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1.
Ca2+-activated K+ channels play an important role in Ca2+ signal transduction and may be regulated by mechanisms other than a direct effect of Ca2+. Inside-out patches of the apical membrane of confluent transformed rabbit cortical collecting duct cells cultured on collagen were subjected to patch clamp analysis. Two types of K+ channel, of medium and high conductance, were observed. The latter channel was characterized by a K+/Na+ permeability ratio of 10, an inwardly rectified current, a conductance of 80 pS at 0 mV, and an open probability dependent on both voltage and Ca2+. Guanosine 5-triphosphate (GTP) but not a guanosine 5-diphosphate (GDP) analogue, adenosine 5-triphosphate (ATP), cytidine 5-triphosphate (CTP), or inosine 5-triphosphate (ITP), inhibited the activity of this Ca2+-activated K+ channel. The inhibitory effect of GTP was dose dependent, with a 50% inhibitory concentration of 10–5
m in the absence of Mg2+. In the presence of Mg2+ (1 mm), which is required for the binding of GTP to G proteins, the 50% inhibitory concentration decreased to 3×10–12
m. Pertussis toxin or cholera toxin (each at 10 ng/ml) did not prevent the inhibitory effect of GTP. After removal of GTP from the medium bathing an inhibited channel, subsequent application of Ca2+ failed to activate the channel. Ca2+-activated K+ channels of smooth muscle cells and proximal tubule cells did not respond to GTP. Thus, the Ca2+-activated K+ channel in the apical membrane of collecting duct cells is inhibited by GTP, which appears to exert its effect via a G protein that is insensitive to both cholera and pertussis toxins. 相似文献
2.
The first enzyme (named GTP cyclohydrolase) in the pathway for the biosynthesis of pteridines has been partially purified from extracts of late pupae and young adults of Drosophila melanogaster. This enzyme catalyzes the hydrolytic removal from GTP of carbon 8 as formate and the synthesis of 2-amino-4-hydroxy-6-(d-erythro-1,2,3-trihydroxypropyl)-7,8-dihydropteridine triphosphate (dihydroneopterin triphosphate). Some of the properties of the enzyme are as follows: it functions optimally at pH 7.8 and at 42 C; activity is unaffected by KCl and NaCl, but divalent cations (Mg2+, Mn2+, Zn2+, and Ca2+) are inhibitory; the K
m for GTP is 22 m; and the molecular weight is estimated at 345,000 from gel filtration experiments. Of a number of nucleotides tested, only GDP and dGTP were used to any extent as substrate in place of GTP, and these respective compounds were used only 1.8% and 1.5% as well as GTP.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929). 相似文献
3.
Calcium transport has been studied using purified endomembrane vesicles from dark-grown roots of Pisum sativum L. Membranes from a mixed microsomal (non-mitochondrial) fraction showed ATP-dependent calcium uptake which was released by the ionophore A 23187, had a pH optimum of 7.2 and required Mg2+ for uptake. Membranes were further purified using a rapid sucrosedensity-gradient technique yielding vesicles suitable for transport studies, and were identified using marker enzymes. Uptake by plasma membrane, tonoplast, endoplasmic reticulum and Golgi apparatus was indicated. Uptake by membranes of low density (predominantly tonoplast) had a pH optimum of 7.2–7.4 and nucleotide specificity ATP> guanosine 5-triphosphate>inosine 5-triphosphate>ADP>, while that by high-density membranes had a pH optimum of 7.5–7.9 and less specificity for ATP. The importance of regulating sucrose concentrations in calcium transport studies was demonstrated.Abbreviations ER
endoplasmic reticulum
- GTP
guanosine 5-triphosphate
- IDPase
inosine diphosphatase
- IIP
inosine 5-triphosphate 相似文献
4.
Summary Crude cell-free extracts of Anabaena cylindrica synthesized adenosine-5-phosphosulphate (AP35S) and 3-phosphoadenosine-5-phosphosulphate (PAP35S) from 35SO4
2- in the presence of Mg2+, ATP and inorganic pyrophosphatase. Maximum AP35S and PAP35S were produced at pH 7.15 and 8.05, respectively. APS kinase was detected in the supernatant of crude cell-free extracts by a spectrophotometric procedure. ATP-Sulphurylase had an absolute requirement for Mg2+ and less than 30% AP35S was formed when Mg2+ was replaced by either Mn2+ or Co2+. Nucleotide triphosphates other than ATP and 2-deoxyATP were ineffective in this reaction. Maximum enzyme activity was observed at equimolar concentrations of Mg2+ and ATP and excess of either of these was inhibitory. Other nucleotide triphosphates, like GTP, UTP, CTP, TTP, ITP, or 2-deoxyATP also inhibited the enzyme activity. Inhibition by GTP was competitive with respect to ATP. ATP-sulphurylase activity was not affected by cysteine, methionine or glutathione.Abbreviations APS
adenosine-5-phosphosulphate
- PAPS
3-phosphoadenosine-5-phosphosulphate 相似文献
5.
ATPase activity in rat heart sarcoplasmic reticulum was stimulated in a concentration-dependent manner by both Ca2+ and Mg2+ in the complete absence of the other cation. Increasing concentrations of Mg2+ produced an apparent inhibition of the Ca2+-dependent ATP hydrolysis. CDTA (trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate) had no effect on these responses. The results indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase in rat heart sarcoplasmic reticulum. However, sarcoplasmic reticulum vesicles transported Ca2+ with a high affinity (K0.5 Ca2+ = 0.41 M) suggesting the presence of a high affinity Ca2+-transporting ATPase. Calmodulin did not stimulate rat heart sarcoplasmic reticulum ATPase activity over a range of Ca2+ and Mg2+ concentrations and failed to stimulate membrane phosphorylation and Ca2+ transport into sarcoplasmic reticulum vesicles. Calmodulin antagonists trifluoperazine and compound 48180 did not affect the ATPase activity. Catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating the ATPase activity. These results suggest the presence of an ATPase activity in rat heart sarcoplasmic reticulum with different properties from the high affinity Ca2+-pumping ATPase previously characterized in dog heart and other species.Abbreviations cAMP
adenosine 3,5-monophosphate
- CaM
calmodulin
- CDTA
trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate
- EDTA
ethylene-diaminetetraacetate
- EGTA
ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate
- PLB
phospholamban
- SR
sarcoplasmic reticulum
- TFP
trifluoperazine 相似文献
6.
The phospholipase C (PLC; EC 3.1.4.3) activity in isolated plasma membranes of light-grown wheat (Triticum aestivum L. cv. Prelude) leaves was investigated. The activity against the polyphosphoinositides was strongly dependent on Ca2+ and was affected by the anionic detergent deoxycholate (DOC). In the presence of 20 M Ca2+ the PLC activity preferred phosphatidylinositol 4,5-bisphosphate (PIP2) over phosphatidylinositol 4-monophosphate (PIP) as a substrate. Instead, with 1 mM Ca2+ the enzyme clearly favoured PIP. In addition, the PIP2-PLC activity was increased by Mg2+ and in the presence of GTP, guanosine 5-(-thio)-triphosphate as well as ATP, CTP, guanosine 5-diphosphate and guanosine 5-(-thio)-diphosphate. Further analysis showed that a molybdate-sensitive phosphatase activity catalysing the dephosphorylation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is also associated with the plasma-membrane vesicles. Dephosphorylation of Ins(1,4,5)P3 was reduced in the presence of GTP or by inclusion of the unspecific phosphatase inhibitor molybdate. The results indicate the presence of a PIP2-PLC activity and the presence of a molybdate-sensitive phosphatase activity in wheat plasma-membrane vesicles.Abbreviations DOC
deoxycholate
- IDPase
inosine 5-diphosphatase
- InsPs
inositol phosphates, the numbering at the end indicates the number of phosphate residues and when their positions on the inositol ring are known they are indicated in parentheses, i.e.
- Ins(1,4,5)P3
inositol 1,4,5-trisphosphate
- PIP
phosphatidylinositol 4-monophosphate
- PIP2
phosphatidylinositol 4,5-bisphosphate
- PLC
phospholipase C
This work was financially supported by grant from the Deutsche Forschungsgemeinschaft (DFG). M. C. Arz gratefully acknowledges the support of a Graduiertenstipendium des Landes Nordrhein-Westfalen (Germany). We wish to thank S. Laden and G.E. Grambow for assistance. 相似文献
7.
V. Stefanovich 《Neurochemical research》1979,4(6):681-687
It was established that microvessels of a bovine cortex exhibit significant cyclic 3,5-adenosine monophosphate phosphodiesterase (cAMP PDE) and cyclic 3,5-guanosine monophosphate phosphodiesterase (cGMP PDE) activities. These activities are dependent on the presence of Mg2+. Absence of Ca2+ was virtually without effect. When both Mg2+ and Ca2+ were absent, PDE activities increased compared with activities observed in the absence of Mg2+. Xanthines (caffeine, theobromine, and theophylline) were better inhibitors of cAMP PDE than of cGMP PDE. Imidazole, in very high concentration (1×10–2M) only, exhibited PDE stimulatory activity at high concentrations of both substrates. Otherwise, it exhibited PDE-inhibitory properties. 相似文献
8.
Rajesh Mahey Michael A. Bridges Sidney Katz 《Molecular and cellular biochemistry》1991,105(2):137-147
Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca2+-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca2+-transporter showed high affinity for Ca2+ (KCa
2+ = 0.04 ± 0.01 M), an apparent requirement for Mg2+ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca2+ or Mg2+ but showed a low affinity for these cations. At low concentrations, Mg2+ appeared to inhibit the Ca2+-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg2+ concentration (0.5–1 mM), a high affinity Ca2+-dependent ATPase activity was observed (KCa
2+ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca2+-transport nor high affinity Ca2+-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca2+ (or Mg+)-ATPase activity, the presence of both a high affinity Ca2+-pump and high affinity Ca2+-dependent ATPase. However, the high affinity Ca2+-ATPase activity does not appear to be the biochemical expression of the Ca2+-pump.Abbreviations Ca2+-ATPase
calcium-activated, magnesium-dependent adenosine triphosphatase
- CaM
calmodulin
- CDTA
trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate
- EDTA
ethylene-diaminetetraacetate
- EGTA
ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate
- NADPH
reduced form of nicotinamide adenine dinucleotide phosphate 相似文献
9.
Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase
In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP
adenosine triphosphate
- -AR
-adrenergic receptors
- AC
adenylyl cyclase
- DHA
dihydroalprenolol
- DPH
diphenylhexatriene
- [Ca2+]i
Ca2+ concentration inside proteoliposomes
- [Ca2+]o
Ca2+ concentration outside proteoliposomes
- cAMP
cyclic adenosine monophosphate
- DTT
Dithiothreitol
- FS
fluorescein sulfonate
- Gs
Stimulatory GTP-binding protein
- GTP
guanosine triphosphate
- GTPS
guanosine 5-O-(3-thiotriphosphate)
- kDa
kilodalton
- SDS
sodium dodecyl sulfate
- Tris
N-tris(hydroxymethyl)aminomethane 相似文献
10.
The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca2+ (EC50=2.4 M). The stable GTP analogue GTPS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca2+ concentrations. At optimal Ca2+ (10 M) the effect of GTPS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A2. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca2+ dependent effects, (ii) phospholipase C was not activated in the absence of Ca2+ and insulin secretion due to the phorbol ester TPA displayed a different Ca2+ dependency, (iii) arachidonic acid did not elicit Ca2+ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca2+ or TPA is attenuated by the inhibitory guanine nucleotide GDPS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.Abbreviations InsP3
inositol trisphosphate
- Ptd-InsP2
phosphatidylinositol 4,5-bisphosphate
- GTPS
guanosine 5-(3-O-thio)triphosphate
- GDPS
guanosine 5-(2-O-thio)diphosphate
- Gpp(NH)p
guanyl-5-yl imidodiphosphate
- TPA
12-O-tetradecanoylphorbol-13-acetate
- OAG
1-oleoyl-2-acetylglycerol
- Hepes
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- EGTA
(ethylenebis(oxyethylenenitrilo)tetraacetic acid
- DAG
diacylglycerol
- [Ca2+]i
cytosolic free Ca2+ concentration 相似文献
11.
Roger R. Lew 《Planta》1994,193(1):67-73
Voltage clamp was used to measure the voltage dependence of cell-to-cell coupling via plasmodesmata between higher-plant cells (root hairs of Arabidopsis thaliana (L.) Heynh.). In addition, ionophoresis was used to introduce a variety of ions [Ca2+, inositol-trisphosphate, Li+, K+, Mg2+, ethylene glycol-bis(-aminoethyl ether)-N,N,N, N-tetraacetic acid (EGTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), H+, and OH–] to examine whether they regulate cell-to-cell coupling. Electrical coupling showed high variability in this single cell type at the same developmental stage; the coupling ratio ranged from near 0% to about 90% with a mean value of 32%. It was voltage independent for intracellular voltage gradients (transplasmodesmatal) of -163 to 212 mV. While Ca2+ closes the plasmodesmatal connections (at concentrations higher than those causing cessation of cytoplasmic streaming), inositol-trisphosphate and lithium are without effect. Apparently, inositol-trisphosphate may not cause increased cytosolic Ca2+ in root hairs. Alkalinization by OH ionophoresis caused a modest decline in cell-to-cell coupling, as did acidification by H+ ionophoresis (to an extent causing the cell to become flacid). Increases in cytosolic K+, Mg2+, and the calcium chelator BAPTA by ionophoresis had no effect on cell-to-cell coupling. The regulation (and lack thereof) reported here for plant plasmodesmata is quite similar to that of gap junctions.Abbreviations BAPTA
1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid 相似文献
12.
Juan M. Lopez 《Archives of microbiology》1982,131(3):247-251
After addition of amino acids to a Bacillus subtilis glucose culture the intracellular guanosine triphosphate (GTP) concentration increased. The growth rate and the rate of RNA accumulation increased too. With mycophenolic acid, an inhibitor of inosinate dehydrogenase, it was possible to adjust the intracellular GTP concentration to values ranging between that of the glucose culture and that of the culture which had received amino acids. This led to intermediate growth rate values. Guanosine abolished the mycophenolic acid inhibition of GTP synthesis. It also counteracted the drug effects on growth rate and RNA accumulation. In cultures growing on Nutrient Sporulation Medium, in which the growth rate decreases as cell density increases, the GTP concentration did correlate with the growth rate.Abbreviations ppGpp
guanosine 5-diphosphate 3-diphosphate
- pppGpp
guanosine 5-triphosphate 3-diphosphate 相似文献
13.
The reaction of the 5 -phosphorimidazolide of adenosine (5-ImpA) with diadenosine pyrophosphate (A5ppA) in the presence of Na+-montmorillonite in aqueous, pH 8 solution results in the regiospecific formation of A5ppA3pA and A5ppA3pA3 pA. The formation of oligomers of general structure (pA)n decreases in the presence of A5ppA. A5ppA3pA is the principal reaction product when a 1:1 ratio of ImpA and A5ppA is used. The yield of A5ppA3pA3pA is optimal when 9:1 or 4:1 ratios of ImpA: A5ppA are used. The overall regiospecificity of formation of 3,5-links is about 80%. The reaction between ImpA and A5ppA on montmorillonite differs from the self-condensation of ImpA in that it proceeds in the absence of Mg2+ and there are only small differences in oligomer yields when Na+, Li+ Ca2+, and NH
4
+
are the exchangeable cations on the montmorillonite. The reaction is inhibited by 0.4 M imidazole but the inhibition is suppressed with 0.4 M Mg2+. Little or no phosphodiester bond formation was observed with Mg2+- or Al3+-montmorillonite. Montmorillonites other than 22A and Volclay exhibited no catalysis for the formation of adducts between ImpA and A5ppA and no catalysis was exhibited in ferrugenous smectite, nontronite, allophane, or sepiolite. 相似文献
14.
Summary Organic pyrophosphates such as UppA and NAD are formed when a solution containing a nucleotide, a nucleoside 5-polyphosphate, Mg2+ and imidazole are allowed to dry out. We suggest that this synthesis may have occured concurrently with oligonucleotide formation.Abbreviations Im
Imidazole
- CDI
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
- EDTA
ethylenediaminetetraacetic acid
- A
adenosine
- U
uridine
- pnA
adenosine 5-poly-phosphate containing n phosphate residues
- pU
uridine 5-phosphate
- AppA
P1,P2-diadenosine 5-pyrophosphate
- UppA
P1-(uridine 5)-P2-(adenosine 5)-pyrophosphate
- ImpA
adenosine 5-phosphorimidazolide
- NMN
nicotinamide mononucleotide
- NAD
nicotinamide-adenine dinucleotide 相似文献
15.
Summary Short oligocytidylates can act as templates for the self-condensation of guanosine 5-phosphorimidazolide. In the absence of a catalytic metal ion or in the presence of Pb2+ a noticeable template effect is already observed with the dimer and the yield of long oligomers reaches a plateau with a hexamer template. Short templates give oligomers longers than the template length. The products are predominantly 2-5 linked for the Pb2+-catalyzed reaction while mixed linkages are observed in the uncatalyzed reaction.In the presence of Zn2+, a template effect is first observed with the pentamer and is maximal by the heptamer. The products are predominantly 3-5 linked. Oligomers shorter than or as long as the template are obtained in substantial yield, and longer products in much lower yields.Abbreviations G
Guanosine
- Gp
guanosine 2(3)-phosphate
- pG
guanosine 5-phosphate
- Gp!
guanosine cyclic 2,3-phosphate
- ImpG
guanosine 5-phosphorimidazolide
- ImpG*
[8-14C]-guanosine 5-phosphorimidazolide
- pGp
5-phosphoguanosine 2(3)-phosphate
- G2pG
guanylyl-[2-5]-guanosine
- G3pG
guanylyl-[3-5]-guanosine
- ImpGpG
5-phosphorimidazolide of GpG
- (pG)n (n = 2,3)
oligomers of pG
- GppG
P1, P2-diguanosine 5-diphosphate
- GppGpG
5-[guanosine 5-pyrophosphate] of GpG
- NH2pG
guanosine 5-phosphoramidate
- (pG)4+
tetramer and higher oligoguanylates with 5 terminal phosphate
- oligo(G)
oligoguanylate
- Cp
cytidine 2(3)-phosphate
- Cp!
cytidine cyclic 2,3-phosphate
- (Cp)n–1 Cp! (n= 2,3,4)
oligocytidylates terminated by 5-OH groups and 2,3-cyclic phosphates
- oligo(C)
oligocytidylate
- poly(C)
polycytidylic acid
- poly(U)
polyuridylic acid
- poly(C,G)
random copolymer of C and G
- BAP
bacterial alkaline phosphatase (E. coli)
- EDTA
ethylenediaminetetraacetic acid
- Rf
chromatographic mobility 相似文献
16.
Parvathi K. Gayathri J. Maralihalli G.B. Bhagwat A.S. Raghavendra A.S. 《Photosynthetica》2000,38(1):23-28
PEP carboxylase (PEPC) in leaves of C4 plants is activated by phosphorylation of enzyme by a PEPC-protein kinase (PEPC-PK). We reevaluated the pattern of PEPC phosphorylation in leaf extracts of Amaranthus hypochondriacus. It was dependent on Ca2+, the optimum concentration of which for stimulation was 10 mM. The extent of stimulation was inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), a Ca2+ chelator. The inhibition by BAPTA was relieved by the addition of Ca2+ but not by the addition of Mg2+. The stimulation by Ca2+ of PEPC phosphorylation was marginally enhanced by calmodulin (CaM), but not by diacylglycerol (DAG). Phosphorylation was strongly restricted by Ca2+ or Ca2+-CaM-dependent protein kinase inhibitors. Thus phosphorylation of PEPC is Ca2+-dependent in leaves of A. hypochondriacus and a calcium-dependent protein kinase (CDPK) may modulate PEPC-PK and subsequently the phosphorylation status of PEPC. 相似文献
17.
Phytochrome is confirmed to be the photoreceptor pigment in the germination response of Onoclea sensibilis L. by demonstrating red-far-red (R-FR) photoreversibility. External Ca2+ is required for this response with a threshold at a submicromolar concentration. Ethylene glycol-bis(-amino-ethyl ether)-N,N,N,N-tetraacetic acid, La3+ and Co2+ reversibly inhibit germination. Lanthanum only inhibits germination when applied before or during irradiation, indicating that the external Ca2+ requirement is transient, although in the absence of Ca2+ the R-stimulated system remains maximally poised to accept the ion for over 4 h after irradiation. The ability to respond to Ca2+ 4.1 h after R-irradiation is not reversed by FR-irradiation, indicating that Ca2+ transport has been uncoupled from phytochrome. Barium and Sr2+, but not Mg2+ can substitute for Ca2+. Artificially increasing the concentration of intracellular free Ca2+ with the ionophore A 23187 stimulates germination in the dark. The Ca2+-calmodulin antagonists, trifluoperizine and chlorpromazine, reversibly inhibit germination. Calcium is required in phytochrome-mediated fern spore germination; it may be acting as a second messenger.Abbreviations EGTA
ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- FR
far-red light
- R
fed light 相似文献
18.
Takenori Miyamoto Diego Restrepo Edward J. Cragoe Jr. John H. Teeter 《The Journal of membrane biology》1992,127(3):173-183
Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP3 or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP3 were blocked by ruthenium red, a blocker of an IP3-gated Ca2+-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP3 in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP3 or cAMP when the buffering capacity for Ca2+ of the pipette solution was increased, when extracellular Ca2+ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca
i
] that opened Ca2+-activated K+ channels. The results suggest that different conductances modulated by either IP3 or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca2+-activated K+ channels contribute to the termination of the IP3 and cAMP responses.Abbreviations ATP
adenosine 5-triphosphate
- BAPTA
(bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid
- cAMP
adenosine cyclic 3,5-monophosphate
- cGMP
guanosine cyclic 3,5-monophosphate
- CTX
charybdotoxin
- DCB
3,4-dichlorobenzamil
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- IP3
inositol-1,4,5-triphosphate
- NMDG
N-methyl-d-glucamine
We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825. 相似文献
19.
Crude Ca2+-activated protein kinase from membranes of apple (Malus domestica L. Borkh., Cox's Orange Pippin) fruit can be partially purified to yield a Ca2+-dependent protein kinase whose activity is apparently not regulated by calmodulin. The autophosphorylating catalytic subunit of this protein kinase shows a Ca2+-dependent mobility shift of approx. 10 kilodaltons (kDa) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis; in the absence of added Ca2+ or ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) its apparent molecular mass is approx. 50 kDa. The Ca2+-dependent protein kinase is inhibited by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide and trifluoperazine with IC50 values of approx. 45 M and 15 M, respectively. These similarities between the protein kinase and calmodulin indicate that the kinase may be a calmodulin-like protein.Abbreviations DEAE
diethylaminoethyl
- EGTA
ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- Hepes
4-(-2-hydroxyethyl)-1-piperazineethanesulphonic acid
- kDa
kilodalton
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulphate
- W7
N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide
- W5
N-(6-aminohexyl)-naphthalenesulphonamide 相似文献
20.
Anastassia Kanavarioti T. Brian Hurley Eldon E. Baird 《Journal of molecular evolution》1995,41(2):161-168
Evidence is presented for complexation of guanosine 5-monophosphate 2-methylimidazolide (2-MeImpG) with polycytidylate (poly(C)) at pH 8.0 and 23°C in the presence of 1.0 M NaCl and 0.2 M MgCl2 in water. The association of 2-McImpG with poly(C) was investigated using UV-vis spectroscopy as well as by monitoring the kinetics of the nucleophilic substitution reaction of the imidazole moiety by amines. The results of both methods are consistent with moderately strong poly(C) · 2-McImpG complexation and the spectrophotometric measurements allowed the construction of a binding isotherm with a concentration of 2-McImpG equal to 5.55 ± 0.15 mM at half occupancy. UV spectroscopy was employed to establish the binding of other guanosine derivatives on poly(C). These derivatives are guanosine 5-monophosphate (5GMP), guanosine 5monophosphate imidazolide (ImpG), and guanosine 5monophosphate morpholidate (morpG). Within experimental error these guanosine derivatives exhibit the same affinity for poly(C) as 2-McImpG. 相似文献