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1.
2.
Trigonopsis variabilis induced for D-amino acid oxidase and catalase was immobilized by entrapment in Polyacrylamide beads obtained by radiation
polymerisation. Permeabilization of the cells was found to be essential for optimal activity of the enzymes in free cells.
However, the process of entrapment itself was found to eliminate the permeability barrier of cells immobilized in Polyacrylamide.
The two enzymes exhibited a differential response on Polyacrylamide entrapment. Thus, D-amino acid oxidase activity was stabilized
to heat inactivation whereas catalase in the same cells showed a destabilization on entrapment in Polyacrylamide. The coimmobilized
enzyme preparation showed an operational half life of 7–9 days after which the D-amino acid oxidase activity remained stable
at a value 35–40% of that of the initial activity for a study period of 3 weeks. Coimmobilization of MnO2 was not effective in enhancing the operational life of the enzyme preparation. 相似文献
3.
Size selection of latex beads by blackfly larvae (Diptera: Simuliidae) in the laboratory 总被引:1,自引:1,他引:0
P. Schröder 《Hydrobiologia》1987,144(2):163-171
In laboratory experiments, blackfly larvae collected from a lake outlet, a woodland and a meadow stream were tested for size
selection of latex beads of < Ito > 100 μm diameters. 3 suspensions of varying proportions for each size category were supplied
to these blackfly larvae in common experiments. Comparisons between the size frequency distributions of particles supplied
and the particle compositions in the larval guts showed intra- and interspecific differences and were quantified by calculating
Jacobs' electivity index. In all species selection of larger particles increased with the larger larval instars. Although
there was a positive selectivity of small particles in some cases, the ingested proportion of large particles increases volumes
and biomasses of gut content and may be more important for larval growth than small particles. 相似文献
4.
N-Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure 总被引:6,自引:5,他引:1
Axel Braun Yves-Alain Barde Friedrich Lottspeich Werner Mewes Hans Thoenen 《Journal of neurochemistry》1987,48(1):16-21
A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala. 相似文献
5.
RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [-33P]UTP in the presence of poly[d(A-T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly843 and Met895 of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerases. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution. 相似文献
6.
The ethanol-oxidizing, proton-reducing Pelobacter acetylenicus was grown in chemostat cocultures with either Acetobacterium woodii, Methanobacterium bryantii, or Desulfovibrio desulfuricans. Stable steady state conditions with tightly coupled growth were reached at various dilution rates between 0.02 and 0.14 h-1. Both ethanol and H2 steady state concentrations increased with growth rate and were lower in cocultures with the sulfate reducer < methanogen < homoacetogen. Due to the higher affinity for H2, D. desulfuricans outcompeted M. bryantii, and this one A. woodii when inoculated in cocultures with P. acetylenicus. Cocultures with A. woodii had lower H2 steady state concentrations when bicarbonate reduction was replaced by the energetically more favourable caffeate reduction. Similarly, cocultures with D. desulfuricans had lower H2 concentrations with nitrate than with sulfate as electron acceptor. The Gibbs free energy (G) available to the H2-producing P. acetylenicus was independent of growth rate and the H2-utilizing partner, whereas the G available to the latter increased with growth rate and the energy yielding potential of the H2 oxidation reaction. The critical Gibbs free energy (Gc), i.e. the minimum energy required for H2 production and H2 oxidation, was-5.5 to-8.0 kJ mol-1 H2 for P. acetylenicus,-5.1 to-6.3 kJ mol-1 H2 for A. woodii,-7.5 to-9.1 kJ mol-1 H2 for M. bryantii, and-10.3 to-12.3 kJ mol-1 H2 for D. desulfuricans. Obviously, the potentially available energy was used more efficiently by homoacetogens > methanogens > sulfate reducers. 相似文献
7.
Françoise Farace Anne-Marie Le Ridant Bernard Escudier Thierry Hercend Frédéric Triebel 《Biotherapy》1992,5(1):11-20
We have attempted to improve negative selection procedures for the large scale purification of human CD
in3
–
CD56+ NK cells. In a series of experiments, purifications of NK cells from 108 PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3+ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 108 cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 109 PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads. 相似文献
8.
Mortimer M. Civan Stephen R. Williams David G. Gadian Enrique Rozengurt 《The Journal of membrane biology》1986,94(1):55-64
Summary Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional31P and19F probes of intracellular pH (pH
c
) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pH
c
on external Na+ concentration (c
Na
o
). pH
c
also depended on intracellular Na+ concentration (c
Na
o
). Increasingc
Na
c
by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducingc
Na
o
produced a larger acid shift in pH
c
than with external K+ present. Comparison of separate preparations indicated that pH
c
was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pH
c
of Swiss mouse 3T3 cells using31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event. 相似文献
9.
Syed M. I. Kazmi Jai Ramwani Lalit K. Srivastava G. Rajakumar Gregory M. Ross Marjorie Cullen Ram K. Mishra 《Journal of neurochemistry》1986,47(5):1493-1502
3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions. 相似文献
10.
Klaus Hallermayer Christine Harmening Herbert Merz Bernd Hamprecht 《Journal of neurochemistry》1983,41(6):1761-1765
The benozomorphan derivative (-)-2-[2-(p-bromoacetamidophenyl)ethyl]-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan (BAB), capable of reacting with nucleophilic groups, acts on neuroblastoma X glioma hybrid cells as a potent, irreversible opiate agonist. Its potency in inhibiting the increase in cellular cyclic AMP, evoked by prostaglandin E1, is comparable to that of Leu-enkephalin. This also applies to its capacity to compete with [3H]D-Ala2-Met-enkephalinamide ([3H]DAEA) in binding on cell membrane preparations. The comparatively lower potency of (-)-2-[2-(p-acetamidophenyl)-ethyl]-5,9 alpha-dimethly-2'-hydroxy-5,7-benzomorphan (AB), which differs from BAB in the substitution of the bromoacetamido group by an acetamido group, is of the same order of magnitude as that of morphine. The covalent interaction of BAB with the opiate receptors is deduced from the observations that (1) it is not possible to wash away this compound from the receptors, (2) the potency of BAB in inhibiting the specific binding of [3H]DAEA increases with prolonged preincubation time, and (3) AB behaves as a reversible agonist. 相似文献