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991.
Summary Requirements for the activation of Cl conductance have been investigated in pig jejunal brush border vesicles. The stability of ATP as a substrate for protein kinase activity, the stability of the phosphoprotein product of protein kinase action, and the choice of buffer system used for vesicle preparation were studied as variables which affected the outcome of in vitro activation attempts. Arsenate was selected as the most effective agent in protecting ATP from hydrolysis by the phosphatase activity in this vesicle system. Brush border vesicle protein appeared to prevent the accumulation of phosphoprotein in a cAMP-dependent protein kinase reaction, and vesicle protein only had phosphate acceptor activity when KF was added as a presumptive inhibitor of phosphoprotein phosphatase.A Cl conductance response to a potassium gradient and valinomycin was present in vesicles prepared in buffers containing tetramethylammonium. Cl– conductance activity was not increased in this system by the addition of ATP, dibutyryl cyclic AMP, and cyclic AMP-dependent protein kinase.There was no Cl conductance response to a potassium gradient in vesicles buffered with imidazolium-acetate. Incorporation of ATP, AsO
4
3–
, and F– into these nonconductive vesicles by homogenization, followed by addition of dibutyryl cAMP, produced substantial conductance activity. Maximal activation of Cl– conductance was obtained with vesicles prepared in imidazolium-acetate buffering, using precautions to stabilize ATP and phosphoprotein prior to conductance measurements. 相似文献
992.
Analysis of the initial rates of 45Ca2+ uptake by rat brain mitochondria in Ca2+-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid buffers indicated that nontelencephalic mitochondria exhibited both a much less pronounced stimulatory effect of spermine and significantly more hyperbolic kinetics of Ca2+ uptake than telencephalic mitochondria. Nontelencephalic mitochondria were also markedly less susceptible to a Ca2+-induced hysteretic allosteric activation of the Ca2+ uniporter. A new Ca2+ loading procedure, which strikingly illustrates differences in mitochondrial Ca2+ buffering characteristics, is also described. In this procedure, low concentrations of Ca2+ (1, 2, or 5 microM) were repetitively added to mitochondria every 30 s while changes in free Ca2+ concentration were recorded. Spermine induced a marked attenuation of the rise in free Ca2+ level under these conditions. Steady-state rates of Ca2+ uptake were determined by a quantitative analysis of the buffering of repetitive Ca2+ additions, and, again, brain regional differences were qualitatively similar to those observed in the initial rate kinetics; Ca2+ uptake by nontelencephalic mitochondria in the steady state was markedly less responsive to stimulation by spermine and appeared to have a more hyperbolic dependence on Ca2+ in the absence of spermine. These results also suggest that there is a lag time in the activation of the uniporter by Ca2+, in addition to the hysteresis that has previously been observed in the deactivation of the uniporter.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
993.
Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii 总被引:3,自引:0,他引:3
APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg2+ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V
max of 360 pkat under optimal reaction conditions declining to v
limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K
m APS: 2 · 10-6 mol · l-1, and K
m ATP: 7 · 10-6 mol · l-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS
Adenosine 5-phosphosulphate
- dATP
2-deoxyadenosine 5-triphosphate
- p-CMB
p-chloromercuribenzoate
- DTE
dithioerythritol
- DTT
dithiothreitol
- -MSH
-mercaptoethanol
- PAPS
3-phosphoadenosine 5-phosphosulphate
- PAP
3-phosphoadenosine 5-phosphate
- SDS
sodium dodecyl sulphate
This work is part of a dissertation submitted by H. G. J., Bochum 1982 相似文献
994.
Three different functionalized bentonites including acid activated bentonite (Ba), organically modified bentonite with cetyltrimethyl ammonium bromide (BCTMAB) and the composite by acid activation and organo-modification (Ba-CTMAB) were prepared, and used for immobilization of lipase from bovine pancreatic lipase by adsorption. The amount of lipase adsorbed on the functionalized bentonites was in the following sequence: Ba > BCTMAB > Ba-CTMAB, showing the strongest affinity of Ba for lipase among the three supports. However, the immobilized lipase on Ba-CTMAB showed the highest activity in the hydrolysis of olive oil by 1.67 times of activity of free lipase due to the hydrophobically interfacial activation and enlarged catalytic interface. While, the activity of immobilized lipase on Ba was lower than 20% of free lipase’s activity due to the absence of hydrophobic activation and negative impact of excessive hydrogen ions on the surface. The Km values for the immobilized lipase on Ba-CTMAB (0.054 g/mL) and BCTMAB (0.074 g/mL) were both lower than that of free lipase (0.115 g/mL), and the Vmax values were higher for the immobilized lipases, exhibiting a higher affinity of the immobilized lipase toward olive oil than free lipase. In comparison to free lipase, the better resistance to heating inactivation, storage stability and reusability of the immobilized lipases on Ba-CTMAB and BCTMAB were also obtained. The results show that the efficient and stable biocatalysts for industrial application can be prepared by using the low-cost bentonite mineral as the supports. 相似文献
995.
996.
Summary To learn more about the ways in which genes silenced by insertion mutations can be reactivated, we have undertaken a systematic investigation of Gal+ revertants of the polar mutant galOP-306::IS1 in Escherichia coli K12. The selective conditions used excluded reversion to wild type by precise excision of IS1. In this system (which resisded on a multi-copy plasmid) reversion to the Gal+ phenotype occurred with a frequency of about 10-7 per cell and per generation. Analysis of the revertants revealed that — with the single exception of the previously published chromosomal mutant sis1 — alterations in the structure of IS1 lead to reactivation of gal operon expression. These events fall into four classes: (I) insertion of IS2 at position 327 in IS1, insertion of IS2 at position 687 in IS1, (III) insertion of a hitherto undetected mobile element, IS150, at position 387, (IV) a 16-bp deletion encompassing IS1 coordinates 553–568. Of some 200 independent reversion events studied, all but one were of types I–III i.e. they involved the intervention of a second mobile element. 相似文献
997.
We showed that applications of cobalt and zinc salicylates lead to restoration of the impulse activity of a PPa1 neuron of
the snail, Helix pomatia, under conditions of the blockade of synaptic transmission by cadmium ions. In the case where a PPa1 neuron demonstrated
no background activity and/or under conditions of total isolation of this cell, the above-mentioned salicylates initiate generation
of action potentials, as well as exert an excitatory effect on “silent” non-identified cells of the parietal and visceral
ganglia. Based on the data obtained, we conclude that the activating effect of cobalt and zinc salicylates on the PPa1 cell
is similar to that of the so-called initiating factor (IF), which initiates generation of the burst activity. These effects
are independent of the inward calcium current. Using an activator of cAMP phosphodiesterase, imidazole, we showed that the
effects of the above salicylates (similar to the effect of IF) are related to the influence of these agents on the system
of cyclic nucleotides.
Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 11–17, January–February, 2006. 相似文献
998.
《Peptides》2016
Due to the cytotoxic effect of antimicrobial peptides (AMP) against several microorganism and tumor cells has been proposed their association with the immune system. However, just a few reports have shown this relationship. In this study, mice were treated with gomesin, a β-hairpin AMP that exhibit high cytotoxicity against bacterial and tumor cells. Different effects in the immune system were observed, such as, decrease of CD3+ in T lymphocytes (Control: 17.7 ± 1.4%; Gomesin: 7.67 ± 1.2%) and in hematopoietic progenitors and increase of hematopoietic stem cell (Control: 0.046 ± 0.004%; Gomesin: 0.067 ± 0.003%), B220+ B lymphocytes (Control: 38.63 ± 1.5%; Gomesin: 47.83 ± 0.48%), and Mac-1+F4/80+ macrophages (Control: 11.76 ± 3.4%; Gomesin: 27.13 ± 4.0%). Additionally, macrophage increase was accompanied by an increase of macrophage phagocytosis (Control 20.85 ± 1.53; Gomesin 31.32 ± 1 Geometric mean), interleukin 6 (Control: 47.24 ± 1.9 ng/mL; Gomesin: 138.68 ± 33.68 ng/mL) and monocyte chemoattractant protein-1 (Control: 0.872 ± 0.093 ng/mL; Gomesin: 1.83 ± 0.067 ng/mL). Thus, this report showed immunomodulatory activity of gomesin in the immune system of mice. 相似文献
999.
Hillary A Hager Ryan J Roberts Emily E Cross Véronique Proux‐Gillardeaux David M Bader 《The EMBO journal》2010,29(3):532-545
Blood vessel/epicardial substance (Bves) is a transmembrane protein that influences cell adhesion and motility through unknown mechanisms. We have discovered that Bves directly interacts with VAMP3, a SNARE protein that facilitates vesicular transport and specifically recycles transferrin and β‐1‐integrin. Two independent assays document that cells expressing a mutated form of Bves are severely impaired in the recycling of these molecules, a phenotype consistent with disruption of VAMP3 function. Using Morpholino knockdown in Xenopus laevis, we demonstrate that elimination of Bves function specifically inhibits transferrin receptor recycling, and results in gastrulation defects previously reported with impaired integrin‐dependent cell movements. Kymographic analysis of Bves‐depleted primary and cultured cells reveals severe impairment of cell spreading and adhesion on fibronectin, indicative of disruption of integrin‐mediated adhesion. Taken together, these data demonstrate that Bves interacts with VAMP3 and facilitates receptor recycling both in vitro and during early development. Thus, this study establishes a newly identified role for Bves in vesicular transport and reveals a novel, broadly applied mechanism governing SNARE protein function. 相似文献
1000.
Caenorhabditis elegans has a single deoxynucleoside kinase-like gene. The sequence is similar to that of human TK1, but besides accepting thymidine as a substrate, the C. elegans TK1 (CeTK1) also phosphorylates deoxyguanosine. In contrast to human TK1, the CeTK1 exclusively exists as a dimer with a molecular mass of ~60 kDa, even if incubated with ATP. Incubation with ATP induces a transition into a more active enzyme with a higher kcat but unchanged Km. This activation only occurs at an enzyme concentration in the incubation buffer of 0.5 μg/ml (8.42 nM) or higher. C-terminal deletion of the enzyme results in lower catalytic efficiency and stability. 相似文献