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1.
(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
2.
(1) +/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO?2 or N2O. Under optimal H+-translocation conditions, the ratios , , for reduction to N2 and for reduction to N2O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO?2 or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. , for reduction to N2 and for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The ratios, mentioned in item 2, were not altered in the presence of and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO?2 and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO?2, N2O or O2 pass two H+-translocating sites. The acceptor ratios predicted by this scheme are in good agreement with the experimental values. 相似文献
3.
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
4.
Commercial [5-14C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-14C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated by ion-exchange chromatography. The artifactual results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the and []cholesterol produced by rat renal cortex slices incubated with purified [5-14C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that is genuinely oxidized to , and that purification of substrate before its use is necessary. Production of and various []lipids from purified [5-14C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described. 相似文献
5.
H. Hauser C.C. Hinckley J. Krebs B.A. Levine M.C. Phillips R.J.P. Williams 《生物化学与生物物理学报:生物膜》1977,468(3):364-377
The interaction of lanthanides and other cations with phosphatidylcholine bilayers present as single bilayer vesicles in 2H2O has been investigated in terms of stoichiometry, apparent binding constants and environmental conditions.Lanthanides are shown to form 2 : 1 (molar ratio) phosphatidylcholine to metal ion complexes.The apparent binding constant varies as a function of the quantity of metal ion bound and as a function of the Cl? concentration. The apparent binding constant at “zero loading” is . It decreases exponentially with increased “loading” expressed as the molar ratio of metal ion bound to effective phosphatidylcholine concentration and increases exponential with Cl? concentration.The interaction of lanthanides and divalent cations such as Ca2+ and Mg2+ is independent of pH in the pH range 3–7+ and 3–10 respectively, but is sensitive to the nature of the anion. The presence of anions enhances the interaction with polyvalent cations, the chaotropic anions showing the largest effect. The order of enhancement is . The nature of the monovalent counterion (cation) has little effect on the enhanced binding of lanthanides in the presence of the above anions.The affinity of other polyvalent cations for phosphatidylcholine bilayers has been determined by competition with lanthanides. The physiologically important divalent cations Ca2+ and Mg2+ both bind less strongly (by about an order of magnitude) to the lipid surface. The order of binding of cations reflects direct binding to the phosphodiester group, with UO22+ showing the highest affinity. 相似文献
6.
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
7.
E.M. Kosower 《Biochemical and biophysical research communications》1980,92(2):356-364
A new mechanism that involves dissociative electron transfer in the energy transducing step is set forward for bacterial luciferase catalyzed light emission. The proposal involves (1) dissociation of the 4a-hydroperoxyflavin to a flavin radical and , accounting for 570 and 620nm absorption, (2) addition to the aldehyde carbonyl to form a peroxyl radical, (3) abstraction of H from an enzyme thiol group to form RCH(OOH)OH, (4) thiyl radical abstraction of the H on C in RCH(OOH)OH, a step which can show a of ca. 4, and (5) dissociative electron- transfer, a highly exothermic step that leads to a protonated flavin excited state, a carboxylic acid and water. 相似文献
8.
W.McD. Armstrong W.R. Bixenman K.F. Frey J.F. Garcia-Diaz M.G. ORegan Jeanie L. Owens 《生物化学与生物物理学报:生物膜》1979,551(1):207-219
Na+, K+ and Cl? concentrations () and activities (), and mucosal membrane potentials () were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na+ and Cl? and 5.4 mM K+. Na+ and K+ concentrations were determined by atomic absorption spectrometry and Cl? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO3. 14C-labelled inulin was used to determine extracellular volume. was measured with conventional open tip microelectrodes, with solid-state Cl?-selective silver microelectrodes and and with Na+- and K+-selective liquid ion-exchanger microelectrodes. The average recorded was ?34 mV. , and were 51, 105 and 52 mM. The corresponding values for , and were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na+ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K+ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl?. significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na+ and Cl? is implicated in intracellular Cl? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na+ and Cl? electrochemical potential differences (Δμ̄Na and Δμ̄Cl). Δμ̄Na (?7000 J · mol?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ̄Cl (1000–2000 J · mol?1). 相似文献
9.
The kinetics of isotopic Na+ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal 22Na+ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na+ flow. The electrical current and mucosal to serosal 22Na+ influx were then measured with transmembrane potential clamped at . Subsequent elimination of active Na+ transport mucosal amiloride permitted calculation of the rates of active Na+ transport and active and passive influx and and . The results indicate that for Dominican toad bladders mounted in chambers only Na+ contributes significantly to transepithelial active ion transport; hence . was abolished at . As Δψ approached , active efflux became demonstrable. At exceeded , so that was negative. Experimental values of agreed well with theoretical values predicted by a thermodynamic formulation: . The dependence of on Δψ is curvilinear. 相似文献
10.
In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes . A minimal concentration of about 6 · 1011 dipicrylamine ions per cm2 of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method is compared with the concentrations obtained from simultaneous electrical relaxation measurements. and agreed at low dipicrylamine concentrations (10?8–10?7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10?6 M). In the saturation range was maximally four times higher than . The significance of this difference is discussed together with general aspects of the saturation phenomenon. 相似文献
11.
A comparison between inosine- and guanosine-containing anticodons in ribosome-free codon-anticodon binding 总被引:1,自引:0,他引:1
Binding of the ribooligomers, AUC, AUU, AUA, AUCA, AUUA, and AUAA to the isoleucine-accepting tRNAs, tRNA (anticodon, -IAU-) and tRNA (anticodon, -GAU-) was measured by equilibrium dialysis. With the aid of Scatchard plots, AUCA was shown to bind to one site per tRNA molecule, presumably the anticodon. AUA and AUAA did not measurably attach to either anticodon in the ribosome-free system. All other oligomers were bound to tRNA about 5 to 13 times stronger than to tRNA. 相似文献
12.
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
13.
The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant () of 13 nM was found in the presence of () and (). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg2+, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K+ and its congeners, by Na+ in the presence of (), and by ATP analogs (ADP-C-P, ATP-OCH3). Ca2+ antagonized the action of K+ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (Rα) and a high affinity conformational state (Rβ). The equilibrium between both states is influenced by ligands of . With 3 mM Mg2+ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot). 相似文献
14.
The interactions between calmodulin, ATP and Ca2+ on the red cell Ca2+ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of by 5–10-fold and the rate of Ca2+-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates along a biphasic concentration curve (), but in stripped membranes the curve is essentially hyperbolic (). In calmodulin-bound membranes Ca2+ activates at low concentrations () in stripped membranes the apparent Ca2+ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E2 and E1 forms of the Ca2+ pump protein. 相似文献
15.
The intrinsic viscosities, weight-average molecular weights (M?w), and radii of gyration [()≈] of Streptococcus salivarius levan in various solvents were respectively obtained from viscosity and light-scattering measurements. The data showed that the levan in water is not aggregated by hydrogen bonds, and that the values of both the refractive index and () are lower in water than in aqueous solutions of urea. Urea may break intramolecular hydrogen-bonds, e.g., between branches, allowing the molecule to expand. 相似文献
16.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts Y.-F. Fu G.A.J. Kuijpers J.J.H.H.M. de Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1984,774(2):277-287
(1) Treatment of from rabbit kidney outer medulla with the labeled thio-analogue of ATP in the presence of and the absence of K+ leads to thiophosphorylation of the enzyme. The value for [γ-S]ATP is 2.2 μM and for Na+ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na+ concentration, yielding a Hill coefficient . (2) The thio-analogue () can also support overall activity, but at 37°C is only h?1 or 0.09% of the specific activity for ATP (). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s?1 vs. 180 s?1), spontaneous dethiophosphorylation (0.04 s?1 vs. 0.5 s?1) and K+-stimulated dethiophosphorylation (0.54 s?1 vs. 230 s?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s?1, ) and is relatively K+-insensitve. The for K+ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E1-conformation. 相似文献
17.
18.
W R Scowcroft A H Gibson J D Pagan 《Biochemical and biophysical research communications》1976,73(2):516-523
Nitrogenase activity in agar cultures of cowpea rhizobia, strain 32H1, was rapidly inhibited by but this was relieved by increased O2 tension. Inhibition was more rapid than that caused by inhibitors of protein synthesis and was not relieved by methionine sulfoximine or methionine sulfone. Under conditions were nitrogenase activity was inhibited by , glutamine synthetase and glutamate synthase were substantially unaffected. Glutamate dehydrogenase was undetected in either nitrogenase active or inhibited cultures. These results indicate that inhibition of nitrogenase activity in strain 32H1 is not effected through glutamine synthetase regulation of nitrogenase synthesis. 相似文献
19.
The relative effectiveness of oxidizing (.OH, H2O2), ambivalent (O2?) and reducing free radicals (e? and CO2?) in causing damage to membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase of resealed erythrocyte ghosts has been determined. The rates of damage to membranebound glyceraldehyde-3-phosphate dehydrogenase ((enz)) were measured and the rates of damage to membranes ((mb)) were assessed by measuring changes in permeability of the resealed ghosts to the relatively low molecular weight substrates of glyceraldehyde-3-phosphate dehydrogenase. Each radical was selectively isolated from the mixture produced during gamma-irradiation, using appropriate mixtures of scavengers such as catalase, superoxide dismutase and formate. .OH, O2? and H2 O2 were approximately equally effective in inactivating membrane-bound glyceraldehyde-3-phosphate dehydrogenase, while e? and CO2? were the least effective. (enz) values of O2? and H2O2 were 10-times and of .OH 15-times that of e?. (mb) values were quite similar for e? and H2O2 (about twice that of O2?), while that of .OH was 3-times that of O2?. Hence, with respect to (mb): , and with respect to (enz): . The difference between the effectiveness of the most damaging and the least damaging free radicals was more than 10-fold greater in damage to the enzyme than to the membranes. Comparison between H2O2 added as a chemical reagent and H2O2 formed by irradiation showed that membranes and membrane-bound glyceraldehyde-3-phosphate dehydrogenase were relatively inert to reagent H2O2 but markedly susceptible to the latter. 相似文献
20.
Peter Nicholls 《BBA》1976,430(1):13-29
1. Formate inhibits cytochrome oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome . The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome ) and in the half-reduced species (). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of minus , free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome ) and azide (which induces peak shifts of cytochrome towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome is faster than its rate of dissociation from , especially in the presence of cytochrome . The for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]2.5. Formate inhibition of ascorbate plus oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion. 相似文献