共查询到20条相似文献,搜索用时 15 毫秒
1.
31P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external Pi. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal Pi peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external Pi and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal Pi increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons. 相似文献
2.
E.A. Shoubridge F.M.H. Jeffry J.M. Keogh G.K. Radda A.-M.L. Seymour 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,847(1):25-32
Rats were fed a diet containing 1% of the creatine substrate analogue β-guanidinopropionic acid for 6–10 weeks. 31P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 μmol/g dry wt in guanidinopropionic acid-fed animals but no change in [Pi], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine → ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 μmol/g dry wt per s in control hearts and decreased 4-fold to 2.5–2.8 μmol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2–7.8 μmol/g dry per s. These rates are 1.5–3-times greater than the rate of the phosphocreatine → ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart. 相似文献
3.
Data from isotopic uptake experiments were used to measure the kinetics of labelling of cellular phosphate, ATP and ADP in the Ehrlich ascites tumor cell. The results show that steady state phosphate exchange flux was 0.333 ± 0.052 (S.E.) μmoles per 107 cells per hour at 37°, and that the specific activity of phosphate was the same as Pγ ATP. Metabolic inhibition reduced the phosphate flux by 30–50%. A model, based on oxidative phosphorylation and the adenylate kinase reaction is used to interpret the labelling sequence of Pβ ATP and Pβ ADP, and its dependence on Pγ ATP. 相似文献
4.
Hyun Dju Kim Martha Baird John Sallis Stewart Nicol Russell E. Isaacks 《Biochemical and biophysical research communications》1984,119(3):1161-1167
The erythrocytes of the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus), which are practically devoid of intracellular ATP content (1), were examined for active Rb86 influx and for the presence of Na+K+Mg ATPase. We found that intact erythrocytes of both species possess the ability to actively transport cations. Ouabain sensitive Rb86 influx in the echidna was approximately 0.17 μmoles/ml cells × hr, whereas the platypus exhibited a higher value of 0.43 μmoles/ml cells × hr. Surprisingly, ouabain sensitive Na+K+Mg ATPase activity of isolated membranes was high amounting to some 15 to 25 fold higher than the human erythrocyte counterpart determined under identical conditions. These findings suggest that a trace amount of ATP is sufficient to maintain active cation transport across the monotreme cell membranes. 相似文献
5.
Nuclear magnetic resonance (NMR) is a non-invasive technique which allows us to examine the biochemical, physiological and metabolic events occurring inside living tissue; such as vascular and other smooth muscles.It has been found that the smooth muscle metabolism is compartmented such that mitochondrial function fuels contraction and that much glycolytic ATP production is used for membrane pumps. Using NMR we have been able to observe the ATP and phosphocreatine (PCr) concentrations and estimate the ADP concentration, as well as flux through the creatine kinase (CK) system. It has also been found that the smooth muscle metabolism is able to maintain ATP concentration in the absence of mitochondrial function (cyanide inhibition). Therefore, the vessels are able to adapt to metabolic demands as necessary.NMR is versatile in the information it can provide because it has also yielded important contributions with regard to the intracellular pH and ionic status. For example, the intracellular free Mg2+ ([Mg2+]i) can be measured with NMR simultaneously with ATP concentrations and NMR has shown us that the [Mg2+]i is highly protected in the muscle (within confined range), but also responds to the environment around it.In this review we conclude that NMR measurements of smooth muscle research is a useful technique for assessing chronic and acute changes that occur in the tissue and during diseases. 相似文献
6.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1987,928(1):36-44
31P-NMR spectra of bullfrog stomach smooth muscle showed peaks for creatine phosphate (4.8 μmol·g−1 wet wt.), ATP (3.6), inorganic phosphate (Pi, 2.4), phosphomonoesters (3.0) and phosphodiesters (3.3). The intracellular pH was 7.3, and calculated from the chemical shift of Pi. 1H-NMR spectra of smooth muscle yielded peaks of 2.9 for lactate, 6.6 for total creatine (creatine phosphate + creatine) and methyl protons of choline tentatively assigned to glycerolphosphorylcholine or to membrane phospholipids. Creatine phosphate and ATP decreased under anaerobic conditions, and intracellular acidification was observed with the concomitant increase in lactate. 31P saturation transfer studies showed that saturation of the γ-ATP resonance reduced the intensity of creatine phosphate to 60% of its control value, and the measured T1 value of creatine phosphate was 2.4 s with saturation. The calculated forward flux of the creatine kinase reaction (decomposition direction of creatine phosphate) was 0.77 μmol·g−1 wet wt.·s−1. The creatine kinase flux was approx. 100-times larger than the ATP turnover rate, calculated from the oxygen consumption rate with the assumption, P/O = 3. In conclusion, the creatine kinase reaction is at equilibrium in resting smooth muscle of bullfrog stomach. 相似文献
7.
The incorporation of 14C-tyrosine into S-RNA catalyzed by a partially purified tyrosine activating enzyme from baker’s yeast was observed. The maximum incorporation was shown in the presence of 5 μmoles of ATP, 10 μmoles of MgCl2 and 10~100 μmoles of KCl in the reaction mixture of total volume of 1ml, at pH 7.8 when 1.2 mg of S-RNA, 0.1 μmole of 14C-tyrosine and 400 μg of enzyme protein were used. Beyond the concentration of ATP, MgCl2 and KCl described above, the tendency of inhibition was observed. The incorporation was strongly inhibited by pCMB and reactivated by cysteine. Manganese and calcium ions were effective as substitutes for magnesium. S-RNA used was prepared from whole baker’s yeast cell with phenol, but S-RNA obtained from the supernatant of the ground yeast had lost its incorporating activity. 相似文献
8.
V.V. Kupriyanov A.Ya. Steinschneider E.K. Ruuge V.N. Smirnov V.A. Saks 《Biochemical and biophysical research communications》1983,114(3):1117-1125
It has been found in experiments with high resolution 31P-NMR spectroscopy (200 MHz) that the phosphocreatine peak is splitted into two different peaks in the mixtures of H2O and D2O and is single but with different chemical shifts in pure H2O and D2O. This phenomenon is explained by substitution of protons of guanidino group in phosphocreatine by deuterium. The effect of splitting disappeared at extreme pH values (>8.5 or <4.0) and at temperatures higher than 45°C due to accelerated proton-deuterium exchange. Creatine kinase added to phosphocreatine solution also lowered its temperature of peaks' collapse by 5°–10°C. A saturation (spin) transfer method was used to show that the phosphoryl group transfer to ADP in creatine kinase active center is slower with deuterium-substituted phosphocreatine than with H-phosphocreatine. The data are taken to show the importance of the proton transfer step in the creatine kinase reaction mechanism and acceleration of phosphocreatine proton-deuterium exchange by creatine kinase. 相似文献
9.
A. Atkinson A.D. Gatenby A.G. Lowe 《Biochimica et Biophysica Acta (BBA)/General Subjects》1973,320(1):195-204
A simple and rapid method is described for determining Pi by spectrophotometric measurement of a soluble complex of phosphomolybdic acid and Cirrasol ALN-WF, a non-ionic detergent formerly known as Lubrol W. The measured complex has a molar extinction coefficient of 4.59 · 103 at 390 nm and little interference is found with relatively high concentrations of chelating agents, salts, and other compounds which interfere with most other Pi assays. Linearity is observed in the range 0–1.2 μmoles Pi and developed assay samples are stable for 8 h at 20 °C or 24 h at 4 °C. The method is suitable for use in the presence of moderate concentrations of protein or ATP.After suitable modification the assay can be used at pH 4.0. Sensitivity is reduced at this pH (εM, 390nm = 2.79 · 103) but linearity is maintained up to 1 μmole Pi and the coloured complex is stable for 4 h at 20 °C. The pH-4 procedure is suitable for measurement of Pi in the presence of very labile phosphate esters such as creatine phosphate.The phosphomolybdic acid-Cirrasol complex can be reduced at ambient temperature in both the above systems. A blue complex results with εM, 820nm of 9.9 · 103 at pH 4.0, and 1.8 · 104 under more acidic conditions. 相似文献
10.
艾氏腹水癌细胞和肉瘤S-180细胞是抗肿瘤药物筛选常用细胞株.实验采用取自小鼠腹腔的第7~8天的艾氏腹水癌和S-180细胞,用31P磁共振谱测得了细胞内小分子含磷代谢成分;计算了细胞内pH值;还用31P谱探讨了作用机制不同的三种抗代谢物:碘乙酸、2,4-二硝基苯酚及棉酚对艾氏腹水癌细胞代谢的影响 相似文献
11.
D. C. Jackson S. J. Warburton E. Arendt Meinertz R. G. Lawler J. S. Wasser 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1995,165(1):77-84
Selected tissues (skeletal muscle, heart ventrical, and liver), sampled from turtles (Chrysemys picta bellii) at 3°C either under normoxic conditions or after 12 weeks of anoxic submergence were quantiaatively analysed for intracellular
pH and phosphorus metabolites using 31P-NMR. Plasma was tested for osmolality and for the concentrations of lactate, calcium, and magnesium to confirm anoxic stress.
We hypothesized that, in the anoxic animals, tissue ATP levels would be maintained and that the increased osmolality of the
body fluids of anoxic turtles would be accounted for by a corresponding increase in the concentrations of phosphodiesters.
The responses observed differed among the three tissues. In muscle, ATP was unchanged by anoxia but phosphocreatine was reduced
by 80%; in heart, both ATP and phosphocreatine fell by 35–40%. The reduction in phosphocreatine in heart tissue at 3°C was
similar to that observed in isolated, perfused working hearts from turtles maintained at 20°C but no decrease in ATP occurred
in the latter tissues. In liver, although analyses of several specimens were confounded by line-broadening, neither ATP nor
phosphocreatine was detectable in anoxic samples. Phosphosdiesters were detected in amounts sufficient to account for 30%
of normoxic cell osmotic concentration in heart and 11% and 12% in liver and muscle, respectively. The phosphodiester levels
did not change in anoxia. Heart ventricular phosphodiester levels in turtles at 3°C were significantly higher than those determined
for whole hearts from turtles at 20°C. 1H, 13C and 31P NMR analyses of perchloric acid extracts of heart and skeletal muscle from 20°C turtles con firmed that the major phosphodiester
observed by NMR in these tissues is serine ethanolamine phosphate. We conclude that the three types of tissues studied differ
substantially in their ability to maintain levels of ATP during anoxia, and that liver may continue to function despite NMR-undetectable
levels of this metabolite. In addition, we conclude that phosphodiesters do not serve as regulated osmolytes during anoxia,
and that the functional significance of their high concentrations in turtle tissues remains uncertain. 相似文献
12.
The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of ampicillin and streptomycin under similar conditions. In the presence of nisin (3–12 μg/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35°C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3–12 μg/ml) and streptomycin (15–60 μg/ml) during the first hour. However, concentrations of ≥3 μg/ml ampicillin and ≥30 μg/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin. 相似文献
13.
P.M. Matthews J.L. Bland D.G. Gadian G.K. Radda 《Biochemical and biophysical research communications》1981,103(3):1052-1059
The steady-state rate of ATP synthesis in the isolated, Langendorff-perfused rat heart was determined using a 31P NMR saturation transfer method. At 37°C and a perfusion pressure of 70 cm H2O the value is 2.8 ± 0.3 (n=5 ± S.E.M.) μmol.s?1. (g. dry wt.)?1. The activity of creatine phosphokinase measured in the same experiments was 14.6 ± 1.0 μ mol.s?1 .(g. dry wt.)?1. From the rate of ATP synthesis and the separately measured oxygen consumption we calculated an apparent mitochondrial ADP:O ratio of 3.5 ± 0.8 in the intact tissue. 相似文献
14.
Javier García-Sancho Mayte Montero Javier Alvarez Rosalba I. Fonteriz Ana Sanchez 《Bioelectromagnetics》1994,15(6):579-588
We have investigated the effects of sinusoidal electromagnetic fields (EMF) on ion transport (Ca2+, Na+, K+, and H+) in several cell types (red blood cells, thymocytes, Ehrlich ascites tumor cells, and HL60 and U937 human leukemia cells). The effects on the uptake of radioactive tracers as well as on the cytosolic Ca2+ concentration ([Ca2+]i), the intracellular pH (pHi), and the transmembrane potentsial (TMP) were studied. Exposure to EMF at 50 Hz and 100–2000 μT (rms) had no significant effects on any of these parameters. Exposure to EMF of 20–1200 μT (rms) at the estimated cyclotron magnetic resonance frequencies for the respective ions had no significant effects except for a 12–32% increase of the uptake of 42K within a window at 14.5–15.5 Hz and 100–200 μT (rms), which was found in U937 and Ehrlich cells but not in the other cell types. © 1994 Wiley-Liss, Inc. 相似文献
15.
T. Hartmund H. Gesser 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1991,160(6):691-697
Summary Twitch force and resting tension of electrically stimulated ventricular strips of rainbow trout were compared with tissue contents of phosphocreatine, creatine, and ATP. The phosphocreatine/total creatine ratio, which was used to assess the cytoplasmic phosphorylation potential, fell with the fraction of cell respiration that was inhibited by sodium cyanide and N2. Concomitantly, twitch force decreased while resting tension tended to increase. This relation between phosphocreatine/total creatine and mechanical parameters became more prominent as glycolysis was increasingly inhibited by sodium iodoacetate. Furthermore, glycolytic inhibition was followed by a decrease in the ATP/phosphocreatine ratio. The latter effect was the same in 1% and 6% CO2. Thus, it cannot be ascribed to an action of intracellular pH on the creatine kinase catalyzed reaction. Notably, resting tension as well as twitch force relative to ATP was augmented by glycolytic inhibition. The main conclusions are that in the presence of a decreased mitochondrial activity, glycolysis protects contractility not only by counteracting a lowering in high energy phosphates but also by supporting the ATP/phosphocreatine ratio. Apparently, the creatine kinase activity is insufficient to maintain ATP in equilibrium with phosphocreatine. In addition, glycolysis seems to elevate the level of free phosphate relative to ATP, so that twitch force development as well as rigor complex formation is counteracted. 相似文献
16.
Kichitaro Kawaguchi Koichi Ogata Tatsurokuro Tochikura 《Bioscience, biotechnology, and biochemistry》2013,77(6):908-918
Effects of various factors including incubation time, water content of airdried cells, concentration and pH of KH2PO4–K2HPO4 mixture, d-glucose concentration, MgSO4 concentration, GMP concentration, cell concentration, aeration and various kinds of carbohydrates on the fermentative production of GDP-mannose, GDP and GTP from 5′-GMP by air-dried cells of baker’s yeast were investigated. The water content of air-dried cells was the most important factor in the fermentation. When the air-dried cells of baker’s yeast (100 mg/ml) were incubated with 5′-GMP (20 μmoles/ml), d-glucose (800 μmoles/ml), potassium phosphate buffer (360 μmoles/ml, pH 7.0), and MgSO4 (20 μmoles/ml), 2-hr incubation gave GDP in 20% yield and GTP in 61.1% yield, GDP-mannose being produced in 45% yield after 8-hr incubation. The phosphorylation of 5′-AMP, 5′-dAMP, 5′-dGMP 5′-CMP and 5′-UMP was also observed in high yields under the same conditions. 相似文献
17.
Brian J. Dewar Kayvan Keshari Rex Jeffries Petras Dzeja Lee M. Graves Jeffrey M. Macdonald 《Metabolomics : Official journal of the Metabolomic Society》2010,6(3):439-450
The goal of this study was to examine metabolic differences between a novel chronic myelogenous leukemic (CML) cell line,
MyL, and a sub-clone, MyL-R, which displays enhanced resistance to the targeted Bcr-Abl tyrosine kinase inhibitor imatinib.
1H nuclear magnetic resonance (NMR) spectroscopy was carried out on cell extracts and conditioned media from each cell type.
Both principal component analysis (PCA) and specific metabolite identification and quantification were used to examine metabolic
differences between the cell types. MyL cells showed enhanced glucose removal from the media compared to MyL-R cells with
significant differences in production rates of the glycolytic end-products, lactate and alanine. Interestingly, the total
intracellular creatine pool (creatine + phosphocreatine) was significantly elevated in MyL-R compared to MyL cells. We further
demonstrated that the MyL-R cells converted the creatine to phosphocreatine using non-invasive monitoring of perfused alginate-encapsulated
MyL-R and MyL cells by in vivo 31P NMR spectroscopy and subsequent HPLC analysis of extracts. Our data demonstrated a clear difference in the metabolite profiles
of drug-resistant and sensitive cells, with the biggest difference being an elevation of creatine metabolites in the imatinib-resistant
MyL-R cells. 相似文献
18.
Marie-Christine Broillet Jean-Daniel Horisberger 《The Journal of membrane biology》1993,134(3):181-188
K+ channels sensitive to intracellular ATP (KATP channels) have been described in a number of cell types and are selectively inhibited by sulfonylurea drugs. To look for the presence of this type of K+ channel in the basolateral membrane of tight epithelia, we have used an amphibian renal cell line, the A6 cells, grown on filters. After the selective permeabilization of the apical membrane with amphotericin B, the basolateral conductance was studied under voltage-clamp conditions. Tolbutamide inhibited 65.8 ± 6.3% of the barium-sensitive current. The tolbutamide-sensitive conductance had an equilibrium potential of ?83 ± 1 mV and was inward rectifying in spite of the outwardly directed K+ gradient. Similar results were obtained with glibenclamide. The half-inhibition constants were 25.7 ± 3.0 μm and 0.114 ± 0.018 μm for tolbutamide and glibenclamide respectively. To study the relation between cellular ATP and the activity of this conductance, A6 cells were treated with glucose (5 mm) and insulin (250 μU/ml). This maneuver significantly increased the cellular ATP and abolished the tolbutamide-sensitive conductance. A sulfonylurea-sensitive K+ conductance is present and active in the basolateral membrane of A6 cells. This conductance appears to be modulated by physiological changes of intracellular ATP. 相似文献
19.
《Chemico-biological interactions》1987,63(3):215-225
Extracts of water blooms of the toxic cyanobacterium Microcystis aeruginosa showed a range of toxicities not related to their ability to lyse mammalian red cells. The HPLC-purified heptapeptide toxin (mol. wt. 1035) from Microcystis did not lyse red cells at up to 500-fold higher concentrations than that required to kill mice. This toxin (LD50 110 μg/kg for male mice) was used to investigate in vitro effects on isolated thymocytes, hepatocytes, mammary alveolar cells, and cultured Swiss 3T3 fibroblasts. Thymocytes were stimulated to progressive Ca2+ entry by toxin (0.1–10 μg/ml), reaching a peak after approx. 5 min. No deformation, intracellular pH change, Trypan Blue entry or cell lysis was seen within 60 min at 37°C. Hepatocytes were grossly deformed by the toxin, with a dose/response relationship between 0.1 and 1.0 μg/ml. No progressive Ca2+ entry was observed on toxin addition, instead a rapid rise in intracellular Ca2+, presumably from intracellular sources. No change in intracellular pH, Trypan Blue exclusion or cell lysis was observed over 60 min. Mammary alveolar cells and 3T3 fibroblasts were unresponsive to toxin at the concentrations tested. No change in protein synthesis or nucleic acid synthesis in thymocytes was observed after culture with 0.5 or 5.0 μg/ml toxin. It was concluded that cytoskeletal changes in deformed hepatocytes (the target cells in vivo) demonstrated the most probable cellular basis for toxicity, rather than changes in membrane permeability or cell metabolism. 相似文献
20.
Abstract: The role of voltage-sensitive Ca2+ channels in mediating Ca2+ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca2+ channels. Concurrent 31P/19F and 23Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca2+] ([Ca2+]i), and intracellular Na+ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na+ content; however, a substantial increase in [Ca2+]i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca2+]i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca2+]i seen during ischemia. These data demonstrate that voltage-gated Ca2+ channels do not mediate significant Ca2+ flux during ischemia in this cell line and suggest an important role for Ca2+i stores, the Na+/Ca2+ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca2+ level during ischemia. 相似文献