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1.
EPR data are used to describe the conformation and identity of the atoms coordinated to Cu(II) in Cu(II)-bleomycin bound to oriented DNA fibers. The fibers were slowly drawn from viscous solutions of Cu(II)-bleomycin-DNA containing one Cu(II)-bleomycin to 200 basepairs. EPR measurements were made at room temperature and 90 K for different orientations of the external magnetic field with respect to the helical axes of the fibers. The g-values (g parallel = 2.21, g perpendicular = 2.04) and the hyperfine constant (A parallel = 175 G) are consistent with values expected for Cu(II) chelated to a square planar array of ligands. In the oriented fibers, the square planar arrays do not all have the same orientations with respect to the fiber axes. At room temperature the chelated ions have rotational freedom in which the normal to the planar array has almost complete freedom of rotation about axes perpendicular to the DNA fiber axes. The normal maintains an angle of 75 degrees with respect to the axis, in the plane of the basepair, about which it rotates. Nine superhyperfine peaks on the high field side of the EPR spectrum were partially resolved. The number and splitting (12 G) of these superhyperfine peaks indicate that four nitrogen atoms are chelated to Cu(II) in a square planar array. These data on Cu(II)-bleomycin bound to DNA give information on the orientation of the metal-containing portion of bleomycin which lies outside to double helix. 相似文献
2.
R S Himmelwright N C Eickman E I Solomon 《Biochemical and biophysical research communications》1978,84(2):300-305
Half hemocyanin is shown to undergo a unique change at the Cu(II)?Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)NNNCu(I) bridging geometry. The effects of CO coordination to half , combined with the presence of a low energy charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half is found to be capable of coordination of a second at the copper(II) site. 相似文献
3.
Under anaerobic conditions, ethyl hydroperoxide functions as a two-electron acceptor in the tyrosinase-catalyzed oxidation of 4-tert-butylcatechol to 4-tert-butyl-o-benzoquinone, apparently by the following mechanism: This is a direct demonstration of the pseudoperoxidase activity of tyrosinase. Ethyl hydroperoxide failed to oxidize either oxy- or deoxyhemocyanin. 相似文献
4.
L R deAlvare K Goda T Kimura 《Biochemical and biophysical research communications》1976,69(3):687-694
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: 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
5.
6.
Luis O. Rodriguez Sidney M. Hecht 《Biochemical and biophysical research communications》1982,104(4):1470-1476
Consistent with a recent literature report (Repine, J. E. (1981) , 1001–1003), the release of [3H]-thymine from PM-2 DNA by Fe(II)-H2O2-generated ·OH was suppressed by dimethyl sulfoxide. In contrast, DMSO did not affect [3H]-thymine release mediated by Fe(II)-bleomycin. Under aerobic conditions in the presence of -butyl phenylnitrone, Fe(II)-BLM produces an epr signal that has been presumed to arise by transfer of ·OH or from the “active complex” of bleomycin to the spin trap. Remarkably, high concentrations (80 mM) of PBN had no effect on the ability of Fe(II)-BLM to solubilize [3H]-thymine, although the ability of authentic ·OH to degrade DNA was completely suppressed under these condition. The suproxide dismutase catalyst tetrakis(4-N-methylpyridyl)porphineiron(III) also failed to suppress BLM-mediated DNA degradation. Moreover, the epr signal observed with 1.6 mM Fe(II)-BLM in the presence of 80 mM PBN was found to be much less intense than that produced by 1.6 mM Fe(II) and 290 mM H2O2, but equivalent in intensity to that obtained with 45 mM Fe(II) and exoess H2O2. We conclude that the fragmentation of DNA produced by Fe(II)-BLM can be due neither to free ·OH nor to . We suggest that DNA degradation is initiated by an “active complex” consisting of BLM, metal and oxygen that functions by abstracting H· from susceptible sites on DNA. 相似文献
7.
Ubisemiquinone radicals from the cytochrome b-c1 complex of the mitochondrial electron transport chain--demonstration of QP-S radical formation 总被引:2,自引:0,他引:2
Y Wei C P Scholes T E King 《Biochemical and biophysical research communications》1981,99(4):1411-1419
Stable ubisemiquinone radical(s) in the cytochrome -II complex of bovine heart was observed following reduction by succinate in the presence of catalytic amounts of succinate dehydrogenase. The radical was abolished by addition of antimycin A, but a residual radical remained in the presence of excess exogenous Q2. The radical showed an EPR signal of g = 2.0046 ± .003 at X band (~9.4 GHz) with no resolved hyperfine structure and had a line width of 8.1 ± .5 Gauss at 23°C. The Q band (35 GHz) spectra showed wellresolved -anisotropy and had a field separation between derivative extrema of 26 ± 1 Gauss. This radical is evidently from QP-C. These observations substantiate that the radical is immobilized and bound to a protein. The QP-S radical was demonstrated in the cytochrome -II complex only in the presence of more than a catalytic amount of succinate dehydrogenase and cytochrome . This signal was not antimycin a inhibitory. The signal amplitude paralleled the reconstitutive enzymic activity of succinate-cytochrome reductase from succinate dehydrogenase and the cytochrome -II complex. 相似文献
8.
A weighted reversal potential, E, was defined as: . The concept was shown to be useful in describing threshold phenomena for single and multiple responses by providing explicit criteria which made possible the classification of responses into regenerative or non-regenerative. Within this framework E was also used to analyse the anodic break response and abolishment experiments. Using zero-duration (8-impulse) stimuli, the end of the absolutely refractory period was determined, according to the developed criteria, to be 3·17 t 0·01 ms after the peak of the spike, in the Hodgkin-Huxley model. 相似文献
9.
The proposal that EPR Signal II in spinach chloroplasts is due to a plastoquinone cation radical (O'Malley, P.J. and Babcock, G.T. (1983) Biophys. J. 41, 315a) has been investigated in further detail. The similarity in spectral shape between Signal II and the 2-methyl-5-isopropylhydroquinone cation radical is shown to arise from hyperfine coupling to one methyl group for both radicals. A well-resolved four line EPR spectrum of approximate relative intensity 1:3:3:1 for membrane orientation parallel and perpendicular to the applied magnetic field direction also indicates that the partially resolved structure of Signal II is due to hyperfine interaction with one methyl group, i.e., the 2-CH3 group of the plastoquinone cation radical. The ENDOR band observed for this coupling is similar to that observed for methyl group bands of model quinone radicals. The principal hyperfine tensor values obtained for the methyl group interactions are A⊥ = 27.2 MHz and . The large isotropic coupling value (28.6 MHz) of the plastoquinone cation radical's 2-methyl group in vivo indicates that the antisymmetric orbital is the sole contributor to the spin-density distribution of Signal II. The orientation data also suggest that the plastoquinone cation radical is oriented such that the C-CH3 bond direction, and hence the aromatic ring plane, lies perpendicular to the membrane plane. 相似文献
10.
Laser flash photolysis techniques have yielded rate constants for physical and reactive quenching modes of by nicotine, nicotinamide adenine dinucleotide (oxidized and reduced forms) and the reduced forms of nicotinamide mononucleotide, nicotinamide adenine dinucleotide phosphate and nicotinamide hypoxanthine dinucleotide. In the case of the last four named compounds, kinetic spectroscopy furnished evidence for one-electron transfers to . Specifically, production of O?2 was demonstrated unequivocally by reaction with 1,4-benzoquinone. Quantitative determinations revealed the extent of reactive quenching to be near 60% in each case. 相似文献
11.
The electron spin relaxation of iron-sulphur centres and ubisemiquinones of plant mitochondria was studied by microwave power saturation of the respective EPR signals. In the microwave power saturation technique, the experimental saturation data were fitted by a least-squares procedure to a saturation function which is characterized by the power for half-saturation () and the inhomogeneity parameter (b). Since the theoretical saturation curves were based on a one-electron spin system, it became possible to differentiate between EPR signals of iron-sulphur centres which have similar g values but different values. If the difference in the values of the overlapped components was small, no significant deviation from these theoretical saturation curves was observed, as shown for the overlapped signals of centre S-3 and the Ruzicka centre of mung bean mitochondria. By contrast, the microwave power saturation data for the g = 1.93 signal (17–26 K) of Arum maculatum submitochondrial particles reduced by succinate could not be fitted using one-electron saturation curves. Reduction by NADH resulted in a stronger deviation. Since the iron-sulphur centres of Complex I were present only in an unusually low concentration in A. maculatum mitochondria, it was proposed that an iron-sulphur centre of the external NADH dehydrogenase contributes to the spectrum of centre S-1. For mung bean mitochondria, the g = 1.93 signal below 20 K could be attributed mainly to centre N-2. The microwave power saturation technique was also suitable for detecting magnetic interactions between paramagnetic centres. From the saturation data of the complex spectrum attributable to centre S-3 and an interacting ubisemiquinone pair in mung bean mitochondria (oxidized state) followed that centre S-3 has a faster electron spin relaxation than the ubisemiquinone molecules. It is noteworthy that the differences in the relaxation rates were maintained despite the interaction between centre S-3 and the ubisemiquinones. Furthermore, a relaxation enhancement was observed for centre S-1 of A. maculatum submitochondrial particles upon reduction of centre S-2 by dithionite. This indicated a magnetic interaction between centres S-1 and S-2. 相似文献
12.
Joseph Y. Cheung David M. Regen Madge E. Schworer Carol F. Whitfield Howard E. Morgan 《生物化学与生物物理学报:生物膜》1977,470(2):212-229
The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of methylglucose entry and exit, and , were 8 and 7 mM, respectively. The equilibrium exchange constant, , and the counterflow constant, , were 9 and 11 mM, respectively. The activity constant for transport, , defined as , was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, , was 2 mM and less than the counterflow constant, (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, , was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants ( and ) for transport were significantly larger than those obtained in anaerobic cells, but the remaining two ( and ) remained unchanged. The values, for were 8, 26, 20 and 8 mM, respectively. The activity constant, , decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane. 相似文献
13.
This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, , is resolved into a fast decaying or kinetic component, , and an infinitely slow decaying or static component, . The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; is proportional to the square of the lipid order parameter. An empirical relation between and is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins. 相似文献
14.
J P Albertini A Garnier-Suillerot L Tosi 《Biochemical and biophysical research communications》1982,104(2):557-563
When Fe(II) is added to a bleomycin. DNA mixture in the presence of air a long-lived EPR silent species (I′) is formed; the circular dichroism and absorption spectra of which have been characterized. This complex slowly decays yielding a ferric complex (III′) analogous to the well known low spin Fe(III). BLM species. 相似文献
15.
Raili Myllylä Lutz M. Schubotz Ulrich Weser Kari I. Kivirikko 《Biochemical and biophysical research communications》1979,89(1):98-102
Four superoxide dismutase active copper chelates, Cu(acetylsalicylate)2, Cu(salicylate)2, Cu(lysine)2 and Cu(tyrosine)2, proved to be inhibitors of prolyl and lysyl hydroxylase. The kinetics of the inhibition are consistent with the proposal that these compounds dismutated at the active site of the enzymes. The data strongly suggest that is the active form of O2 in the prolyl and lysyl hydroxylase reactions. 相似文献
16.
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
17.
The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent Km values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g1 = 2.007 and g11 = 2.051, which are characteristic of . The generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes. 相似文献
18.
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
19.
The risetime of EPR signal IIvf (S IIvf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction (). These data are consistent with a model where the species Z responsible for S IIvf is the immediate electron donor to P-680+ in spinach chloroplasts. A new, faster decay component of S IIvf has also been detected in these experiments. 相似文献
20.
The kinetics of fructose uptake was determined in perfused rat liver during steady-state fructose elimination. On the basis of the corresponding values of fructose concentration in the affluent and in the effluent medium, and the fructose and ATP concentration in biopsies, the kinetics of membrane transport and intracellular phosphorylation in the intact organ was calculated according to a model system. Carrier-mediated fructose transport has a high (67 mM) and (30 μmoles · min?1 ·g?1). The calculated kinetic constants of the intracellular phosphorylation were compared with values obtained with an acid-treated rat liver high speed supernatant (values given in parentheses). with fructose 1.0 mM (0.7 mM), with ATP 0.54 mM (0.37 mM), 10.3 μmoles · min?1 · g?1 (10.1 μmoles · min?1 · g?1, calculated on the basis of the highest measured rate of fructose uptake correcting the ATP concentration to saturating values). The kinetics of fructose uptake reveals that at Physiological fructose concentrations the membrane transport limits the rate of fructose uptake, thus protecting the liver from severe depletion of adenine nucleotides. 相似文献