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1.
Homogenates of adult Schistosoma mansoni (blood flukes), isolated from the porto-mesenteric veins of infected mice, contain substantial activities of adenylyl cyclase, cyclic AMP phosphodiesterase, and a cyclic AMP stimulated protein kinase. The adenylyl cyclase, which is largely sedimentable at 10,000xg, is stimulated 20-fold by 10mM sodium fluoride and 1.4 to 2-fold by serotonin, glucagon, prostaglandins E1, E2 or B1. The phosphodiesterase, which is largely sedimentable at 10,000xg, is inhibited by both aminophylline and papaverine but is not influenced by 10mM sodium fluoride. The protein kinase, which is present in the 10,000xg supernatant is stimulated 4 to 8-fold by either cyclic AMP or cyclic GMP. There is a preference for cyclic AMP (K12 = 1.1×10?7M) over cyclic GMP (K12 = 4.5×10?6M). If intact worms are incubated in a glucose free medium there is a mobilization of glycogen stores which is preceded by a rise in cyclic AMP concentration. In a medium with 5mM glucose there is neither a rise in cyclic AMP nor mobilization of glycogen.  相似文献   

2.
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 KADPm = 1.4 × 10?5m and KPim = 2.2 × 10?4m for free chromatophores and KADPm = 2.3 × 10?4m and KPim = 5.6 × 10?4m 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   

3.
The electronic structure of 19 established and potential biological oxidants has been studied by semiempirical all-valence-electron quantum-chemical methods. Electronic ground and excited states of O2, HO2, HO, H2O2, H3O, H4O2 and their (radical) ions have been investigated in order to get information on the geometry, vertical ionization potentials, vertical electron affinities and low-lying electronic excited states. The actual aim has been (i) to arrange the studied species according to their oxidizing power as given by gas-phase electron affinity.
9·HO·OH2O12>(1?+g).·OH>O12(1δ+g) >HO12(2A′)>O12(2A′)>O2(3?-g>HO·2)
and (ii) to contribute to the thermodynamics of early changes of the O2 molecule
O2+e→O?2·;O?2·+H+→HO·2
. Moreover, it has been found theoretically that the hydrated form of the hydroxyl radical (·HO.OH2) should be a relatively stable species with very high electron affinity (2·4 eV, INDO method). This circumstance and the theoretically predicted, extraordinarily low-lying, excited doublet state of the peroxyl radical (about 6000 cm?1) could be of biological significance.  相似文献   

4.
Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (I50 = 6·10?7M). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity (Kd1 = 1.2·10?7M) to 8.5·105 sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity (Kd2 = 10?4M) to a second set of sites (4.6·107 per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.  相似文献   

5.
To determine whether changes in unsaturation of fatty acids in rat liver plasma membranes might alter activities of membrane-associated enzymes, liver plasma membranes were prepared from rats fed purified diets lacking or supplemented with essential fatty acids. Two methods of membrane purification were used. A similar degree of purification was obtained with both methods for both depleted and control membranes, as indicated by marker enzyme purification. The proportion of essential fatty acids of the linoleate series was significantly lower in phospholipids from depleted rats. The specific activity of 5′-nucleotidase was lower, and the activity, V and apparent Km for total (Na++K++Mg2+)-ATPase were higher in the depleted liver plasma membranes. Arrhenius plots of total ATPase activity showed a discontinuity at the same temperature for both the depleted and control membranes. Activity with the depleted membranes was higher at all temperatures tested. Supplementation of deficient rats with a source of essential fatty acids (corn oil) restored V and apparent Km values to normal. Adenylate cyclase activity in the presence of fluoride, glucagon or glucagon plus GTP was significantly lower in the depleted plasma membranes.  相似文献   

6.
Nitrogenase activity in agar cultures of cowpea rhizobia, strain 32H1, was rapidly inhibited by NH4+ 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 NH4+, glutamine synthetase and glutamate synthase were substantially unaffected. Glutamate dehydrogenase was undetected in either nitrogenase active or NH4+ inhibited cultures. These results indicate that NH4+ inhibition of nitrogenase activity in strain 32H1 is not effected through glutamine synthetase regulation of nitrogenase synthesis.  相似文献   

7.
8.
M1 cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells (M1?) to mature macrophages (M1+) within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While M1? cells have no phagocytic activity nor Fc receptor (FcR), M1+ cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on M1+ cells is resistant to trypsin and pronase. M1+ cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. M1? cells lack this macrophage-substituting capacity. Mm1 cells, mutant cells from M1 cells, having FcR and higher phagocytic activity than M1+ cells, are also devoid of this capacity.  相似文献   

9.
A general procedure for the isolation of 3′-linked fragments derived from tRNA molecules is described. Purified N-2-naphthoxyacetylglycyl derivatives of the tRNA1Gly and tRNA2Gly of yeast were exhaustively digested with RNase T1 and the 3′-linked fragments (bearing the derivative) were separated from other degradation products (lacking the derivative) by stepwise chromatography on BD-cellulose. Subsequent chromatographic resolution and base-composition analysis allowed tentative identification of the 3′-terminals of tRNA1Gly and tRNA2Gly as Gp(Cp,Ap)CpCpA and Gp(Cp,Cp,Up,Ap)CpCpA, respectively. The potential utility of this procedure for development of a novel approach to nucleic acid sequence analysis is discussed.  相似文献   

10.
The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of 125I-labeled toxin B-IV by native toxin B-IV have shown specific binding of 125I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [3H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association (k+1=7.3·105M?1·s?1) and dissociation (k? 1=2·10?3s?1) shows a nearly identical Kd of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.  相似文献   

11.
Na+, K+ and Cl? concentrations (cji) and activities (aji), and mucosal membrane potentials (Em) 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. Em was measured with conventional open tip microelectrodes, aCli with solid-state Cl?-selective silver microelectrodes and aNai and aKi with Na+- and K+-selective liquid ion-exchanger microelectrodes. The average Em recorded was ?34 mV. cNai, cKi and cCli were 51, 105 and 52 mM. The corresponding values for aNai, aKi and aCli 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?. aCli 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   

12.
Isolation and characterization of isocitrate lyase of castor endosperm   总被引:1,自引:0,他引:1  
Isocitrate lyase (threo-DS-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: At = A02 · e?k1t + A02 · e?k2t, where A0 and At are activities at time zero at t, and k1 and k2 are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg2+ (5.0 mm) to a solution of this protein. Addition of Ba2+ or Mn2+ brings about partial recovery. Other metal ions are not effective.  相似文献   

13.
The rates of electron exchange between ferricytochrome c (CIII)3 and ferrocytochrome c (CII) were observed as a function of the concentrations of ferrihexacyanide (FeIII) and ferrohexacyanide (FeII) by monitoring the line widths of several proton resonances of the protein. Addition of FeII to CIII homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, CIII·FeII, in the over-all reaction:
CIII+FeII?k?1k1CIII·FeII?k?2k2CII·FeIII?k?3k3CIII+FeII
Values for k1k?1 = 0.4 × 103m?1and k2 = 208 s?1, respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of CIII in the presence of FeII and either KCl or FeIII suggest that complexation is principally ionic, that FeIII and FeII compete for a common site. Addition of saturating concentrations of FeIII to CIII produced only minor changes in the nuclear magnetic resonance spectrum of CIII suggesting that complexation occurs on the protein surface.Addition of FeIII to CII in the presence of excess FeII (to retain most of the protein as CII) increased the line width of the methyl protons of ligated methionine 80. A value for k?2 ≈ 2.08 × 104 s?1 was calculated from the dependence of linewidth on the concentration of FeII at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k2 and k?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k2 and k?2, respectively.  相似文献   

14.
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 ?O2?, accounting for 570 and 620nm absorption, (2) ?O2? 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 kHkD 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   

15.
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 I and mucosal to serosal 22Na+ influx were then measured with transmembrane potential clamped at Δψ = 0, 25, 50, 75 or 100 mV. Subsequent elimination of active Na+ transport mucosal amiloride permitted calculation of the rates of active Na+ transport JNaa and active and passive influx JNaNa and JNaa and JNap. The results indicate that for Dominican toad bladders mounted in chambers only Na+ contributes significantly to transepithelial active ion transport; hence JNaa = Ja. Ja was abolished at Δψ = E = 96.3 ± 1.9 (S.E.) mV. As Δψ approached E, active efflux Ja became demonstrable. At Δ = 100 mV, Ja exceeded Ja, so that Ja was negative. Experimental values of Ja agreed well with theoretical values predicted by a thermodynamic formulation: Jexpa = 0.985 Jtheora (r = 0.993). The dependence of Ja on Δψ is curvilinear.  相似文献   

16.
Labeling of intact erythrocytes with galactose oxidaseNaB[3H]4 resulted in the incorporation of radioactivity into the monosaccharide transporter. When the purified labeled protein was subjected to SDS gel electrophoresis, the peak of radioactivity migrated more slowly than the peak of Coomassie Blue-staining material. Endo-β-galactosidase treatment of the purified labeled transporter led to partial loss of the label, sharpening of the stain profile, and a change in the apparent molecular mass of the polypeptide from 55,000 to 46,000 daltons. Approximately 50% of the transporter bound to a column of Ricinus communis agglutinin I-agarose. These findings demonstrate that the transporter is heterogeneously glycosylated and, in conjunction with other data, show that it is a transmembrane protein and probably a source of erythroglycan.  相似文献   

17.
2 experiments were designed to study the reversion rate values of the a1+a1a2+a2 system of tobacco over the Lagravette uranous outcrop for dose rates ranging close to the background level, and so to detect an eventual threshold effect. Both experiments apparently led to the same conclusion: for the genetic system involved, there seemed to be a threshold somewhere between 0.291 and 0.449 mrd/h, i.e., 2.55 and 3.93 rd/y. It also appeared that, above the threshold, the dose—response relationship could be linear.  相似文献   

18.
The oxidation of unsaturated fatty acid micelles by the superoxide free radical (O?2), during γ irradiation in the presence of formate, is kinetically distinct from oxidation by hydroxyl free radicals (HO.). The evidence suggests that a direct reaction between (O?2) and lipid hydroperoxide initiates a chain oxidation process in the micelles. While tetranitromethane, which reacts rapidly with (O?2), protects the micelles from oxidation, active superoxide dismutase is no more effective than its apoprotein, due to lack of penetration of the micellar environment. We discuss these findings in the light of recent literature, and with reference to their possible significance for biological systems.  相似文献   

19.
The mechanism of the reaction of bis-(salicylato)-copper(II) with superoxide anion has been studied by utilizing electron paramagnetic resonance and polarographic techniques. The proposed reaction sequence is as follows:
Cu(II) + O2?Cu(II)O2?Cu(I)O2?Cu(I) + O2
Cu(I) + O2?Cu(I)O2?Cu(II)O22?2H+Cu(II)O + H2O2
Using the xanthine-xanthine oxidase system as a superoxide generator, it was found that the concentration of this copper complex for 50% inhibition of the xanthine-cytochrome c reductase activity was about 1000 times more per mole of copper than that of erythrocyte superoxide dismutase.  相似文献   

20.
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