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1.
The oxygen dependence of cellular energy metabolism. 总被引:14,自引:0,他引:14
D F Wilson M Erecińska C Drown I A Silver 《Archives of biochemistry and biophysics》1979,195(2):485-493
Suspensions of cultured C 1300 neuroblastoma cells, sarcoma 180 ascites tumor cells, and Tetrahymena pyriformis cells were used to study the oxygen dependence of cellular energy metabolism. Cellular respiration was found to be almost independent of oxygen tension to values of less than 20 μm with an apparent Km for oxygen of less than 1 μm. In contrast, the reduction of mitochondrial cytochrome c was found to be dependent on oxygen tension at all values from 240 μm downward. Oxygen dependence was also observed in terms of cellular energy metabolism expressed as adenosine triphosphate and adenosine diphosphate concentrations. These data provide direct evidence that in intact cells mitochondrial oxidative phosphorylation is oxygen dependent throughout the physiological range of oxygen tension (air saturation and below). The respiratory rate is maintained constant when the oxygen tension is lowered by decreasing values of the cytosolic and intramitochondrial because these regulatory parameters adjust to maintain a constant rate of ATP synthesis. The lack of oxygen dependence in the respiratory rate means that the rate of cellular ATP utilization is essentially oxygen independent until the mitochondria can no longer synthesize ATP at the required rate and . 相似文献
2.
The reduction-oxidation reactions of NADH and cytochrome to incipient oxygen insufficiency caused by nitrogen ventilation or hemorrhagic hypotension were examined in the exposed cerebral cortex of the cat. A comparison of the onset of redox changes with each procedure shows that cytochrome reduction precedes the reduction of mitochondrial NAD. This constitutes evidence that, in the living brain, NADH maintains its resting oxidation state at lower cellular oxygen tensions than cytochrome does, consistent with the differences in oxygen affinity these respiratory chain components exhibit during oxygen titration . 相似文献
3.
Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K+ concentration () which greatly exceeds that of the growth medium, and a low Na+ concentration (). Unlike varies with cell aging.The K+ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K+ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, decreases and is partially compensated by a gain in Na+. This substitution completely reverses when metabolic substrate is added (K+ reaccumulation process). Kinetic analysis of K+ movement in cells with steady K+ level shows that most of K+ influx is mediated by an autologous K+-K+ exchange mechanism. On the other hand, during K+ reaccumulation by K+-depleted cells, a different mechanism (a K+ uptake mechanism) with higher transport capacity and affinity drives the net K+ influx. Both mechanisms are energy-dependent.Ouabain and anoxia have no effect on K+ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg2+-dependent ATPase activity. 相似文献
4.
Quercetin inhibited a dog kidney ( preparation without affecting for ATP or for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the ( activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the for ATP and the for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on , for substrate, and for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations. 相似文献
5.
The transport of sucrose by selected mutant and wild-type cells of Streptococcus mutans was studied using washed cocci harvested at appropriate phases of growth, incubated in the presence of fluoride and appropriately labelled substrates. The rapid sucrose uptake observed cannot be ascribed to possible extracellular formation of hexoses from sucrose and their subsequent transport, formation of intracellular glycogen-like polysaccharide, or binding of sucrose or extracellular glucans to the cocci. Rather, there are at least three discrete transport systems for sucrose, two of which are phosphotransferases with relatively low apparent values and the other a non-phosphotransferase (non-PTS) third transport system (termed TTS) with a relatively high apparent . For strain 6715-13 mutant 33, the values are 6.25·10?5 M, 2.4·10?4 M, and 3.0·10?3 M, respectively; for strain NCTC-10449, the values are 7.1·10?5 M, 2.5·10?4 M and 3.3·10?3 M, respectively. The two lower systems could not be demonstrated in mid-log phase glucose-adapted cocci, a condition known to repress sucrose-specific phosphotransferase activity, but under these conditions the highest system persists. Also, a mutant devoid of sucrose-specific phosphotransferase activity fails to evidence the two high affinity (low apparent ) systems, but still has the lowest affinity (highest ) system. There was essentially no uptake at 4°C indicating these processes are energy dependent. The third transport system, whose nature is unknown, appears to function under conditions of sucrose abundance and rapid growth which are known to repress sucrose-specific phosphotransferase activity in S. mutans. These multiple transport systems seem well-adapted to S. mutans which is faced with fluctuating supplies of sucrose in its natural habitat on the surfaces of teeth. 相似文献
6.
G.E. Breitwieser 《生物化学与生物物理学报:生物膜》1982,689(3):457-463
(1) A membrane fraction enriched in (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an specific activity of approx. 2 units/mg and was ouabain-sensitive. (2) The had a for ATP of and a pH optimum of 7.0. It was inhibited by ouabain with a of . (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with . (4) The Mg2+ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the for Mg2+ was , and at 6 mM ATP, the was . High levels of Mg2+ caused inhibition of hydrolysis. (5) The interactions of Na+ and K+ were examined over a range of conditions. K+ levels caused modulations in the Na+ dependence in the range of 1–150 mM. (6) The prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves. 相似文献
7.
Chana Vinkler 《Biochemical and biophysical research communications》1981,99(4):1095-1100
The of ADP for photophosphorylation in lettuce chloroplasts was measured both at various light intensities and in the presence of various uncoupler (nigericin + K+) concentrations. Lowering the light intensity results in both, a decrease in the rate of phosphorylation and a several fold in the of ADP for the reaction. However, when increasing concentrations of the uncoupler nigericin + K+ are employed, the rate of photophosphorylation is decreased but a several-fold in the of ADP for the reaction is observed. The results are discussed in terms of the chemiosmotic hypothesis. It is suggested that these effects might indicate the existence of a mechanism controlling the rate of ATP formation which is different than the formation of the electrochemical gradient. 相似文献
8.
John A. Jacquez 《生物化学与生物物理学报:生物膜》1973,318(3):411-425
The Michaelis-Menten parameters, and of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na+ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, , decreased with decrease in extracellular Na+ for both α-aminoisobutyric acid and phenylalanine but the for α-aminoisobutyric acid increased markedly as the Na+ concentration fell whereas the for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na+-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na+-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na+-independent but it has a high and is not additive with the Na+-dependent flux. 相似文献
9.
(1) A quantitative study has been made of the binding of ouabain to the ( in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ( will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the ) of ouabain to the M. sexta brain ( under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ( has a higher for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ( can bind ouabain. 相似文献
10.
G.D. Holman 《生物化学与生物物理学报:生物膜》1980,599(1):202-213
Glucose transport in human erythrocytes is characterized by a marked asymmetry in the and values for entry and for exit. In addition, they show a high and a high for equilibrium exchange but low values for infinite cis and for infinite trans exit and entry. An allosteric pore model has been proposed to account for these characteristics. In this model, substrate-induced conformational changes destabilize the interfaces between protein subunits (the pore gates).Pores doubly occupied from inside destabilize the transport gates and result in high and high transport parameters. This effect is less marked when pores are doubly occupied from outside and therefore transport asymmetry results. 相似文献
11.
J.J. Schrijen A. Omachi W.A.H.M. Van Groningen-Luyben J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1981,649(1):1-12
(1) The total phospholipid content of a gradient purified ()-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The ()-ATPase and K+ stimulated activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, value for ATP and temperature dependence of the gastric ()-ATPase, but slightly decreases the value for K+. (5) Phospholipase C treatment lowers the binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules. 相似文献
12.
A method is described to measure the oxygen diffusion-concentration product, , at any locus that can be probed or labeled using nitroxide radicals. The method is based on the dependence of the spin-lattice relaxation time of the spin label on the bimolecular collision rate with oxygen. Strong Heisenberg exchange between spin label and oxygen contributes directly to of the spin label, while dipolar interactions are negligible. Both time-domain and continuous wave saturation methods for studying are considered. The method has been applied to phospholipid liposomes using fatty acid spin labels. A discontinuity in at the main phase transition was observed. 相似文献
13.
14.
Kevin H. Mayo 《Journal of molecular biology》1981,146(4):589-609
Precise oxygen equilibrium curves for carp hemoglobin were determined at 15 °C in bis-Tris buffer, and in phosphate buffer in the presence and absence of P6-inositol, and at various temperatures in phosphate buffer. Parameters of the Koshland, Némethy and Filmer (1966) (KNF) simple, sequential models (square and tetrahedral) were estimated by non-linear least-square fit of the experimental data to Hill plots. Non-, negative and positive co-operativity can be fitted by the KNF models. Considering equilibrium arguments, , the KNF parameter governing the co-operativity of the system, predicts a symmetry conserved mode of action in regions of high, positive co-operativity, and a symmetry non-conserved mode of action in regions of low, non- or negative co-operativity. The simple, sequential, square KNF model fits better the Hill plots than does the simple, sequential, tetrahedral KNF model. From the effect of temperature on carp hemoglobin in phosphate buffer, the heats and entropies of the subunit interaction parameter, , and of the oxygenation parameters, KBBKxBKtAB and , for the square and tetrahedral models, respectively, were calculated and show the square model to account well for previously published data on the carp hemoglobin molecule. This study indicates that the KNF model, in its simplest form, is capable of explaining many of the functional properties of cooperative systems, as opposed to the Monod, Wyman and Changeux (1965) model which seems only to be a special case of the KNF model in regions of high, positive co-operativity. 相似文献
15.
1. In mitochondrial particles antimycin binds to two separate specific sites with dissociation constants and , respectively.2. The concentrations of the two antimycin binding sites are about equal. The absolute concentration for each binding site is about 100 – 150 pmol per mg of mitochondrial protein.3. Antimycin bound to the stronger site mainly inhibits NADH- and succinate oxidase. Binding of antimycin to the weaker binding site inhibits the electron flux to exogenously added cytochrome c after blocking cytochrome oxidase by KCN.4. Under certain conditions cytochrome b and c1 are dispensible components for antimycin-sensitive electron transport.5. A model of the respiratory chain in yeast is proposed which accounts for the results reported here and previously. (Lang, B., Burger, G. and Bandlow, W. (1974) Biochim. Biophys. Acta 368, 71–85). 相似文献
16.
Diethelm Kleiner 《生物化学与生物物理学报:生物膜》1982,688(3):702-708
Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent , per g dry weight at 15°C) is competitively inhibited by ammonium (apparent ). The low value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase. 相似文献
17.
18.
The ionization of fatty acids, fatty amines and acids incorporated in phosphatidylcholine single-walled vesicles has been measured. The guest molecules have been specifically enriched with 13C and titrated by using NMR spectroscopy. The apparent p of fatty acids in phosphatidylcholine bilayers is 7.2–7.4 and those of fatty amines are approx. 9.5. These p values depend on many different parameters related to the structure of the lipid/ solution interface, to the composition of the aqueous medium and to the localization of the ionizable groups. A special sensitivity to the ionic strength and to the surface charge has been found. A positive surface charge decreases the p value whereas a negative one increases it, the total range of variation being 2.5–3 units. In a qualitative macroscopic interpretation, it is proposed that p is essentially determined by the low polarity of the lipidic matrix. 相似文献
19.
G.D. Holman 《生物化学与生物物理学报:生物膜》1979,553(3):489-494
Infinite cis uptake of cyclic AMP into red blood cell ghosts has been measured. The is calculated from two different integrated rate equations that are applicable when the substrate concentration is unsufficient to cause volume changes. Values of 0.69 mM and 0.66 mM are obtained for the infinite cis at 30°C using these procedures. These values are only slightly higher than that predicted from zero trans net flux experiments.Lowering the temperature reduces from 0.69 mM at 30°C to 0.478 mM at 20°C, 0.108 mM at 10°C and 0.072 mM at 4°C (). The for activation of influx permeability of 10?5 M cyclic AMP is 1.55. 相似文献
20.
The immobilization of Rhodopseudomonas capsulata chromatophores by entrapment in an alginate gel is described. Alginate beads were prepared with Ba2+, Sr2+ and Ca2+ as gel-forming agents and compared for their mechanical strength, chemical resistance against disruption by phosphate-induced swelling, and yield of photophosphorylation activity. Barium alginate beads proved to have better physico-chemical properties than the more commonly used calcium alginate beads. After embedding in barium alginate gel, R. capsulata chromatophores retained a high yield (up to 70%) of their photophosphorylation capacity. Alginate entrapment did not cause a large increase in the Michaelis constant for ADP and phosphate, the substrates of adenosinetriphosphatase (ATPase). These constants were and for free chromatophores and and for chromatophores entrapped in barium alginate gel. However, embedding gave no additional protection against rapid inactivation of chromatophores upon storage at 3°C. Preliminary results with a batch reactor for continuous ATP regeneration are presented. The barium alginate method has two features which are not generally encountered at the same time, extremely mild conditions for entrapment and excellent physical properties of the gels beads, which make this method a suitable tool for the construction of bioreactors with immobilized cells or organelles. 相似文献