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51.
A method is presented for the rapid enzymatic determination of acetate in spent bacterial culture supernatants. The assay is based on a previously published assay for acetate kinase [Bergmeyer et al. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 1, pp. 425-426, Verlag Chemie-Academic Press, New York/London], and is sufficiently sensitive to detect acetate levels of 50 microM. The assay is cheaper than commercially available assays and is particularly useful for occasional use by laboratories not equipped for routine acetate analysis using gas chromatography. The application of the assay to the measurement of acetate in bacterial cultures is described, though it should also be applicable to other biological fluids and foodstuffs. 相似文献
52.
Microbial production of L-[15N]glutamic acid and its gas chromatography-mass spectrometry analysis 总被引:1,自引:0,他引:1
L-[15N]Glutamic acid was prepared in high yields via a fermentative process. Brevibacterium lactofermentum, growing on a medium containing 97% enriched 15NH4Cl as a sole isotopic precursor, excreted mostly L-[15N]glutamic acid. The L-[15N]glutamic acid was purified and identified. Gas chromatography-mass spectrometry analysis was performed to demonstrate its usefulness in clinical studies. 相似文献
53.
Christian J. Le Peuch Danielle A.M. Le Peuch Sidney Katz Jacques G. Demaille Maxwell T. Hincke R. Bredoux J. Enouf S. Levy-Toledano Jacques Caen 《生物化学与生物物理学报:生物膜》1983,731(3):456-464
Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a activity of about 10 nmol (min·mg)?1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min·mg)?1. When incubated in the presence of Mg[γ-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (). Some minor calmodulin-binding proteins were enriched in the membrane fractions (). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation. 相似文献
54.
Characterization of a fluorescent substrate for the adenosine 3',5'-cyclic monophosphate-dependent protein kinase 总被引:1,自引:0,他引:1
A synthetic tetradecapeptide derived from the phosphorylation site of the beta-subunit of phosphorylase kinase (Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val-Tyr-Glu-Pro-Leu-Lys-Ile) is a highly efficient substrate for the cAMP-dependent protein kinase, exhibiting a 36% decrease in the intrinsic tyrosine fluorescence on phosphorylation. The fluorescence changes in continuous assays were monitored to demonstrate the roles of protein kinase effectors (cAMP, the type II regulatory subunit, and the 8000-Da heat-stable inhibitor) in the regulation of the enzyme and to determine Km and Vmax. The phosphorylation reaction requires 1 mol ATP/mol peptide. Amino acid analysis demonstrates the presence of phosphoserine in the phosphorylated peptide. Auxiliary experiments show that tyrosine phosphorylation can also be detected fluorometrically and distinguished from serine or threonine phosphorylation. 相似文献
55.
Err-Cheng Chan Peter P. Ueng Karri L. Eder Li Fu Chen 《Journal of industrial microbiology & biotechnology》1989,4(6):409-417
Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively. 相似文献
56.
Adam Bertl 《The Journal of membrane biology》1989,109(1):9-19
Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K+-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K+ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na+ and Cl–. However, addition of Na+ decreases the K+ currents. TheI–V relationships of the open channel as measured at various K+ concentrations with or without Na+ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data. 相似文献
57.
Bart M. Nicolaï Jan F. Van Impe Peter A. Vanrolleghem Joos Vandewalle 《Antonie van Leeuwenhoek》1992,62(4):273-283
The mathematical model for the penicillin G fed-batch fermentation proposed by Heijnen et al. (1979) is compared with the model of Bajpai & Reuß (1980). Although the general structure of these models is similar, the difference in metabolic assumptions and specific growth and production kinetics results in a completely different behaviour towards product optimization. A detailed analysis of both models reveals some physical and biochemical shortcomings. It is shown that it is impossible to make a reliable estimation of the model parameters, only using experimental data of simple constant glucose feed rate fermentations with low initial substrate amount. However, it is demonstrated that some model parameters might be key factors in concluding whether or not altering the substrate feeding strategy has an important influence on the final amount of product.It is illustrated that feeding strategy optimization studies can be a tool in designing experiments for parameter estimation purposes. 相似文献
58.
Edward J. B. Beeley P. A. Bennett L. G. I. Poland J. S. 《Biological trace element research》1990,(1):53-61
A microcomputer-controlled irradiation and measurement system and a microprocessor-controlled sample changer have been installed
at the SLOWPOKE-2 Facility at the Royal Military College of Canada (RMC). These systems can provide the gamut of instrumental
neutron activation analysis (INAA) techniques for the analyst. Custom software has been created for system control, data acquisition,
and off-line spectral analysis using programs that incorporate Gaussian peak-fitting methods of analysis. The design and use
of the equipment is discussed, and the performance is illustrated with results obtained from the analysis of marine sediment
and biological reference materials. 相似文献
59.
60.
Mark Phillippe Trevania Saunders Shrikar Bangalore 《In vitro cellular & developmental biology. Plant》1990,26(4):369-378
Summary The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification
of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium,
rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated
myocytes were maintained in culture in 75-cm2 flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10−8
M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells
was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal
anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western
blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes,
both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates.
Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine,
an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic
receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can
be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.
This research was supported by grant HD-22063 from the National Institutes of Health, Bethesda, MD. 相似文献