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1.
S.F. Pedersen J. Prenen G. Droogmans E.K. Hoffmann B. Nilius 《The Journal of membrane biology》1998,163(2):97-110
A Ca2+-activated (I
Cl,Ca) and a swelling-activated anion current (I
Cl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying
currents were activated by an increase in the free intracellular calcium concentration ([Ca2+]
i
), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular
Cl− concentration. I
Cl,Ca current density increased with increasing [Ca2+]
i
, and this current was abolished by lowering [Ca2+]
i
to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA). In contrast, activation of I
Cl,vol did not require an increase in [Ca2+]
i
. The kinetics of I
Cl,Ca and I
Cl,vol were different: at depolarized potentials, I
Cl,Ca as activated in a [Ca2+]
i
- and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I
Cl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials.
The deactivation of I
Cl,vol was dependent on the extracellular Mg2+ concentration. The anion permeability sequence for both currents was I
− > Cl− > gluconate. I
Cl,Ca was inhibited by niflumic acid (100 μm), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μm) and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 100 μm), niflumic acid being the most potent inhibitor. In contrast, I
Cl,vol was unaffected by niflumic acid (100 μm), but abolished by tamoxifen (10 μm). Thus, in Ehrlich cells, separate chloride currents, I
Cl,Ca and I
Cl,vol, are activated by an increase in [Ca2+]
i
and by cell swelling, respectively.
Received: 12 November 1997/Revised: 5 February 1998 相似文献
2.
OATP1B1 and 1B3 are related transporters mediating uptake of numerous compounds into hepatocytes. A putative model of OATP1B3
with a “positive binding pocket” containing conserved positively charged amino acids was predicted (Meier-Abt et al. J Membr
Biol 208:213–227, 2005). Based on this model, we tested the hypothesis that these positive amino acids are important for OATP1B1
function. We made mutants and measured surface expression and uptake of estradiol-17β-glucuronide, estrone-3-sulfate and bromosulfophthalein
in HEK293 cells. Two of the mutants had low surface expression levels: R181K at 10% and R580A at 30% of wild-type OATP1B1.
A lysine at position 580 (R580K) rescued the expression of R580A. Mutations of several amino acids resulted in substrate-dependent
effects. The largest changes were seen for estradiol-17β-glucuronide, while estrone-3-sulfate and bromosulfophthalein transport
were less affected. The wild-type OATP1B1 K
m value for estradiol-17β-glucuronide of 5.35 ± 0.54 μM was increased by R57A to 30.5 ± 3.64 μM and decreased by R580K to 0.52 ± 0.18 μM.
For estrone-3-sulfate the wild-type high-affinity K
m value of 0.55 ± 0.12 μM was increased by K361R to 1.8 ± 0.47 μM and decreased by R580K to 0.1 ± 0.04 μM. In addition, R580K
reduced the V
max values for all three substrates to <25% of wild-type OATP1B1. Mutations at intracellular K90, H92 and R93 mainly affected
V
max values for estradiol-17β-glucuronide uptake. In conclusion, the conserved amino acids R57, K361 and R580 seem to be part
of the substrate binding sites and/or translocation pathways in OATP1B1. 相似文献
3.
Krystyna Konopka Barbara Dorocka-Bobkowska Senait Gebremedhin Nejat Düzgüneş 《Antonie van Leeuwenhoek》2010,97(4):413-417
Candida-associated denture stomatitis has a high rate of recurrence. Candida biofilms formed on denture acrylic are more resistant to antifungals than planktonic yeasts. Histatins, a family of basic
peptides secreted by the major salivary glands in humans, especially histatin 5, possess significant antifungal properties.
We examined antifungal activities of histatin 5 against planktonic or biofilm Candida albicans and Candida glabrata. Candida biofilms were developed on poly(methyl methacrylate) discs and treated with histatin 5 (0.01–100 μM) or fluconazole (1–200 μM).
The metabolic activity of the biofilms was measured by the XTT reduction assay. The fungicidal activity of histatin 5 against
planktonic Candida was tested by microdilution plate assay. Biofilm and planktonic C. albicans GDH18, UTR-14 and 6122/06 were highly susceptible to histatin 5, with 50% RMA (concentration of the agent causing 50% reduction
in the metabolic activity; biofilm) of 4.6 ± 2.2, 6.9 ± 3.7 and 1.7 ± 1.5 μM, and IC50 (planktonic cells) of 3.0 ± 0.5, 2.6 ± 0.1 and 4.8 ± 0.5, respectively. Biofilms of C. glabrata GDH1407 and 6115/06 were less susceptible to histatin 5, with 50% RMA of 31.2 ± 4.8 and 62.5 ± 0.7 μM, respectively. Planktonic
C. glabrata was insensitive to histatin 5 (IC50 > 100 μM). Biofilm-associated Candida was highly resistant to fluconazole in the range 1–200 μM; e.g. at 100 μM only ~20% inhibition was observed for C. albicans, and ~30% inhibition for C. glabrata. These results indicate that histatin 5 exhibits antifungal activity against biofilms of C. albicans and C. glabrata developed on denture acrylic. C. glabrata is significantly less sensitive to histatin 5 than C. albicans. 相似文献
4.
The protein bCblC (bCblCpro) is a bovine homolog of a human B12 trafficking chaperone that is responsible for the processing of vitamin B12 and its escorted delivery in intracellular B12 metabolism. In this study, we found that bCblCpro is highly thermolabile with a T
m = 42.0 ± 0.2 °C as shown for the human homolog, suggesting thermal regulation of these proteins. Binding of the reduced form
of glutathione (GSH) that is a predominant cellular thiol increased the T
m of bCblCpro from 42 °C to ~45 °C (ΔT
m max = 3.1 ± 0.2 °C and AC50 = 2.1 ± 0.5 mM). Binding of vitamin B12 and its derivatives also stabilized bCblCpro increasing the T
m to a different extent and vitamin B12 (cyanocobalamin, CNCbl) was the least efficient (ΔT
m max = 4.3 ± 0.3 °C and AC50 = 291 ± 36 μM). However, the stabilizing effect of CNCbl was significantly greater for GSH-bound bCblCpro (ΔT
m max = 12.8 ± 0.6 °C and AC50 = 9.3 ± 1.6 μM) than for GSH-free bCblCpro. In addition, the stabilizing effect of GSH was also greater for CNCbl-bound bCblCpro
(ΔT
m max = 9.3 ± 0.3 °C and AC50 = 57.0 ± 6.8 μM). Limited proteolysis revealed that thermal stabilization of bCblCpro is derived from conformational changes
of the protein induced by binding of the ligands. The results in this study indicate that GSH cooperates with vitamin B12 in thermal stabilization of bCblCpro and is a positive regulator of the protein. 相似文献
5.
Protective Effect of Retinal Ischemia by Blockers of Voltage-dependent Calcium Channels and Intracellular Calcium Stores 总被引:1,自引:0,他引:1
Massote PD Pinheiro AC Fonseca CG Prado MA Guimarães AL Massensini AR Gomez MV 《Cellular and molecular neurobiology》2008,28(6):847-856
In the present study, the neuroprotective effect of blockers of voltage-dependent calcium channels (VDCC) and intracellular
calcium stores on retinal ischemic damage induced by oxygen deprivation-low glucose insult (ODLG) was investigated. Retinal
damage induced by ODLG was dependent on the calcium concentration in the perfusion medium. When incubated in medium containing
2.4 mM CaCl2, cell death in ischemic retinal slices treated with blockers of VDCC, ω-conotoxin GVIA (1.0 μM), ω-conotoxin MVIIC (100 nM)
and nifedipine (1.0 μM), was reduced to 62 ± 2.3, 46 ± 4.3 and 47 ± 3.9%, respectively. In the presence of blockers of intracellular
calcium stores, dantrolene (100 μM) and 2-APB (100 μM), the cell death was reduced to 46 ± 3.2 and 55 ± 2.9%, respectively.
Tetrodotoxin (1.0 μM), reducing the extent of the membrane depolarization reduces the magnitude of calcium influx trough VDCC
causing a reduction of the cell death to 55 ± 4.3. Lactate dehydrogenase content of untreated ischemic retinal slices was
reduced by 37% and treatment of ischemic slices with BAPTA-AM (100 μM) or 2-APB (100 μM) abolished the leakage of LDH. Dantrolene
(100 μM) and nifedipine (1.0 μM) partially blocked the induced reduction on the LDH content of retinal ischemic slices. Histological
analysis of retinal ischemic slices showed 40% reduction of ganglion cells that was prevented by BAPTA-AM or dantrolene. 2-APB
partially blocked this reduction whilst nifedipine had no effect, p > 0.95. Conclusion Blockers of VDCC and intracellular calcium-sensitive receptors exert neuroprotective effect on retinal ischemia. 相似文献
6.
Hanana H Talarmin H Pennec JP Droguet M Gobin E Marcorelle P Dorange G 《Cytotechnology》2011,63(3):295-305
Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the
plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic
cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural
characteristics and their reactivity with antibodies against sarcomeric α-actinin, sarcomeric tropomyosin, myosin and troponin
T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium
current (I
K slow) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (I
K fast) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (I
KCa) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes:
L-type calcium current (I
Ca) inhibited by verapamil at 5 × 10−4 M, T-type Ca2+ current, rapidly activated and inactivated, and sodium current (I
Na) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential
in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 μM)
but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2′-deoxyuridine).
Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology. 相似文献
7.
Icilin is recognized as a chemical agonist of nociceptors and can activate TRPM8 channels. However, whether this agent has
any effects on immune cells remains unknown. In this study, the effects of icilin on ion currents were investigated in RAW
264.7 murine macrophage-like cells. Icilin (1–100 μM) increased the amplitude of nonselective (NS) cation current (I
NS) in a concentration-dependent manner with an EC50 value of 8.6 μM. LaCl3 (100 μM) or capsazepine (30 μM) reversed icilin-induced I
NS; however, neither apamin (200 nM) nor iberiotoxin (200 nM) had any effects on it. In cell-attached configuration, when the
electrode was filled with icilin (30 μM), a unique population of NS cation channels were activated with single-channel conductance
of 158 pS. With the use of a long-lasting ramp pulse protocol, increasing icilin concentration produced a left shift in the
activation curve of NS channels, with no change in the slope factor of the curve. The probability of channel opening enhanced
by icilin was increased by either elevated extracellular Ca2+ or application of ionomycin (10 μM), while it was reduced by BAPTA-AM (10 μM). Icilin-stimulated activity is associated with
an increase in mean open time and a reduction in mean closed time. Under current-clamp conditions, icilin caused membrane
depolarization. Therefore, icilin interacts with the TRPM8-like channel to increase I
NS and depolarizes the membrane in these cells. 相似文献
8.
Effects of Cannabinoids on Caffeine Contractures in Slow and Fast Skeletal Muscle Fibers of the Frog
Miguel Huerta Mónica Ortiz-Mesina Xóchitl Trujillo Enrique Sánchez-Pastor Clemente Vásquez Elena Castro Raymundo Velasco Rocío Montoya-Pérez Carlos Onetti 《The Journal of membrane biology》2009,229(2):91-99
The effect of cannabinoids on caffeine contractures was investigated in slow and fast skeletal muscle fibers using isometric
tension recording. In slow muscle fibers, WIN 55,212-2 (10 and 5 μM) caused a decrease in tension. These doses reduced maximum
tension to 67.43 ± 8.07% (P = 0.02, n = 5) and 79.4 ± 14.11% (P = 0.007, n = 5) compared to control, respectively. Tension-time integral was reduced to 58.37 ± 7.17% and 75.10 ± 3.60% (P = 0.002, n = 5), respectively. Using the CB1 cannabinoid receptor agonist ACPA (1 μM) reduced the maximum tension of caffeine contractures by 68.70 ± 11.63% (P = 0.01, n = 5); tension-time integral was reduced by 66.82 ± 6.89% (P = 0.02, n = 5) compared to controls. When the CB1 receptor antagonist AM281 was coapplied with ACPA, it reversed the effect of ACPA on caffeine-evoked tension. In slow and
fast muscle fibers incubated with the pertussis toxin, ACPA had no effect on tension evoked by caffeine. In fast muscle fibers,
ACPA (1 μM) also decreased tension; the maximum tension was reduced by 56.48 ± 3.4% (P = 0.001, n = 4), and tension-time integral was reduced by 57.81 ± 2.6% (P = 0.006, n = 4). This ACPA effect was not statistically significant with respect to the reduction in tension in slow muscle fibers.
Moreover, we detected the presence of mRNA for the cannabinoid CB1 receptor on fast and slow skeletal muscle fibers, which was significantly higher in fast compared to slow muscle fiber expression.
In conclusion, our results suggest that in the slow and fast muscle fibers of the frog cannabinoids diminish caffeine-evoked
tension through a receptor-mediated mechanism. 相似文献
9.
G. Szücs G. Buyse J. Eggermont G. Droogmans B. Nilius 《The Journal of membrane biology》1996,149(3):189-197
We have measured the kinetic and pharmacological properties of volume-activated Cl− currents (I
Cl,vol) in endothelial cells, and tried to correlate them with those of the already described volume-activated current I
Cln. Both conductances show a similar permeability sequence for monovalent anions, and they are blocked by extracellular ATP.
In the present report, we demonstrate by Western blot and RT-PCR that cultured endothelial cells from bovine pulmonary artery
(CPAE) contain pI
Cln. The expression of this protein has been shown to be closely associated with the I
Cln current.
I
Cl,vol showed however, in contrast with I
Cln, no striking inactivation at positive potentials. This property is also at variance with that of the volume-activated current
related to MDR-1. Activation of I
Cl,vol at potentials more negative than −80 mV was not time dependent, which excludes a major contribution of a ClC-2 related current.
The antiviral nucleoside analogue AZT (3′-azido-3′-deoxythymidine) inhibited I
Cl,vol by 21 ± 2.7% (n = 10), at a concentration of 100 μm. Another antiviral drug, acyclovir (ACV, 9-[(2-hydroxyethoxy)methyl]guanine) blocked I
Cl,vol by 27 ± 6.2% at 100 μm (n = 11). Both blocking effects are much smaller than those reported for I
Cln.
The phenol derivative gossypol, which blocks I
Cln-related currents, efficiently inhibited I
Cl,vol in CPAE cells (67 ± 2.1% at 1 μm, n = 7, K
I
= 0.4 μm).
The presence of pI
Cln in CPAE cells and the similar qualitative pharmacological profile of I
Cl,vol and I
Cln support the hypothesis that pI
Cln is a good molecular candidate for I
Cl,vol in endothelial cells. The discrepant kinetic properties may indicate that these time-dependent currents at high positive
or negative potentials are not intrinsic properties of the channels, but are caused by time-dependent depletion/accumulation
phenomena due to the large amplitudes of these currents.
Received: 8 May 1995/Revised: 12 October 1995 相似文献
10.
The incidence rates of long QT syndrome (LQTS) and drug-induced torsades de pointes (TDP) are higher in women than men. Although
gonadal steroids are assumed to play an important role in the gender-based differences in cardiac electrophysiological properties,
the underlying mechanisms of the gender-based differences are not fully understood. In particular I
Kr, which comprises the repolarization phase of the action potential, has not been well understood in its modulation by sex
hormones. To assess this, we examined the effects of the female sex hormone β-estradiol on the human ether-a-go-go-related gene (hERG)-encoded potassium current stably expressed in human embryonic kidney-293 (HEK) cells. We demonstrated that hERG currents
were inhibited by β-estradiol maximally to 62% of control with an IC50 of 1.3 μM and a Hill coefficient of 0.87, which might account for the sex-related differences in LQTS. We also examined whether
estrogen modulated drug-induced blocking effects on hERG currents or not. With simultaneous application of 10 μM erythromycin,
which is known to block hERG currents but not in low doses, the blocking effects of β-estradiol on hERG currents were enhanced.
Namely, hERG currents were inhibited maximally to 45.8% of control with an IC50 of 59 nM (P < 0.02) by β-estradiol with 10 μM erythromycin. We conclude here that a significant block of hERG currents by β-estradiol
may account for the sex-related differences in LQTS and the synergic effects of β-estradiol and erythromycin indicate a higher
risk of drug-induced TDP in women than men. 相似文献
11.
The genus Dierama comprises plants with a potential to be developed as ornamentals. D. erectum seeds were decontaminated and germinated on 1/10th strength Murashige and Skoog (Physiol Plant 15:473–497, 1962) (MS) media
without plant growth regulators or sucrose. In an experiment investigating the effects of 6-benzyladenine (BA), meta-Topolin (mT), kinetin (KIN) and zeatin (Z) with or without α-naphthaleneacetic acid (NAA), the highest shoot number per hypocotyl (4.20 ± 0.51)
was obtained from MS medium supplemented with 1.0 μM Z after 8 weeks. This was followed by a combination of 2.0 μM KIN and
2.0 μM NAA with 3.67 ± 0.81 shoots per explant. BA treatments produced 3.20 ± 0.22 shoots per hypocotyl explant when 2.0 μM
was combined with 1.0 μM NAA, while mT gave 3.09 ± 0.99 shoots per explant when 2.0 μM mT was combined with 2.0 μM NAA. Adventitious shoot regeneration was optimised when shoots were grown under a 16-h photoperiod
at 100 μmol m−2 s−1 on MS medium supplemented with 1.0 μM BA. This resulted in an average of 12.73 ± 1.03 shoots per hypocotyl explant. Various
concentrations of ancymidol, activated charcoal and sucrose did not promote in vitro corm formation of this species. Plants
rooted successfully after 8 weeks on MS medium supplemented with 1.0 μM indole-3-butyric acid (IBA) and had an average root
number of 2.73 ± 0.40. After 2 months of acclimatisation, plants had formed corms. The largest corms (of diameter 0.45 ± 0.03 cm)
were produced in plants pre-treated with 0.5 μM IBA. The highest plant survival percentage of 73% was also associated with
this treatment. 相似文献
12.
Christopher A. Dieni Kenneth B. Storey 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2010,180(8):1133-1142
Glucose-6-phosphate dehydrogenase (G6PDH) and the pentose phosphate pathway play a key role in reductive biosynthesis and
antioxidant defense, while diverting glucose from other cellular functions. G6PDH was isolated from liver of the wood frog,
Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K
m and V
max) of G6PDH showed a significant increase in K
m G6P (from 98.2 ± 3.8 to 121 ± 5.3 μM) and K
m NADP+ (from 65.5 ± 2.3 to 89.1 ± 4.8 μM) in frogs following freezing exposure, indicating lower affinity for G6PDH substrates in
this state. Subsequent analyses indicated that differential phosphorylation of G6PDH between the two states was responsible
for the altered kinetic properties. Thus, two differentially charged forms of G6PDH were resolved by DEAE ion-exchange chromatography
and, compared with controls, the proportion of G6PDH activity in peak I decreased and in peak II increased in liver from frozen
frogs. G6PDH in peak I had a K
m G6P of 94.1 ± 1.1 μM and K
m NADP+ of 61.2 ± 3.5 μM, whereas Peak II G6PDH showed higher values (K
m G6P was 172 ± 4.3 μM, K
m NADP+ was 98.2 ± 3.3 μM). G6PDH from each peak was incubated with ions and second messengers to stimulate the actions of protein
kinases with results indicating that G6PDH can be phosphorylated by protein kinase G, protein kinase C, AMP-activated protein
kinase, or calmodulin-dependent protein kinase. The data indicate that in control frogs, G6PDH is in a high phosphate form
and displays a high substrate affinity, whereas in frozen frogs G6PDH is less phosphorylated, with lower substrate affinity. 相似文献
13.
An efficient regeneration protocol for rapid multiplication of Melia azedarach, an economically as well as medicinally important timber-yielding tree, was developed. Nearly 90% of the culture exhibited
axillary bud sprouting and multiple shoot formation from nodal segments derived from 20-year-old candidate plus tree on Murashige
and Skoog (MS) medium supplemented with 5 μM 6-benzyladenine (BA). The highest shoot regeneration frequency (92%), maximum
number of multiple shoots (19.7 ± 0.31) as well as shoot length (4.9 ± 0.08 cm) was induced from nodal explants on MS medium
amended with 5.0 μM BA, 0.5 μM indole-3-acetic acid (IAA) and 30 μM adenine sulfate (AdS). Addition of 250 mg l−1 ammonium sulphate, (NH4)2SO4, and 100 mg l−1 K2SO4, prevented defoliation and tip burning without affecting the number of shoots. The explant harvest period also influenced
the bud break and shoot sprouting from nodal segments. Repeated subculturing of nodal explants on fresh MS medium containing
lower concentration of BA (2.5 μM) along with IAA (0.5 μM), AdS (30 μM) and additives was found most suitable growth regulator
regime for achieving 1.2-fold increase in shoot multiplication rate. The percentage of shoot multiplication as well as the
number of shoots per node remained the same during first three subculture passages, afterwards a decline was recorded. About
90% of the in vitro regenerated shoots were successfully rooted ex vitro by giving a pulse treatment of 250 μM indole-3-butyric
acid for 15 min, followed by their transfer to thermocol cups containing soilrite. The raised plantlets were successfully
acclimatized first under culture room conditions, then to green house with 85% survival rate. 相似文献
14.
Montiel-Herrera M Zaske AM García-Colunga J Martínez-Torres A Miledi R 《Molecules and cells》2011,32(5):397-404
Xenopus laevis oocytes are commonly used to study the biophysical and pharmacological properties of foreign ion channels and receptors,
but little is known about those endogenously expressed in their enveloping layer of follicular cells (FCs). Whole-cell recordings
and the perforated patch-clamp technique in cultured FCs held at −60 mV revealed that ATP (20–250 μM) generates inward currents
of 465 ± 93 pA (mean ± standard error) in ∼60% of the FCs studied, whereas outward currents of 317 ± 100 pA were found in
∼5% of the cells. The net effect of ATP on the FCs was to activate both mono- and biphasic inward currents, with an associated
increase in membrane chloride conductance. Two-microelectrode voltage-clamp recordings of nude oocytes held at −60 mV disclosed
that ATP elicited biphasic inward currents, corresponding to the well-known Fin and Sin-like currents. ATP receptor antagonists like suramin, TNP-ATP, and RB2 did not inhibit any of these responses. On the other
hand, when using wholecell recordings, 1 μM Ang II yielded smooth inward currents of 157 ± 45 pA in ∼16% of the FC held at
−60 mV. The net Ang II response, mediated by the activation of the AT1 receptor, was a chloride current inhibited by 10 nM ZD7155. This study will help to better understand the roles of ATP and
Ang II receptors in the physiology of X. laevis oocytes. 相似文献
15.
Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which
had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids
had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots
of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear.
The K
m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures
was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence
of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by
NADH was linear competitive with respect to acetyl-CoA (K
is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K
is = 56 ± 3.8 μM and K
ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD+ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when
the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment
of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely
to be relieved by NAD+ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle.
Received: 14 July 1999 / Accepted: 20 September 1999 相似文献
16.
Solntseva EI Bukanova JV Marchenko EV Rossokhin AV Skrebitsky VG 《Cellular and molecular neurobiology》2009,29(2):219-224
Earlier, we have shown a strong inhibitory effect of donepezil on K+-current of molluscan neurons (Solntseva et al., Comp Biochem Physiol 144, 319–326, 2007). In the present work, a possible
interaction of donepezil with the external mouth of the channel was examined using, as a tool, tetraethylammonium (TEA), a
classical antagonist of potassium channels. Experiments were conducted in isolated neurons of snail Helix aspersa using the
two-microelectrode voltage-clamp technique. A high-threshold slow-inactivating K+-current involving Ca2+-dependent (I
C) and Ca2+-independent (I
K) components was recorded. The I
C was estimated at 30 mV, and I
K at 100 mV. The IC50 values for blocking effect of donepezil on I
C varied from 5.0 to 8.9 μM in different cells. Corresponding values for I
K varied from 4.9 to 9.9 μM. The IC50 values for blocking effect of TEA on I
C lied in the range of 200 to 910 μM, and on I
K lied in the range of 100 to 990 μM. The comparison of the effects of donepezil and TEA on the same cells revealed significant
correlation between IC50 values of these effects. The value of Spearman coefficient of correlation (r) was 0.77 for I
C (P < 0.05), and 0.82 for I
K (P < 0.05). In the presence of TEA, the effect of donepezil, both on I
C and I
K, appears significantly weaker than in control solution. Dose–response curves of donepezil effect both on I
C and I
K were shifted right along horizontal axis when donepezil was applied in combination with TEA. Results suggest that TEA interferes
with donepezil and precludes the occupation by donepezil of its own site. We suppose that the site for donepezil is situated
near the TEA site with possible overlap. 相似文献
17.
Characterization of a thermostable lipase showing loss of secondary structure at ambient temperature
Sharma PK Singh K Singh R Capalash N Ali A Mohammad O Kaur J 《Molecular biology reports》2012,39(3):2795-2804
A gene encoding extracellular lipase was cloned and characterized from metagenomic DNA extracted from hot spring soil. The
recombinant gene was expressed in E. coli and expressed protein was purified to homogeneity using hydrophobic interactions chromatography. The mature polypeptide consists
of 388 amino acids with apparent molecular weight of 43 kDa. The enzyme displayed maximum activity at 50°C and pH 9.0. It
showed thermal stability up to 40°C without any loss of enzyme activity. Nearly 80% enzyme activity was retained at 50°C even
after incubation for 75 min. However above 50°C the enzyme displayed thermal instability. The half life of the enzyme was
determined to be 5 min at 60°C. Interestingly the CD spectroscopic study carried out in the temperature range of 25–95°C revealed
distortion in solution structure above 35°C. However the intrinsic tryptophan fluorescence spectroscopic study revealed that
even with the loss of secondary structure at 35°C and above the tertiary structure was retained. With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K
m
, V
max
and K
cat
of 0.73 ± 0.18 μM, 239 ± 16 μmol/ml/min and 569 s−1 respectively. Enzyme activity was strongly inhibited by CuCl2, HgCl2 and DEPC but not by PMSF, eserine and SDS. The protein retained significant activity (~70%) with Triton X-100. The enzyme
displayed 100% activity in presence of 30% n-Hexane and acetone. 相似文献
18.
Membrane electroporation (MEP) induces a drastic change in membrane conductance and permeability. However, the underlying
mechanisms by which MEP-induced currents (I
MEP) are generated or resealed remain unclear. In this study, we investigated how the fluctuations of I
MEP might be elicited in different types of cells, including pituitary GH3 cells, NG108-15 neuronal cells, and RAW 264.7 macrophages. We applied the detrended fluctuation analysis (DFA) to analyze
the current signals in response to large hyperpolarizations. The DFA exponents from the current signals at 10 s preceding
the start of the initial I
MEP (I
Pre) in GH3 cells exhibited two components (short time lag [α1] and long time lag [α2]) with a crossover threshold of about 7 ms. The α1 value was 0.46 ± 0.04 (n = 7), whereas the α2 value with 0.62 ± 0.05 (n = 7) indicated the presence of long-term correlations of current signals. However, during maximal I
MEP, the slope of double logarithmic plot was linear and estimated to be 0.99 ± 0.02 (n = 8) with no clear crossover. Upon changes in membrane polarization, neither short- nor long-range correlation was altered.
Chloroquine (CQ), a lysosomotropic agent, decreased the I
MEP amplitude with an IC50 value of 46 μM; however, it had no effects on the scaling exponents of I
Pre or I
MEP. Although CQ or membrane polarization altered the amplitudes of I
MEP, no changes in correlation properties of this current were detected. The scaling exponents derived from I
Pre exhibit long-range correlations in these different types of cells, indicating there is a correlated character of the electropore
dynamics that may be allowed to predict the MEP process. 相似文献
19.
Jin-Ha Jeong Young-Dong Jeon O-Mi Lee Jeong-Do Kim Na-Ri Lee Geun-Tae Park Hong-Joo Son 《Biodegradation》2010,21(6):1029-1040
In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and
plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease
were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO3, 0.1% (w/v) K2HPO4, 0.06% (w/v) KH2PO4 and 0.04% (w/v) MgCl2·6H2O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production
was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml).
After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted
in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was
15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the
concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM)
were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA),
phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium
produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA
by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of
feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil. 相似文献
20.
中国科学 《中国科学:生命科学英文版》2000,43(6):655-662
Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric
acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural
nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA-and muscimol (Mus)-activated currents (Igaba and IMus), respectively in the Mg2+-free external solution containing 1 μmol/L glycine at a holding potential (V
H
) of −40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 γmol/L),
inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of Igaba; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 μmol/L), the inhibitory effect of NMDA on IGAba disappeared. Cd2+ (10 μmol/L) or La3+ (30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of Igaba by NMDA application; (iii) the suppression of IGAba by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated
that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition. 相似文献