共查询到20条相似文献,搜索用时 15 毫秒
1.
G W Kidder 《Biochemical and biophysical research communications》1982,107(1):381-388
Adenosine kinase was demonstrated in the soluble fraction of . Magnesium is required for activity. ATP and GTP are efficient phosphate donors while is without activity. The pH optimum is high (8.0), it is heat labile and is stabile to freezing (?20° or ?80°). It is substrate inhibited, does not survive dialysis and its stability to gel filtration is enhanced by the presence of ATP or GTP. Time curves are parabolic only if the enzyme is preincubated with ATP (or GTP); sigmoid when preincubated with adenosine. 相似文献
2.
The transmembrane electropotential of microsomal vesicles from pea internode segments, monitored by equilibrium distribution of the permeant anion SCN?, is strongly hyperpolarized when ATP is present in the incubation medium.The stimulation of SCN? uptake by ATP is rather specific with respect to the other nucleoside di- and triphosphates tested: ADP, GTP, CTP and UTP. ATP-stimulated SCN? uptake is strongly inhibited by ATPase inhibitors such as and and by 2.5% toluene/ethanol (1 : 4, v/v), the latter being a treatment which makes the vesicles permeable. On the contrary, oligomycin is almost ineffective in influencing ATP-induced SCN? uptake. The proton conductor carbonyl cyanide strongly inhibits ATP-stimulated SCN? uptake. The effect of ATP on SCN? uptake depends on the pH of the medium, the maximum being reached at about pH 7.0.These data support the view that microsomal fractions from pea internodes contain membrane vesicles endowed with a membrane-bound ATPase coupling ATP hydrolysis to electrogenic transport of ions, probably H+. 相似文献
3.
The oxygen equilibria of the hemoglobins of one holostean fish, the spotted gar (Lepisosteus osculatus), and of four teleost fish, the carpsucker (Carpiodes carpio), the small mouth buffalo fish (Ictiobus bubalus), the Rio Grande cichlid (Cichlasoma cyanoguttatum), and the redear sunfish (Lepomis microlophus), have been measured as a function of pH in the presence and absence of ATP. The oxygen equilibria of the teleost hemoglobins in the presence of 200 μm ATP can be superimposed within experimental error upon the data obtained in the absence of ATP by a simple downward shift of the pH scale by 0.5 unit. Thus the effects of proton and ATP binding appear equivalent: Both can be interpreted in terms of a two-state allosteric model in which binding occurs preferentially to the low-affinity T-state. The oxygen affinities of each of the teleost hemoglobins approach asymptotically a maximal value at high pH. Although these high affinities are associated with decreased cooperativity of oxygen binding, as reflected by the Hill coefficient n, the asymptotic value of n never appears lower than 1.2 to 1.4. This indicates that the data cannot be completely described in terms of a single high-affinity R-state in alkaline solution: At least two different conformations are required. The oxygen affinity of the spotted gar hemoglobin, like that of each of the teleost hemoglobins, reaches a maximal value (minimal value of log PO2 for half-saturation) above pH 8, but unlike teleost hemoglobins, the Hill coefficient reaches maximal values of 2.6 to 2.7 at high pH. The data in the absence of ATP are superimposable on the data in its presence by a downward shift of the pH scale by 0.25 unit. The measurement of the Bohr effect () in the presence and absence of ATP shows that the Bohr effect in each of the hemoglobins is substantially enhanced by organic phosphates, as it is in mammalian hemoglobins. The extent of the enhancement of the Bohr effect by 200 μm ATP for each of the hemoglobins is approximately the same in the range pH 6.7 to 7.3 (increase in ). This is a direct consequence of the equivalence of the linked-function relationship to the effects of ATP and proton binding on oxygenation. 相似文献
4.
ADP and Pi-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with . ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔGp) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na+ or K+ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low . 相似文献
5.
E Mendez A O Grubb C Lopez B Frangione E C Franklin 《Archives of biochemistry and biophysics》1982,213(1):240-250
L-929 cell surface membranes have been assayed in vitro and found to contain significant protein kinase activity. A steady-state kinetic analysis indicated that at least two distinct protein kinases were present. Plots of reaction velocity (v) against substrate (ATP) concentration were distinctly biphasic, as were Lineweaver-Burk plots of versus . Michaelis constants of the two enzymes were calculated to be 22 and 173 μm, respectively. Sodium dodecyl sulfate polyacrylamide gel analysis of the phosphorylated membrane proteins provided additional support for the existence of more than one protein kinase. Different endogenous proteins were phosphorylated at 1 μm ATP compared to 1 μm ATP. Further studies of the low Km (22 μm) enzyme suggested that it is a typical cyclic 3′,5′-AMP-independent protein kinase. Its activity was dependent on the presence of Mg2+, but it was not affected by cyclic 3′,5′-AMP, cyclic 3′,5′-GMP, or the heat-stable inhibitor of cyclic 3′,5′-AMP-dependent protein kinases. ATP and GTP, but not other nucleoside triphosphates, could serve as phosphoryl donor and maximum kinase activity was expressed at pH 7.0. Phosvitin and casein were superior to histones as exogenous substrates for the low Km enzyme. 相似文献
6.
A M Vaccaro Torracca R Raschetti R Salvioli G Ricciardi K H Winterhalter 《Biochimica et biophysica acta》1977,496(2):367-373
Both ATP and GTP are present in considerable amounts in red cells of the common goldfish Carassius auratus. They both influence the Root effect of the single major fish hemoglobin, but GTP is, depending on pH, 2-6 times more effective than ATP. The two triphosphates account for 3/4 of the effect of trichloroacetic acid supernatant obtained from hemolysate which contains still some compound(s) which can influence the shift of the Root effect toward higher pH. 相似文献
7.
Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Line-weaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25°C, this transport activity was easily lost during the storage of vesicles at 4°C. The for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation of transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37°C, this transport activity was stable during storage of vesicles at 4°C for several days. The for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the , and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes. 相似文献
8.
C.R. Bridges A.C. Taylor R.J.A. Atkinson 《Journal of experimental marine biology and ecology》1982,59(1):51-60
The respiratory properties of the whole blood of the burrowing red band fish Cepola rubescens L. were investigated. Oxygen dissociation curves constructed at 15°C were found to be close to hyperbolic in shape with a mean value for the cooperativity coefficient at half-saturation (n50) of 1.56. Half-saturation oxygen tension (P50) for pH = 7.56 (mean in vivo pH of venous blood) was 27 Torr. The blood showed a marked Bohr effect () and also a Root effect which at the in vivo pH reduced oxygen carrying capacity by 20%. The PvCO2 was 3.2 Torr and the buffering power of the blood was low, the buffer value of true plasma averaging 5.43 mmol · 1?1 · pH?1. It is suggested that the large Bohr effect coupled with the low buffer value confers on the haemoglobin a flexibility, in terms of oxygen affinity, to withstand changes which occur in environmental oxygen tensions. 相似文献
9.
1. The aerobic uptake of inorganic ions, such as 86Rb+ or 125I?, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of ΔpH, the transmembrane pH differential, and Δψ, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the ΔpH and Δψ values from organic and inorganic ions is large at high but not at low ion/protein ratios.2. In the absence of weak bases and strong acids the values of , the proton electrochemical potential difference, are close to 100 mV and the magnitude of ΔpH and Δψ are similar. Weak bases decrease ΔpH and enhance Δψ. Strong acids decrease Δψ and enhance ΔpH. Interchangeability of ΔpH with Δψ occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of .3. Concentrations of weak bases capable of abolishing ΔpH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing Δψ affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of ΔpH + Δψ inhibit ATP synthesis.4. Depression of is paralleled by inhibition of ATP synthesis and decline of ΔGp, the phosphate potential. Abolition of ATP synthesis occurs only when is below 20 mV. The ratio increases hyperbolically with the decrease of . 相似文献
10.
Peter J.M. Van Haastert 《Biochemical and biophysical research communications》1984,124(2):597-604
D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (Kd = 15 nM, ). A second class is fast dissociating ( about 1 s) and is composed of high affinity binding sites H (Kd ≈ 60 nM), and low affinity binding sites L (Kd = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors. 相似文献
11.
In the presence of [γ32-P] ATP or [γ32-P] GTP 4 non ribosomal proteins (Mr 110,000; 105,000; 89,000 and 25,000) of the native 40S subunit became phosphorylated. The protein kinase responsible for this phosphorylation could be removed by treatment with 0.5 KCl. Sucrose density gradient analysis showed that the endogenous enzyme activity sedimented with approx. 7.5S. 相似文献
12.
Efficiencies of oxidative phosphorylation ( ratios), intracellular high-energy phosphate pools (ATP and ADP) under aerobic and anaerobic dark conditions, and photosynthetic oxygen evolution measured with intact cells of Anacystis nidulans were found to be specifically depressed by NaCl, but not by KCl. A scheme is proposed which explains the deleterious effect of sodium on the energy metabolism of A. nidulans by competition for protons between ATP synthesis and active sodium extrusion. 相似文献
13.
Thiol modulation of the chloroplast protonmotive ATPase (CF0-CF1) by preillumination of broken chloroplasts in the presence of dithiothreitol (or preillumination of intact chloroplasts in the absence of added thiols) had the following effects on photophosphorylation. (1) When assayed at pH 8 and saturating light, the initial rate of photophosphorylation was increased by 10–40%. There was an accompanying increase in the rate of coupled electron transport with no significant change in the overall ratio. (2) On lowering the pH of the assay medium to pH 7, the stimulatory effect of thiol modulation on photophosphorylation and coupled electron flow was enhanced. At pH 7, there was also a small increase in ratio. (3) Addition of a non-saturating amount of uncoupler to the assay medium enhanced the stimulatory effect of thiol modulation on photophosphorylation. In the presence of 1 mM NH4Cl, there was only a small increase in coupled electron flow and a correspondingly larger increase in ratio. (4) Lowering the light intensity, or inhibiting electron transport, diminished the stimulatory effect of thiol modulation on photophosphorylation, coupled electron transport and ratio. (5) Under all the above conditions, the ΔpH maintained across the thylakoid membrane was lower after thiol modulation, even when photophosphorylation markedly increased in rate. (6) Thiol modulation of CF0-CF1 increased the observed Michaelis constant for ADP (Km(ADP)) and the apparent maximum rate (Vapp of photophosphorylation by the same factor, so that ratio was not altered. was also unaffected by changing the medium pH, but was significantly decreased upon addition of uncouplers to the medium. These results indicate that the observed rate of ATP synthesis catalysed by thiol demodulated chloroplasts is limited kinetically by the fraction (α) of enzyme molecules that are active during photophosphorylation. A model based on a dual pH optimum requirement for activation of CF0-CF1 is presented to explain the dependence of α on ΔpH. Thiol modulation of CF0-CF1 is proposed to stimulate photophosphorylation by causing the enzyme to become active over a lower range of ΔpH, thereby reducing the kinetic limitation on ATP synthesis imposed by the activation process. 相似文献
14.
Methanol at 35% () overcomes the latency of spinach thylakoid ATPase. Activation is immediate and reversible involving changes in the Vmax, not the Km of the enzyme, MgATP is a much better substrate than CaATP; free Mg2+ noncompetitively inhibits activity. This inhibition can be overcome by the addition of Na2SO3. While both MgATP and MgGTP act as substrates, free ATP and GTP both inhibit activity. ADP and MgADP are also inhibitory. Insensitivity to certain inhibitors indicates that methanol neither induces the same conformational changes in CF1 as illumination does, nor does it lead to coupling between H+ movement through CF0 and ATP hydrolysis. Methanol activation provides a much improved method for assaying thylakoid ATPase. 相似文献
15.
Reinhard Krämer 《生物化学与生物物理学报:生物膜》1983,735(1):145-159
Various modulating influences of negative and positive membrane charges on binding and transport properties of the reconstituted ADP/ATP carrier from mitochondria were investigated. The results are interpreted in terms of functional and structural asymmetries of the adenine nucleotide carrier embedded in the liposomal membrane. The surface potential of liposomes was measured directly either by potential-dependent adsorption of the fluorescent dye 6-sulfonate (TNS) or by the shift of the lipophilic pH indicator pentadecylumbelliferone. These results were correlated with the following observations. (1) Negative surface potentials increase the apparent dissociation constant, , for binding of the negatively charged inhbitor carboxyatractylate to the reconstituted carrier protein. (2) Surface potentials modulate the apparent transport affinity, , of the reconstituted adenine nucleotide carrier for ADP and ATP. The interaction of surface charges with the transport function was investigated with carrier proteins oriented both right-side-out and inside-out. Thus the influence of the surface potential on the function of the ADP/ATP carrier could be determined for the internal and external active sites of the translocator on the outer side of the membrane. Large discrepancies were observed not only between the potentials measured directly (fluorescent dyes) and those measured indirectly (binding and transport affinities), but also between the different surface potentials determined from the influence on the alternatively oriented carrier proteins. The effect of surface charges was rather weak on the cytosolic side of the translocator, whereas there was a strong influence of surface charges on the active site at the matrix side. The most obvious explanation, i.e., screening of negative membrane charges by positively charged amino acid residues at the protein surface, could be ruled out. Besides the modulation of binding affinities for substrates and inhibitors, an additional side-specific effect of surface charges on the transport velocity was observed. Again, the influence on the internal active site of the ADP/ATP carrier was found to be much higher than that on the cytosolic site. The observed effects can be explained by a definite structural asymmetry of the carrier embedded in the liposomal membrane. That site which is physiologically exposed to the cytosol is located at a considerable distance from the plane of the membrane, whereas the opposite site seems to be in close proximity to the membrane surface. Moreover, a spatial equivalence of carboxyatractylate binding site and nucleotide binding site at the external side of the carrier protein was concluded. 相似文献
16.
The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na+,+)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a ; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na+ and K+; (3) Mg2+ binds with an association constant of while ATP binds with an apparent for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl2, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg2+ concentrations. There is no ATP binding in the absence of Mg2+. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is . In the presence of ATP a reduction in size is observed. 相似文献
17.
《Comparative biochemistry and physiology. A, Comparative physiology》1979,62(1):219-226
- 1. The two hemoglobins, Hb I and II, of the obligate air-breathing catfish,Hoplosternum littorale have been isolated.
- 2. The unfractionated stripped hemoglobin has a high oxygen affinity, a normal alkaline Bohr effect, and a Root effect.
- 3. Both the Bohr and Root effects are enhanced by 1 mM ATP.
- 4. Stripped Hb I has a relatively high oxygen affinity, a reversed Bohr effect between pH 6.0 and 8.0 (Δlog P502DpH> 0), but no Root effect. Addition of 1 mM ATP to Hb I causes a marked reduction in the oxygen affinity, a change to a normal alkaline Bohr effect (Δlog P50ΔpH< 0), but no Root effect.
- 5. Stripped Hb II has a lower oxygen affinity at low pH and a higher oxygen affinity at high pH than does Hb I. Hb II shows a large alkaline Bohr effect which is only slightly increased by 1 mM ATP and a Root effect at low pH which is enhanced by 1 mM ATP.
- 6. The observed rates of O2 dissociation and of CO combination with Hbs I and II show differences which parallel those observed in the oxygen equilibrium measurements.
18.
d-Gluconate uptake was studied in whole cells of Arthrobacter pyridinolis; the uptake activity was inducible, mutable and showed saturation kinetics (). Uptake of d-gluconate was not mediated by a phosphoenolpyruvate: hexose phosphotransferase system, nor was it directly energized by ATP. A transmembrane pH gradient, ΔpH, of ?63 mV was generated by A. pyridinolis cells at pH 6.5, while at pH 7.5, . Addition of 8 μM d-gluconate significantly reduced the ΔpH. The transmembrane electrical potential, Δψ, which was ?87 mV over a range of pH from 5.5 to 7.5, was unaffected by the presence of substrate. d-Gluconate accumulated at the same rate and as the protonated solute, at both pH 6.5 and 7.5. Experiments in which a diffusion potential was generated in cyanide-treated cells, indicated that the Δψ did not energize transport. Rather, the rate of d-gluconate uptake correlated with and appeared to be determined by the rate of d-gluconate metabolism: (a) treatment of cells with valinomycin or nigericin, under conditions in which there was a loss of intracellular potassium, inhibited both d-gluconate uptake and the metabolism of pre-accumulated d-gluconate; (b) the effects of cyanide and azide on d-gluconate uptake were much more severe at pH 6.5 than pH 7.5, a pattern which paralleled the effects of these inhibitors on d-gluconate metabolism; (c) extraction and chromatography of intracellular label from d-gluconate uptake revealed that accumulation of unaltered d-gluconate was negligible; (d) a series of mutant strains with lower d-gluconate kinase activities also exhibited low rates of d-gluconate uptake; (e) spontaneous revertants of these mutant strains consistently regained both d-gluconate kinase activity and wild type levels of uptake. 相似文献
19.
Masaharu Miyake Toshihiko Innami Yasuo Kakimoto 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,760(2):206-214
An enzyme responsible for the deacylation of β-citryl-L-glutamate to citrate and glutamate has been characterized in rat testis. The enzyme required manganese ion for full activity and was strongly inhibited by nucleotides such as ATP or GTP. The activity was localized in the particulate fractions. The enzyme favored in a decreasing order. The amidohydrolyase activity was highest in the testis and lung, a moderate activity was detected in heart, kidney and intestine, and low in brain, thymus, stomach, skeletal muscle, spleen and liver. These findings suggest that the amidohydrolase is different from any of amidohydrolases reported so far, amidohydrolase I (EC 3.5.1.14), II (EC 3.5.1.15), III, N-acetyl-lysine deacylase (EC 3.5.1.17) and N-acetyl-β-alanine deacetylase (EC 3.5.1.21), and various peptidases. 相似文献
20.
An adenine nucleotide translocase in the procaryote Methanobacterium thermoautotrophicum 总被引:7,自引:0,他引:7
H J Doddema C A Claesen D B Kell C van der Drift G D Vogels 《Biochemical and biophysical research communications》1980,95(3):1288-1293
ATP synthesis, ATP hydrolysis and ADP uptake by membrane vesicles of Methanobacterium thermoautotrophicum are inhibited by inhibitors of mitochondrial translocases. Atractyloside binds to one of the membrane proteins. These data demonstrate the presence of an eucaryotic type of translocase in a procaryotic microorganism and stress the unique position of methanogenic bacteria in evolution. 相似文献