 m thick. The mass distribution was fairly homogeneous within the chromatids and neither a hollow nor a dense core was observed. The distribution and course of fibers observed are most consistent with a looping model of chromosome structure. 相似文献
8.
Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin 总被引:1,自引:0,他引:1
The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[3H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (Mr) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an Mr of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[3H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([3H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an Mr of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [3H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC
[(4-azidobenzoyl)-ethylenediamine]-fusicoccin
- ABE-NHS
(4-azidobenzoyl)-N-hydroxysuccinimide ester
- FC
fusicoccin
- FCBP
fusicoccin-binding protein
- FCol
9-norfusicoccin-8-alcohol
- MAB
monoclonal antibody
- Mega-9(10)
nonanoyl(decanoyl)-N-methylglucamide
- Mr
apparent molecular mass
- PMSF
phenylmethyl-sulfonyl fluoride
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol 相似文献
9.
Paul G. Layer Regina Alber Patrick Mansky Günter Vollmer Elmar Willbold 《Cell and tissue research》1990,259(2):187-198
Summary We report here that similar to E6-chicken retinal cells, dissociated cells from 5.5-day-old (E5.5) quail retinae reaggregate in rotary culture, multiply about tenfold and reestablish histotypical areas. These cellular aggregates include all nuclear layers either with inversed or correct laminar polarity, depending on the local origin of the cells (called rosetted and laminar in-vitro-retinae (IVR), respectively; Layer and Willbold 1989). In combined cultures, chick and quail cells are evenly mixed only during the first two days of culture. Along with the assembly of single cells into rosettes and then into discrete laminae, sectors of chick and quail cells begin to segregate. They are delineated by borders running radially through all three nuclear layers. Thus, interspecies migration of cells at this advanced stage of differentiation is strongly inhibited. Concomitant with this segregation, coherent radial columns spanning all three layers but containing cells from either species only, can be traced histologically. We conclude that a weak segregation of chick and quail retinal cells takes place already at the single cell level, but that the permanent segregation of entire tissue parts must be due to clonal cellular proliferation within the IVR in conjunction with some developmental-structural mechanism retaining clonal progenies within a columnar order.Abbreviations
ECM
extracellular matrix
-
E5.5
days of embryonic age
-
GCL
ganglion cell layer
-
GC's
ganglion cells
-
i.c.
in culture
-
INL
inner nuclear layer
- rosetted in-vitro-retina
retinal cell organoid aggregated from single cells of the central retina
-
IPL
inner plexiform layer
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MRE
marginal retinal epithelium
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ONL
outer nuclear layer
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OPL
outer plexiform layer
-
OS
ora serrate
-
PR
photoreceptor cell
- laminated in-vitro-retina
fully laminated retinal cellorganoid resembling an E15-retina aggregated from cells of the eye periphery including RPE
-
RPE
retinal pigment epithelium 相似文献
10.
l-Methionine-dl-sulfoximine (MSX) stimulated nitrate uptake but inhibited14CO2 fixation and O2 evolution inAnabaena doliolum. Nitrate uptake was inhibited by ammonium (NH
4
+
) in the absence of MSX, but not in the presence of MSX. Glutamine or a derivative of it appears to be the actual negative effector of nitrate utilization. In presence of nitrate, MSX-treated cells ofA. doliolum evolve more O2 than do untreated cells. Our results suggest a close relation between photoassimilation of carbon and utilization of nitrogen. 相似文献