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51.
In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca2+-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca2+-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca2+ uptake, and intracellular Ca2+ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 M veratridine (from 67±2.3 mV resting membrane potential to 50.7±2.5 mV) was not significantly influenced by vesamicol (1–20 M). Vesamicol (1–20 M) had no effect on either the overall curve of the veratridine-evoked45Ca2+ uptake or the amount of Ca2+ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca2+ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 M). The K+-evoked (40 mM) increase of Ca2+ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 M) vesamicol inhibited both the fall in membrane potential and the elevated Ca2+ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na+ channels at this concentration. Vesamicol, in lower concentration (20 M) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [14C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [14C]choline. The results suggest that vesamicol does not interfere with veratridine-induced changes in isolated nerve terminals other than with the release of acetylcholine, thus further supporting the involvement of a vesamicol-sensitive vesicular transmitter pool in Ca2+-dependent veratridine-elicited acetylcholine release. 相似文献
52.
Radiolabelled calmodulin has previously been used to screen cDNA expression libraries to isolate calmodulin-binding proteins.
We have modified this technique for the isolation of plant calmodulin-binding proteins. [35S]-methionine was used instead of the inorganic [35S]-sulfate, or125I used in previous methods. In addition, theE. coli pET expression system was chosen to obtain high levels of recombinant calmodulin at the time of labelling. The procedure
thus takes into account both the specific activity of the probe and the amount of protein necessary for screening a large
number of filters. Here we describe in detail a procedure for the production and purification of [35S]-recombinant calmodulin and the use of the radiolabelled protein as a probe to screen plant cDNA expression libraries. The
[35S]-labeled calmodulin probe easily detects the λICM-1 phage encoding a partial mouse calmodulin-dependent protein kinase II
that was previously isolated using a [125I]-calmodulin probe (Sikela and Hahn, 1987). Subsequently, a tobacco root cDNA expression library was screened and a positive
clone encoding a calcium-dependent calmodulin-binding protein was isolated. 相似文献
53.
Carol Anelli Sheppard Marcia J. Loeb 《Archives of insect biochemistry and physiology》1992,21(3):179-193
A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [45Ca]2+ as a tracer, Ca2+ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca2+ (apparent Km for [Ca2+ free] 1 Abbreviations used: [Ca2+free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. = 22 nM). Ca2+ transport was abolished upon addition of the calcium ionophore, A23187. Ca2+-stimulated, Mg2+-dependent ATPase activity peaked between 100 and 200 nM Ca2+free. Ca2+-Mg2+-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca2+ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca2+; effect of certain ions and inhibitors on Ca2+ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc. 相似文献
54.
A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium. 相似文献
55.
56.
Dicotyledonous plants subjected to Fe-deficiency stress can decrease pH in the rhizosphere by proton excretion and reduce ferric iron by an activated reduction system in the plasma membranes of the root or by reductants released from the roots. The efficiency by which these plants take up Fe may strongly depend on their cation-anion balance. This study presents results of two experiments conducted to evaluate the effect of K, growth stage and cultivar on ionic balance and Fe acquisition of peanut (Arachis hypogaea L.) plants.Potassium applications to the high calcareous soil (30.3% CaCO3) favoured proton release, but did not ameliorate plant Fe acquisition. At the earliest stages of plant growth, anion uptake exceeded cation uptake due to intensive N uptake. With time, a shift in the ionic balance was observed as a result of predominant cation uptake. It appears that the relationship between H/OH-ion release and Fe nutrition of peanut plants is actually a complex phenomenon under soil conditions and depends on some soil parameters, such as CaCO3 content. Even by enhanced H-ion release Fe nutrition of plants can be impaired if soil CaCO3 is too high. 相似文献
57.
The physiological basis of plant reaction to and tolerance of aluminium (Al) is poorly understood. We review the results of
investigations into Al toxicity and root physiology to develop a theoretical basis for explaining the reaction of the root
to Al, including suggested roles for Ca2+, mucilaginous cap secretions and endogenous growth regulators in mediating a transmitted response between Al-damaged cap
cells and the interacting cell populations of the cap and root.
This information is used to identify possible mechanisms of Al tolerance, notably involving signal transduction, Al uptake
pathways and root morphogenesis; and to briefly discuss how procedures selecting for Al tolerance may be improved by incorporating
the concept of stimulus-response coupling.
Similarities in the responses of roots to Al and other signals (e.g. gravity, light, mechanical impedance) are used to develop the hypothesis that roots respond to environmental signals by way
of a common regulatory system. New research prospects for extending our perception of Al tolerance mechanisms are identified. 相似文献
58.
A field experiment was conducted on an Ultisol in Malaysia to assess changes in soil solution composition and their effects
on maize and groundnut yields, resulting from limestone and gypsum application. The results showed that soil solution Ca in
the lime treatment remained mainly in the zone of incorporation, but in the gypsum treatment some Ca moved into 15–30 cm zone.
Al3+ and AlSO4
+ were dominant Al species in the soil solution of nil treatment. Liming decreased Al3+ and AlSO4
+, but increased hydroxy-Al monomer activities. However, gypsum application resulted in an increase of AlSO4
+ activity and in a decrease of Al3+ activity.
Relative maize and groundnut yields were negatively correlated with Al3+, Al(OH)2+ and Alsum activities. Likewise, relative yields were negatively correlated with Al concentration and the Al concentration ratio and
positively correlated with soil solution Mg concentration and Ca/Al ratio. 相似文献
59.
The lower part (4 cm) of the witloof chicory tap-root (15 cm) was immersed in a complete nutrient solution for 21 days, in the darkness at 18°C and at high RH. This process of forcing which leads to the emergence of an etiolated bud (chicon) was associated with a decrease in root dry weight. Although the amount of calcium in the root and the root cationic exchange capacity remained constant during forcing, the net uptake of calcium, negligible at the onset of forcing, progressively increased to a rate after ten days of 45 mol day–1. Absorption of 45Ca remained at a constant high rate, while the initially low upward migration of 45Ca within the root and the chicon accelerated markedly. This upward migration was associated with a progressive decline in the release of newly absorbed 45Ca. The data support the hypothesis that calcium acquisition by witloof chicory root is predominantly determined by calcium efflux. As the forcing progressed, the influx remained almost constant while a large decrease in the efflux led to a net uptake of calcium. Upward translocation was probably linked to the formation of new negative exchange sites within the growing chicon. The hypothesis that calcium movement occurred along a preferential pathway (xylem vessels) or involved a mass movement through the root is discussed. 相似文献
60.
We have found and characterized an antigen associated with crystal-containing cells in the stomium and connective tissue of the anthers of Nicotiana tabacum L. (tobacco). The antigen, defined by the monoclonal antibody NtF-8B1, localizes to subcellular regions surrounding the crystals. At the light-microscope level, the antigen is detectable just after the first appearance of crystals in the connective tissue of the anther, and at approximately the same time as the appearance of crystals in the stomium. The antigen is not detectable on a Western blot, and gave inconclusive results on a test of periodate sensitivity. It is not the crystals themselves, nor is the presence of the crystals required for antibody recognition. The antigen is sensitive to heat and protease treatment, indicating that it is a protein. The antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Chemical tests indicate that the druse crystals in the stomium are calcium oxalate.Abbreviations ELISA
enzyme-linked immunosorbent assay
- FITC
fluorescein isothiocyanate
This research was supported by a National Science Foundation postdoctoral fellowship to B.L.H., by National Science Foundation grants DMB-87-15799 and to W.E.F. BSR-88-18035, and by U.S. Department of Agriculture grant GAM-89-01056. The authors thank Phillip T. Evans (Louisiana State University, Baton Rouge, USA), Wilma L. Lingle, Harry T. Horner, Jr. (Iowa State University), and A. Jack Fowler, Jr., for advice and helpful discussions. 相似文献