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1.
Sandbichler AM Pelster B 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2004,174(8):601-610
Mechanisms of acid release and intracellular pH (pHi) homeostasis were analysed in goldfish (Carassius auratus) gill cells in primary culture. The rate of acid secretion was measured using a cytosensor microphysiometer, and pHi was determined using the fluorescent probe 2,7-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF). Amiloride, a Na+ channel and Na+/H+ exchanger (NHE) inhibitor, had no effect on pHi, but acid secretion of the gill cells was significantly impaired. In the presence of amiloride, the intracellular acidification (achieved using the NH4Cl pulse technique) was more severe than in the absence of amiloride, and recovery from the acidosis was slowed down. Accordingly, acid secretion of gill cells was severely reduced in the absence of extracellular Na+. Under steady-state conditions, 4,4-diisothiocyanatodihydro-stilbene-2,2-disulfonic acid (DIDS), a HCO3–-transport inhibitor, caused a slow acidification of pHi, and acid secretion was significantly reduced. No recovery from intracellular acidification was observed in the presence of DIDS. Bafilomycin A1, an inhibitor of V-ATPase, had no effect on steady-state pHi and recovery from an intracellular acidification, whereas the rate of acid secretion under steady-state conditions was slightly reduced. Immunohistochemistry clearly revealed the presence of the V-ATPase B-subunit in goldfish gill lamellae. Taken together, these results suggest that a Na+-dependent HCO3– transport is the dominant mechanism besides an NHE and V-ATPase to control pHi in goldfish gill cells.Communicated by G. Heldmaier 相似文献
2.
Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H+-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that thein situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl– transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl– uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl– concentration and is inhibited by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparentK
m
for Cl– of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail. 相似文献
3.
Masaaki Satoh Hiroichi Asagami Dongchon Kang Shigeki Minakami Koichiro Takeshige 《Molecular and cellular biochemistry》1995,152(2):159-165
A chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced an acidification of cytosol by about 0.05 pH units in 30 sec followed by an alkalinization in human neutrophils. The quantitative contribution of acid production to the acidification was studied. The superoxide (O2
–) production stimulated by fMLP was not involved in the acidification because the production of acids in neutrophils from patients with chronic granulomatous disease who do not produce O2
–, was the same as that in normal neutrophils. The intracellular acidification was completely inhibited by deoxyglucose, suggesting that energy metabolism enhanced upon stimulation by fMLP might be the main source of the acidification. Although enhancement of the lactate formation by fMLP was 0.8 nmol/106 cells, which could lower intracellular pH by 0.08 pH units, the lactate production could not explain the initial acidification because the production of lactate started at 1 min after the stimulation while the intracellular acidification began immediately after the stimulation. Mitochondrial respiratory inhibitors such as KCN and rotenone had no effects on the fMLP-induced intracellular acidification. The fMLP-induced production of CO2 in 30 sec through the hexose monophosphate shunt was only 2.6 pmol/106 cells, which was calculated to decrease intracellular pH by only 0.0014. Thus, changes of energy metabolism induced by fMLP does not explain the acidification.Abbreviations fMLP
N-formyl-methionyl-leucyl-phenylalanine
- BCECF-AM
2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester
- PMA
phorbol 12-myristate 13-acetate
- CGD
chronic granulomatous disease
- HMP
hexose monophosphate
- pHi
intracellular pH 相似文献
4.
N. C. Jørgensen 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1995,165(6):450-457
Simultaneous net uptake of Na+ and net extrusion of H+, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na+ and H+ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na+ and H+ with an exchange ratio of 1:1 Na+/H+ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO2/HCO
3
-
-buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na+.Abbreviations
DIDS
4,4-diisothiocyanatostilbene-2,2-disulphonate
-
DMSO
dimethylsulphoxide
-
IU
international unit
-
pH
e
extracellular pH
-
pH
i
intracellular pH
-
RBC
red blood cell 相似文献
5.
The transport activities of two primary ATP-dependent organic-anion transporters in the tonoplast of isolated barley (Hordeum vulgare L. cv. Klaxon) vacuoles have been characterised with N-ethylmaleimide glutathione (NEM-SG) and taurocholate as substrates. The transporters showed different sensitivities to organic anions and a variety of transport inhibitors and drugs. The vacuolar uptake of NEM-SG was inhibited by carbonylcyanide 4-trifluoromethoxyphenylhydrazone, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), S-(2,4-dinitrophenyl)glutathione, alkyl-S-glutathione derivatives and taurocholate but stimulated by probenecid. The uptake of taurocholate was inhibited by vinblastine, DIDS and probenecid. Both transporters were unaffected by verapamil. The kinetic properties of the transporters indicate a general preference for amphiphilic anions with some substrate overlap. These characteristics of the transporters are similar to those displayed by the multidrug resistance protein of mammalian drug-resistant cells. We suggest that these vacuolar transporters be described as plant multispecific organic anion transporters (pMOATs).Abbreviations Bm-S
bimane S-glutathione
- DIDS
4,4-diisothiocyanatostilbene-2,2-disulfonic acid
- DNP-SG
S-(2,4-dinitrophenyl)glutathione
- FCCP
carbonylcyanide 4-trifluoromethoxyphenylhydrazone
- LTC4
cysteinyl leukotriene
- MDR
multidrug transporter
- MRP
multidrug resistance protein
- NEM-SG
N-ethylmaleimide glutathione
We thank Prof E. Martinoia for technical advice on the uptake experiments and Prof J. Palmer for helpful discussions and suggestions. M.B.-K. was partially sponsored by a grant from Stichting VSB Fonds, The Netherlands. IACR receives grant-aided support from the Biotechnology and Biological Science Research Council of the United Kingdom 相似文献
6.
Summary (3H)DIDS (4,4-diisothiocyano-2,2-ditritiostilbene-disulfonate) was used as a convalent label for membrane sites involved in anion permeability. The label binds to a small, superficially located population of sites, about 300,000 per cell, resulting in almost complete inhibition of anion exchange. The relationship of biding to inhibition is linear suggesting that binding renders each site nonfunctional. In the inhibitory range less than 1% of the label is associated with lipids but at higher concentrations of DIDS, the fraction may be as high as 4%. In ghosts, however, treatment with (3H)DIDS results in extensive labeling of lipids. In cells, a protein fraction that behavens on SDS acrylamide gels as thought its molecular weight is 95,000 daltons (95K) is predominatly labeled by (3H)DIDS. The only other labeled protein is the major sialoglycoprotein which contains less than, 5% of the total bound (3H)DIDS. Because of the linear relationship of binding to inhibition and the unique architecture of the site, it is suggested that the (3H)DIDS-binding site of the 95K protein is the substrate binding site of the anion transport system. The 95K protein is asymmetrically arranged in the membrane with the sites arranged on the outer face accessible to agent in the medium. In leaky ghost, only a few additional binding sites can be reached from the inside of the membrane in the 95K protein, in contrast to the extensive labeling of other membrane proteins in ghosts as compared to cells.Abbreviations DADS
4,4-Diamino-2,2-dihydrostilbene disulfonic acid
- DIDS
4,4-Diisothiocyano-2,2-stilbene disulfonic acid
- (3H)DADS
4,4-Diamino-2,2-ditritiostilbene disulfonic acid
- (3H)DIDS
4,4-Diisothiocyano-2,2-ditritiostilbene disulfonic acid 相似文献
7.
Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60c-src, with a Ki of 24 M. This peptide inhibitor exhibited selectivity for pp60c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal
l-1-naphthylalanine
- 2-Nal
l-2-naphthylalanine
- BOP
benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate
- BSA
bovine serum albumin
- cAPK
cyclic AMP-dependent protein kinase
- DIEA
diisopropylethylamine
- EGFR
epidermal growth factor receptor
- Fmoc
fluorenylmethoxycarbonyl
- HOBt
1-hydroxybenzotriazole
- MES
2-[N-morpholino]ethanesulfonic acid
- PBS
phosphate-buffered salts
- pCl
l-p-chlorophenylalanine
- pF
l-p-fluorophenylalanine
- PTK
protein tyrosine kinase
- TLC
thin-layer chromatography 相似文献
8.
The rate of CO2- and p-benzoquione-dependent photosynthetic O2 evolution by Anabaena variabilis cells remained unaltered and the rate of O2 uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O2 concentration was increased from 200 to 400 M. Photosystem-I-linked O2 uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O2 concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O2 evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N
3
-
, CN- and NH2OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O2 evolution. The molar ratio of cytochromes b6, f, c-553, aa3 and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea
- DNP-INT
2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether
- Hepes
4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid
- TMPD
N,N,NN-tetramethyl-p-phenylenediamine 相似文献
9.
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
10.
The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C3 plant) and maize (Zea mays L.) (C4 plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [3H2]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, Mr, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [3H2]DIDS. However, when broken chloroplasts were incubated with [3H2]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [3H2]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C3 and C4 chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB
Coomassie brilliant blue
- DIC
differential interference contrast optics
- DIDS
4,4-diisothiocyanatostilbene-2,2-disulfonic acid
- [3H2]DIDS
1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane
- kDa
kilodalton
- Mr
relative molecular mass
- PGA
3-phosphoglycerate
- Pitranslocator
phosphate translocator
- SDS
sodium dodecyl sulfate 相似文献
11.
Summary Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rates of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of35SO4 efflux on the intracellular SO4 concentration, the pH dependence and the inhibition of35SO4 efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of36Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higherV
max andK
1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H2DIDS, where DIDS is 4,4-diisothiocyano-2,2 stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H2DIDS. 相似文献
12.
Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton. 相似文献
13.
D. Siebers C. Lucu K. Böttcher K. Jürss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1994,164(1):16-22
Isolated posterior gills (no. 7) of shore crabsCarcinus maenas acclimated to brackish water of a salinity of 10 S were bathed and perfused with 50% sea water (200 mmol·l-1 Na+), and the internal perfusate collected during subsequent periods of 5 min. During a single passage through the gill the pH of the perfusion medium decreased from ca. 8.1 to ca. 7.7, a result implying that the gill possesses structures which recognize unphysiologically high pH values in the haemolymph and regulates them down to physiological values of ca. 7.7. The calculated apparent proton fluxes from the epithelial cells into the haemolymph space amounted to 17.9 mol·g fw-1·h-1, a value of only 3.8% of net Na+ fluxes observed under comparable conditions. When 0.1 mmol·l-1 KCN, an inhibitor of mitochondrial cytochrome oxidase, or 5 mmol·l-1 ouabain, a specific inhibitor of Na+/K+-ATPase were applied in the internal perfusate, down-regulation of pH was no longer observed and the gill was completely depolarized, i.e. transepithelial potential differences dropped from-7.8 to 0 mV (haemolymph space negative to bath). Regulation of pH was completely inhibited by antagonists of carbonic anhydrase (0.1 mmol·l-1 acetazolamide or 0.01 mmol·l-1 ethoxyzolamide) applied in the perfusate. Inhibitors of Na+/H+ exchange, 0.1 mmol·l-1 amiloride applied in the external bathing medium or in the internal perfusate, and symmetrical 0.01 mmol·l-1 5-(N-ethyl-N-isopropyl)amiloride, as well as inhibitors of Cl-/HCO3
- exchange and Na+/HCO3
- cotransport, 0.5 mmol·l-1 4,4-diisothiocyanatostilbene-2,2-disulphonate or 0.3 mmol·l-1 4-acetamido-4-isothiocyanatostilbene 2,2-disulphonate applied on both sides of the gill, and inhibitors of H+-ATPase, 0.05 mmol·l-1
N-ethylmaleimide and 0.1 mmol·l-1
N,N-dicyclohexylcarbodiimide —applied on both sides of the gill — did not alter the acidification of the perfusate observed in controls. Using artificial salines buffered to pH 8.1 with 0.75 mmol·l-1 tris (hydroxymethyl) aminomethane instead of 2 mmol·l-1 HCO3
-, apparent proton fluxes were reduced to 11% of controls, a result suggesting that pH regulation by crab gills needs the presence of HCO3
-. The findings obtained suggest that pH regulation by crab gills depends on the oxidative metabolism of the intact branchial epithelium and that carbonic anhydrase plays a central role in this process. Na+/H+ exchange, anion exchange or cotransport and active proton secretion seem not to be involved. While unimpaired active ion uptake is a prerequisite for pH regulation, ion transport itself is independent of it.Abbreviations acetazolamide
(N-[sulphamoyl-1, 3, 4-thiadiazol-2-yl]-acetamide)
- amiloride
3,5-diamino-6-chloropyrazinoyl-guanidine
- CA
carbonic anhydrase
- DBI
dextrane-bound inhibitor thiadiazolesulphonamide
- DCCD N
N dicyclohexylcarbodiimide
- DIDS
4,4-diisothiocyanato-stilbene-2,2-disulphonate
- EIPA
5-(N-ethyl-N-isopropyl) amiloride
- ethoxyzolamide
6-ethoxy-2-benzothiazole-sulphonamide
- fw
fresh weight
-
J
H
+
apparent proton flux
- NEM
N-ethylmaleimide
- PD
transepithelial potential difference
- PEG-STZ
polyethylene-glycol-thiadiazolesulphonamide
- STTS
4-acetamido-4-isothiocyanatostibene 2,2-disulphonate
- SW
sea water
- TRIS
tris(hydroxymethyl)aminomethane 相似文献
14.
Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii 总被引:3,自引:0,他引:3
APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg2+ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V
max of 360 pkat under optimal reaction conditions declining to v
limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K
m APS: 2 · 10-6 mol · l-1, and K
m ATP: 7 · 10-6 mol · l-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS
Adenosine 5-phosphosulphate
- dATP
2-deoxyadenosine 5-triphosphate
- p-CMB
p-chloromercuribenzoate
- DTE
dithioerythritol
- DTT
dithiothreitol
- -MSH
-mercaptoethanol
- PAPS
3-phosphoadenosine 5-phosphosulphate
- PAP
3-phosphoadenosine 5-phosphate
- SDS
sodium dodecyl sulphate
This work is part of a dissertation submitted by H. G. J., Bochum 1982 相似文献
15.
Summary Recently we proposed that cytoplasmic acidification of low K+ (LK) sheep erythrocytes may stimulate ouabain-resistant Cl–-dependent K+ flux (K+Cl– cotransport), also known to be activated by cell swelling, treatment with N-ethylmaleimide (NEM), or removal of cellular bivalent cations. Here we studied the dependence of K+ transport on intracellular and extracellular pH (pH
i
, pH
o
) varied either simultaneously or independently using the Cl–/HCO
3
–
exchange inhibitor 4,4, diisothiocyanatostilbene-3,2-disulfonic acid (DIDS). In both control and NEM-treated LK cells volumes were kept near normal by varying extracellular sucrose. Using DIDS as an effective pH clamp, both K+ efflux and influx of Rb+ used as K+ congener were strongly activated at acid pH
i
and alkaline pH
o
. A small stimulation of K+ (Rb+) flux was also seen at acid pH
i
in the absence of DIDS, i.e., when pH
i
pH
o
. Anti-L
l
serum, known to inhibit K+Cl– cotransport, prevented the pH
i
-stimulated K+ (Rb+) fluxes. Subsequent to NEM treatment at pH 6, K+ (Rb+) fluxes were activated only by raising pH, and thus were similar to the pH activation profile of K+ (Rb+) fluxes in DIDS-treated cells with pH
o
varied at constant physiologic pH
i
. Anti-L
l
, which inhibited NEM-stimulated K+ (Rb+) fluxes, failed to do so in NEM-plus DIDS-treated cells. Thus, NEM treatment interferes with the internal but not with the external pH-sensitive site. 相似文献
16.
S. Biagionia G. Scarsella L. Settimi M. E. Traina 《Molecular and cellular biochemistry》1982,43(3):183-190
Summary Inhibition of growth of PY815 mouse mastocytoma cells in vitro by N6, O2-dibutyryladenosine 3,5 cyclic monophosphate (DB cyclic AMP) was accompanied by increases in intracellular cyclic AMP and histamine and minor changes in cytosolic cyclic AMP-dependent histone kinase activity. However, DEAE-cellulose chromatography revealed substantial changes in the relative proportions of the principal cyclic AMP-dependent protein kinases and in free cyclic AMP-binding protein after DB cyclic AMP treatment. The activity of cytosolic cyclic AMP-dependent protein kinase type I (PKI) decreased relative to cyclic AMP-dependent protein kinase type II (PKII) and there was an increase in a cytosol cyclic AMP-binding protein with little associated protein kinase activity. The relative changes in activity of PKI, PKII and cyclic AMP binding protein after DB cyclic AMP treatment may reflect events important in the regulation of growth and differentiation of mast cells.Abbreviations DB cyclic AMP
N6,O2-dibutyryladenosine-3, 5-cyclic monophosphate
- cyclic AMP
adenosine 3,5-cyclic monophosphate
- PKI
type I cyclic AMP-dependent protein kinase
- PKII
type II cyclic AMP-dependent protein kinase 相似文献
17.
Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [14C]5FU, but no carrier. The [14C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [3H]uracil, and [3H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway. 相似文献
18.
Saul Ship Yehezkel Shami William Breuer Aser Rothstein 《The Journal of membrane biology》1977,33(1):311-323
Summary The potent and specific inhibitor of anion permeability, 4,4-diisothicyanostilbene-2,2-disulfonic acid (DIDS) was synthesized in tritiated form ([3H]DIDS) from tritiated 5-nitrotoluene-o-sulfonic acid. Its reactions with and effects on red blood cells were compared with those of a reduced form ([3H]H2DIDS), previously used as a tracer for DIDS. The rate of covalent reaction of [3H]DIDS was substantially faster than that of [3H]H2DIDS at all temperatures tested. With both agents, the rate of reaction was increased in alkaline media, although the response occurred at a lower pH with [3H]DIDS. On the other hand, the relationship of irreversible membrane binding to the degree of inhibition of sulfate fluxes was linear and virtually the same for both agents, with 100% inhibition associated with the binding of approximately 1.2×106 molecules per cell. About 90% of the binding for each probe was to a particular membrane protein, known as band 3, equivalent to about 1 mole of agent per mole of protein. 相似文献
19.
The intracellular concentration of cAMP in the green alga Chlorella fusca was in the range of 2 · 10-9 to 10-8 moles/g dry weight and was strongly dependent on the growth conditions. The cAMP level was high with high light intensity, low nitrate or glucose concentration. Intracellular cAMP increased only by factor of 2 when high amounts (up to 10-3 M) of cAMP were added to the medium. Most of the given cAMP was converted to 5-AMP.Addition of cAMP had little effect on the chlorophyll content of the cells, only at 10-6 M some enhancement in photoautotrophic cultures was observed. On the other hand high amounts of cAMP in the medium increased the growth rate. DBcAMP* showed a positive effect on chlorophyll synthesis and growth rate at much lower concentrations compared to cAMP.Stimulation effects of exogenous cAMP on the synthesis of chlorophyll were also observed in mixotrophic cultures with a high glucose/nitrate ratio, conditions where chlorophyll synthesis is repressed. Similar to autotrophic conditions DBcAMP was more effective than cAMP.These data indicate that cAMP may act in a system controlling the chlorophyll content of the cells in response to nutrients or light.Abbreviation DBcAMP*
N6-2-O-dibutyryl-adenosine-35-monophosphate 相似文献
20.
Tadako Murayama Isao Uno Kimihiko Hamamoto Tatsuo Ishikawa 《Archives of microbiology》1985,142(2):109-112
A cpk mutant of Neurospora crassa with morphological alteration was obtained spontaneously during the cross between the wild-type and a glycerol utilizing cr-l strain. The growth rate of cpk was intermediate between the wild-type and cr-1 mutant strains. The cpk conidia contained a reduced level of carotenoid pigments as compared to the wild-type conidia. The cpk mutant had no detectable amount of cyclic adenosine 3,5-monophosphate (cAMP)-binding protein at all stages of growth tested. On a DEAE-Sephacel column chromatogram, protein kinase activity of the wild type was eluted at two peaks; the first peak was cAMP-dependent, and the second one was not. In contrast, the cpk strain had two peaks of cAMP-independent enzymes. It is suggested that cAMP-dependent protein kinase may be altered in the cpk mutant into a cAMP-independent type by an alteration of the regulatory subunit of this enzyme.Abbreviations cAMP
Cyclic adenosine 3,5-monophosphate
- 8-N3-[3H] cAMP
8-azido-[3H]cyclic adenosine 3,5-monophosphate 相似文献