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1.
We have evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca 2+ accumulation by chick intestinal mitochondria. Ca 2+ accumulation appears to occur in two phases: an early, transient accumulation into an Na +-labile pool followed by an ATP-dependent accumulation into an Na +-resistant pool. Ca 2+ accumulation is extensive at free Ca 2+ concentrations greater than 3 · 10 ?6 M in the presence of ATP. Ruthenium red and dinitrophenol block Ca 2+ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH) 2D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca 2+, or the rate and extent of ATP-supported Ca 2+ accumulation. Intestinal cytosol stimulated Ca 2+ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca 2+-binding protein. 30 ng/ml 1,25(OH) 2D-3 stimulated Ca 2+ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 > 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca 2+ concentration from 3 · 10 ?6 to 1 · 10 ?5 M increased Ca 2+ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH) 2D-3 in vitro increased Ca 2+ accumulation less than 2-fold, we conclude that 1,25(OH) 2D-3 influences mitochondrial accumulation of Ca 2+ in vivo primarily by altering cytosol concentrations of free Ca 2+. 相似文献
2.
Ca 2+ stimulates the uptake of α-aminoisobutyric acid (AIB) into excised or intact Phaseolus vulgaris L. roots by a factor of two. In roots depleted of Ca 2+ by preincubation with ethylenediaminetetraacetate, ethyleneglycol-bis(β-aminoethyl ether)- N,N′-tetraacetic acid, or streptomycin, the stimulatory effect is 7- to 10-fold. In the presence of Ca 2+, roots accumulate AIB more than 100-fold; Ca 2+-depleted roots only equilibrate with AIB. Radioautography shows [ 14C]AIB to be present in all cells after 90 min. Although Ca 2+-depleted roots lose accumulated [ 14C]AIB about 10 times faster than roots supplied with Ca 2+, this increased efflux is not the main cause for the decrease in net uptake observed. The latter is rather due to a less negative membrane potential Δψ in Ca 2+ depleted roots (−120 mV → −50 mV). The basic feature explaining all the results of Ca 2+ deficiency is an increase in general membrane permeability. No indication of a specific regulatory function of Ca 2+ in membrane transport of roots has been obtained. 相似文献
4.
The influence of ozone on Ca 2+ transport in plant membranes from pinto bean ( Phaseolus vulgaris L. var Pinto) leaves was investigated in vitro by means of a filtration method using purified vesicles. Two transport mechanisms located at the plasma membrane are involved in a response to ozone: (a) passive Ca 2+ influx into the cell and (b) active Ca 2+ efflux driven by an ATP-dependent system, which has two components: a primary Ca 2+ transport directly linked to ATP which is partially activated by calmodulin and a H +/Ca 2+ antiport coupled to activity of a H +-ATPase. The passive Ca 2+ permeability is increased by ozone. A triangular pulse of ozone stimulates a higher influx of Ca 2+ than does a square wave, even though the total dose was the same (0.6 microliter per liter × hour). Leaves exposed to a square wave did not exhibit visible injury and were still able to recover from oxidant stress by activation of calmodulin-dependent Ca 2+ extrusion mechanisms. On the other hand, leaves exposed to a triangular wave of ozone, exhibit visible injury and lost the ability of extruding Ca 2+ out of the cell. 相似文献
5.
The characteristics of Ca 2+ transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca 2+ and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0. The Ca2+-dependent modulation protein, calmodulin, was tested for its effect on Ca2+ transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca2+ transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca2+ into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca2+ transport activity. The inhibition of Ca2+ transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca2+ uptake into root endoplasmic reticulum. 相似文献
6.
To cast light upon the role of Ca 1+ and calmodulin on photosynthetic rate (P n), dark respiration (R D) and amino acid and protein contents in salinity stressed and non-stressed Chlorella cultures, the Ca 2+ chelator EGTA [ethylene glycol-bis-(2-aminoethyl ether)- N,N- tetraacetate] and the calmodulin antagonist TFP (trifluperazine) were used. TFP markedly inhibited P N while EGTA exerted a slight, if any, effect on P N. NaCl tolerance, on the other side, was markedly abolished by TFP that inhibited P N and lowered rate of proline accumulation. Calmodulin might be involved in osmoregulation and salt tolerance of Chlorella. R D, however, was markedly enhanced by EGTA and Ca 2+-free medium and hence the Ca 2+ deprivation increased stress severity exerted by NaCl. Combinations of Na + and Ca 2+ enhanced P N, decreased R D and proline content in comparison with an osmotically equivalent reference culture containing only NaCl. Addition of Ca 2+ to TFP treated cultures failed to reactivate calmodulin for proline synthesis. However, when Ca 2+ was added to EGTA-treated cultures, only relatively reduced proline contents were recorded. 相似文献
7.
The purpose of the present study was to use zebrafish as a model to investigate how vitamin D and its receptors interact to control Ca 2+ uptake function. Low-Ca 2+ fresh water stimulated Ca 2+ influx and expressions of epithelial calcium channel ( ecac), vitamin D-25-hydroxylase ( cyp2r1), vitamin D receptor a ( vdra), and vdrb in zebrafish. Exogenous vitamin D increased Ca 2+ influx and expressions of ecac and 25-hydroxyvitamin D3-24-hydroxylase (cyp24a1), but downregulated 1α-OHase ( cyp27b1) with no effects on other Ca 2+ transporters. Morpholino oligonucleotide knockdown of VDRa, but not VDRb, was found as a consequence of calcium uptake inhibition by knockdown of ecac, and ossification of vertebrae is impaired. Taken together, vitamin D-VDRa signaling may stimulate Ca 2+ uptake by upregulating ECaC in zebrafish, thereby clarifying the Ca 2+-handling function of only a VDR in teleosts. Zebrafish may be useful as a model to explore the function of vitamin D-VDR signaling in Ca 2+ homeostasis and the related physiological processes in vertebrates. 相似文献
8.
Background and aims Liming is considered normal agricultural practise for remediating soil acidity and improving crop productivity; however recommended lime applications can reduce yield. We tested the hypothesis that elevated xylem sap Ca 2+ limited gas exchange of Phaseolus vulgaris L. and Pisum sativum L. plants that exhibited reduced shoot biomass and leaf area when limed. Methods We used Scholander and whole-plant pressure chamber techniques to collect root and leaf xylem sap, a calcium-specific ion-selective electrode to measure xylem sap Ca 2+, infra-red gas analysis to measure gas exchange of limed and unlimed (control) plants, and a detached leaf transpiration bioassay to determine stomatal sensitivity to Ca 2+. Results Liming reduced shoot biomass, leaf area and leaf gas exchange in both species. Root xylem sap Ca 2+ concentration was only increased in P. vulgaris and not in P. sativum. Detached leaves of both species required 5 m M Ca 2+ supplied to via the transpiration stream to induce stomatal closure, however, maximum in vivo xylem sap Ca 2+ concentrations of limed plants was only 1.7 m M and thus not high enough to influence stomata. Conclusion We conclude that an alternative xylem-borne antitranspirant other than Ca 2+ decreases gas exchange of limed plants. 相似文献
9.
The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca 2+ ions. The inhibitors of Ca 2+/calmodulin and Ca 2+/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca 2+-dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca 2+/calmodulin and Ca 2+/phospholipid-dependent STPK reduced the Ca 2+-induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria. 相似文献
10.
The effect of Ca 2+ level in the growth medium on the response of germination and early seedling growth of Phaseolus vulgaris to NaCl salinity was investigated. When NaCl concentration was increased germination and early seedling growth was decreased. The addition of Ca 2+ to the media increased both germination percentage and seedling growth. Chloride concentrations were not affected by the level of Ca 2+. Potassium and Ca 2+ concentrations and transport from roots to shoots were decreased by NaCl, but were restored by increasing Ca 2+ in the medium. The opposite was true for Na +. Leakage of NO 3
- and H 2PO 4
- was increased by salinity and reduced by high Ca 2+ in the medium. The results are discussed in terms of the beneficial effects of calcium for plant growth under saline conditions. 相似文献
11.
In vitro phosphorylation of several membrane polypeptides and soluble polypeptides from corn ( Zea mays var. Patriot) coleoptiles was promoted by adding Ca 2+. Ca 2+-promoted phosphorylation of the membrane polypeptides was further increased in the presence of calmodulin. Both Ca 2+-stimulated and Ca 2+- and calmodulin-stimulated phosphorylations of membrane polypeptides were inhibited by chlorpromazine, a calmodulin antagonist. Ca 2+-stimulated phosphorylation of soluble polypeptides increased with increasing Ca 2+ concentration. The calmodulin antagonists chlorpromazine and trifluoperazine inhibited the Ca 2+-promoted phosphorylation of soluble polypeptides. Added calmodulin promoted the Ca 2+-dependent phosphorylation of a 98 kilodaltons polypeptide. Both Ca 2+-dependent and Ca 2+-independent phosphorylations required Mg 2+ at an optimal concentration of 5 to 10 millimolar. Cyclic AMP was found to have no stimulatory effect on protein phosphorylation. Sodium molybdate, an inhibitor of protein phosphatase, increased the net phosphorylation of several polypeptides. Rapid loss of radioactivity from the phosphorylated polypeptides following incubation in unlabeled ATP indicated the presence of phosphoprotein phosphatase activity. 相似文献
13.
Ca 2+ enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The
number of transformants showed a marked increase with increasing concentration of CaCl 2 upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that
are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01–0.04
mM along with optimal CaCl 2 concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca 2+-stimulated transformation. The results of present investigation suggest the involvement of a Ca 2+-dependent protein activator in the development of Ca 2+-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic
spore forming thermophilic actinomycete.
Received: 21 May 2002 / Accepted: 21 June 2002 相似文献
14.
A soluble Ca 2+- and Ca 2+—calmodulin-activated protein kinase was partially purified from wheat germ. The phosphorylation of histones and casein catalyzed by this enzyme is largely Ca 2+-dependent. After repeated gel filtration of the protein kinase in the presence of 1 mM EGTA, the phosphorylation of casein and histones by the enzyme is activated 3-fold and up to 16-fold, respectively, by added calmodulin (12.5 μM). Such activation of the protein kinase by calmodulin is Ca 2+-dependent. The protein kinase binds to calmodulin—Sepharose 4B in a Ca 2+-dependent fashion. This type of Ca 2+-activated protein kinase may be involved in stimulus—response coupling in plants. 相似文献
15.
The effects of Ca 2+ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [ 32P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca 2+. Maximal incorporation of [ 32P] into the protein was achieved with free Ca 2+ concentrations of 90 μM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 μM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [ 32P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca 2+ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca 2+ concentrations between 10 −6 – 10 −5 M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca 2+ and calmodulin. 相似文献
16.
Calmodulin, the ubiquitous and multifunctional Ca 2+-binding protein, mediates many of the regulatory effects of Ca 2+, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca 2+] i transient via the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca 2+/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca 2+. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca 2+/calmodulin or indirectly by phosphorylation catalysed by Ca 2+/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca 2+ across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca 2+/calmodulin, e.g. the sarcolemmal Ca 2+ pump and the ryanodine receptor/Ca 2+ release channel, and other proteins which indirectly regulate [Ca 2+] i via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues. 相似文献
17.
TRPM3 proteins assemble to Ca 2+-permeable cation channels in the plasma membrane, which act as nociceptors of noxious heat and mediators of insulin and cytokine release. Here we show that TRPM3 channel activity is strongly dependent on intracellular Ca 2+. Conceivably, this effect is attributed to the Ca 2+ binding protein calmodulin, which binds to TRPM3 in a Ca 2+-dependent manner. We identified five calmodulin binding sites within the amino terminus of TRPM3, which displayed different binding affinities in dependence of Ca 2+. Mutations of lysine residues in calmodulin binding site 2 strongly reduced calmodulin binding and TRPM3 activity indicating the importance of this domain for TRPM3-mediated Ca 2+ signaling. Our data show that TRPM3 channels are regulated by intracellular Ca 2+ and provide the basis for a mechanistic understanding of the regulation of TRPM3 by calmodulin. 相似文献
18.
Mesophyll cells in discs cut from primary leaves of Phaseolus vulgaris L. were exposed to a concentration of phaseolotoxin that inhibited ornithine carbamoyltransferase (OCTase) measured in an extract of the tissue. This treatment also blocked incorporation of exogenous [ 14C] ornithine into protein-arginine of the mesophyll cells. By contrast more than 80% of the [ 14C]ornithine supplied to untreated tissue was incorporated into protein-arginine in 565 minutes. Protein synthesis in mesophyll cells was unaffected by phaseolotoxin because treated tissue continued to incorporate [ 14C]leucine into protein at the same rate as the untreated control. The phaseolotoxin-treated tissue should therefore remain metabolically competent and this prediction was reinforced by the finding that the rate of photosynthetic O 2 evolution per unit chlorophyll was similar for tissue from the phaseolotoxin-induced chlorosis and from green healthy tissue. Phaseolotoxin also blocked OCTase but not protein synthesis in exponentially growing cell suspension cultures. Phaseolotoxin rapidly inhibited growth of Escherichia coli and this effect was rapidly reversed by arginine. Thus, the toxic effects of phaseolotoxin may be attributed to the inhibition of OCTase which, in turn, blocks arginine synthesis. Protein accumulation is blocked as a consequence, but protein synthesis is unaffected. Chlorosis is due to reduced chlorophyll synthesis and this is presumably a consequence of the lower protein level in affected tissue. 相似文献
19.
To examine possible calcium (Ca 2+)-mediated prefertilization events in male gametes of higher plants, we studied protein phosphorylation and the Ca 2+-binding proteins, calmodulin and calreticulin, in sperm cells isolated from maize ( Zea mays L.) pollen in the presence and absence of Ca 2+. Using immunoblotting, we detected calmodulin and calreticulin and Ca 2+-induced variations. Exposure of sperm cells to 1 mM Ca 2+ for 1 h increased calmodulin content by 136% compared with the control. Ca 2+ had little effect on calreticulin at 1 h, but induced a 34% increase after 3 h. Phosphorylation of proteins was low in 1 h-control
and Ca 2+-treated cells. However, a 13-fold increase in phosphorylation of a 18-kDa protein was found at 12 h in the presence of Ca 2+. Ca 2+-induced changes in calmodulin, calreticulin and protein phosphorylation observed in maize sperm cells may reflect prefertilization
changes in vivo that facilitate sperm cell fusion with egg and central cells.
Received: 26 July 1996 / Revision accepted: 7 February 1997 相似文献
20.
Activation of Ca 2+-calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic β-cell. To study the properties of such kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [γ- 32P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca 2+ plus calmodulin or by cyclic AMP. The major effect of Ca 2+ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53 100±500 ( n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 μM free Ca 2+ and 0.7 μM calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a protein of similar molecular weight could be enhanced to a lesser extent in the absence of Ca 2+ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55 000 and 70–80 000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion. 相似文献
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