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1.
Infrared spectra of N2O in a variety of solvents and in the brain of a dog under typical conditions of halothane-N2O anesthesia have been determined. The appearance or disappearance of N2O in the brain was readily followed as N2O was administered or withdrawn. The sites in brain were of two major types; one, with ν3 = 2229.8 ± 0.4 cm?1 and Δν12 = 13.0 ± 0.6 cm?1, is rather like the polar site in water and the other, with ν3 = 2216.8 ± 0.8 cm?1 and Δν12 = 9.6 ± 1.0 cm?1, is non-polar and is probably associated with membrane lipid. The significant variations in the antisymmetric stretch (ν3) of N2O as the polarity and other properties of the medium (solvent) vary make possible the characterization of in tissue sites occupied by this anesthetic.  相似文献   

2.
α-Chymotrypsin, converted to the acetyl enzyme by the p-nitrophenyl esters of CH3COOH, CH2DCOOH, CHD2COOH, and CD3COOH, undergoes deacetylation at pH 7.6 (phosphate buffer) and 25°C with secondary isotope effects of k(CH3)k(CH2D) = 0.985 ± 0.006, k(CH3)k(CHD2) = 0.971 ± 0.010, and k(CH3)k(CD3) = 0.956 ± 0.008. These isotope effects obey the simple additivity rule (“Rule of the Geometric Mean”) to within 20 J/mol, corresponding to about 5–6% of the maximum isotope effect for carbonyl addition. Thus, to this level, the three hydrogenic sites of the acetyl group are not rendered distinct in their contributions to the overall isotope effect even in the chiral environment of the chymotrypsin active site.  相似文献   

3.
R.L. Pan  S. Izawa 《BBA》1979,547(2):311-319
NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100–250 μequiv · h?1 · mg?1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 → Photosystem II → dimethylquinone reaction supports phosphorylation with a Pe2 ratio of 0.25–0.35 and proton uptake with H+e values of 0.67 (pH 8)–0.85 (pH 6). These are close to the Pe2 value of 0.3–0.38 and the H+e values of 0.7–0.93 found in parallel experiments for the H2O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I?, ferrocyanide).  相似文献   

4.
The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. GM1 and GD1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris—HCI buffer (pH 6.8) at 37°C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 Å argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c0) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius RH, and also contained information on the polydispersity of the sample. We find co < 1 × 10?6 M for both GM1 and GD1a, M = 532 000 ± 50 000 and RH = 63.9 ± 2 A? for GM1, and M = 417 000 ± 40 000 and RH = 59.5 ± 2 A? for GD1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range. and, in particular of Ca2+, did not influence significantly the values of co and the main features of the micelle.  相似文献   

5.
The Rotational Isomeric States model is applied to calculate dipole moments of polypeptides of the twenty natural α-amino acids in the random coil state. Dipole moments of each repeat unit (μi), are evaluated using a quantum mechanics procedure. Dipole moment ratios (Dx = 〈μ2xμi2, x = number of repeat units) of homopolypeptides are calculated and extrapolated to x →?. With a few exceptions, D? = 0.36 ± 0.1. Ten actual proteins and three enzymes are also studied; their dipole ratios (Dx′ =〈μ〉/x) range from 7.34 to 10.57 in 10?59 C2 m2 (6.6–9.5 D2). Diffferences in the values of Dx′ are due mainly to the different contributions, μi, of the amino acid residues contained in each polymer, whereas the sequence of amino acids has a very minor effect.  相似文献   

6.
Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]0 ? [S]0 and [S]0 ? [E]0 conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant (k2Ks)max = 4.2 × 107 M?1 s?1. The limiting values of Ks′ (dissociation constant of E · S), K2 (acylation rate) and k3 (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10?5 m, 360 ± 15 s?1 and 29.3 ± 0.8 s?1, respectively. Likewise small values were observed for Ki of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and Km of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a k?1k2 value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k2 value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.  相似文献   

7.
A method for calculating the rate constant (KA1A2) for the oxidation of the primary electron acceptor (A1) by the secondary one (A2) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of KA1A2 obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: (4.5±1.4) · 103s?1 and (6.9±1.2) · 103 s?1, respectively.The proposed method has also been used for the estimation of the KA1A2 value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P+ at a rate exceeding this for the transfer of electron from A1 to A2. The method of Parson cannot be used in this case. The value of KA1A2 has been found to be (2.7±0.8) · 103 s?1.The activation energies for the A1 to A2 electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.  相似文献   

8.
Hydrolysis of benzyloxycarbonyl-GlyGlyPhe by nitro(Tyr 248)carboxypeptidase A over the pH range 4.88–8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of Km is scarcely affected. The peptidase activity has a pH dependence characterized by the following parameters: pKE1 of 6.37 ± 0.19 and pKE2 of 6.60 ± 0.17 in kcatKm, and apparent pK of 5.59 ± 0.06 in Kcat. A spectroscopic pK of 6.75 ± 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pKE2 in the kcatKm profile, which appears to be shifted from a higher value, pKE2 of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the kcat profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, based on chemical modification and crystallographic evidence for the enzyme, is described.  相似文献   

9.
10.
An investigation of the effect of PGF2α induced parturition alone or in combination with neostigmine (NEO) on subsequent productivity was conducted in a total confinement swine production unit. Forty-five crossbred gilts and 106 sows were randomly assigned to one of six treatments: (1) 0 mg PGF2 α0 mg NEO, (2) 10 mg PGF2 α0 mg NEO, (3) 0 mg PGF2 α5 mg NEO, (4) 10 mg PGF2 α5 mg NEO, (5) 0 mg PGF2 α10 mg NEO, (6) 10 mg PGF2 α10 mg NEO. PGF2α injections (IM) were given 2 days prior to the mean expected farrowing day for this farm (day 112). NEO injections (IM) were given after the fourth pig born in gilt litters and after the fifth pig born in sow litters. Females injected with 0 or 10 mg PGF2α farrowed 71.7 ± 3.7 and 31.4 ± 3.4 hours after treatment, respectively (P<0.01). No other parameters measured were significantly affected by injection of PGF2α. These data show that PGF2α effectively induced parturition and did not adversely affect subsequent litter productivity. NEO was found to be ineffective in reducing total farrowing time (P>0.05).  相似文献   

11.
《Inorganica chimica acta》1986,115(2):169-172
2-(Methylamino)pyridine reacts with RuCl2(CO)3 to give a carbamoyl complex, [Ru(C(O)N(CH3)(C5H4N)Cl(CO)2], which yields with pyridine (py) and acetylacetone (Hacac), respectively, [Ru(C(O)N(CH3)C5H4N)Cl(CO)2(py)] and [Ru(C(O)N(CH3)C5H4N)(CO)2(acac)]. These complexes are characterized spectroscopically. The amino group of the ligand is carbonylated and the resulted carbamoyl ligand is chelating through a pyridine ring-N and a carbamoyl-C atom. 2-Aminopyridine and 2-aminopyrimidine react similarly with RuCl2(CO)3 to give the corresponding carbamoyl complexes.  相似文献   

12.
A precise continuous photometric assay has been devised and utilized for mechanistic studies of chicken and rat liver microsomal epoxide hydrolase (EH). The assay is based on monitoring the hydration of p-nitrostyrene oxide (PNSO) at 310 nm. Rat liver EH hydrates S-(+)- and R-(?)-PNSO differentially, the Km and V values for the former being ca. four times those for the latter; in contrast, enantiomeric differences are negligible with chicken liver EH. With rat EH V increases slightly from pH 7 to 8 and then falls rapidly from pH 8 to 9.5; Km remains constant from pH 7 to 8 and then increases steadily from pH 8 to 9.5. In 86 mol% D2O the solvent isotope effect on V (H2OD2O) is 1.103 ± 0.015. Both rat and chicken EH show a 3% inverse isotope effect for the hydration of [7-2H]PNSO and a 4% normal isotope effect for the hydration of [8-2H2]PNSO. These observations are discussed in terms of the possible participation of acid as well as base catalysis in the enzymatic mechanism.  相似文献   

13.
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   

14.
(1) H+/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 H+O, H+N2O, H+NO?2 for reduction to N2 and H+NO?2 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. H+N2O, H+NO?2 for reduction to N2 and H+NO?2 for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The H+oxidant ratios, mentioned in item 2, were not altered in the presence of valinomycinK+ 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 H+electron acceptor ratios predicted by this scheme are in good agreement with the experimental values.  相似文献   

15.
Using guanidinium and n-butylammonium cations (C+) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H2A, three association complexes may exist: K1M = [CHA][C+] [HA?]; K1D = [CA?][C+] [A2?]; K2D = [C2A][C+] [CA?]. For guanidinium ion and phosphate, K1M = 1.4, K1D = 2.6, and K2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pKa = 4.8, K1M = 0.37; formate, pKa = 3.8, K1M = 0.32; and chloroacetate, pKa = 2.9, K1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.  相似文献   

16.
Electron paramagnetic resonance (EPR) spectroscopy of the iron-semiquinone complex in photosynthetic bacterial cells and chromatophores of Rhodopseudomonas viridis is reported. Magnetic fields are used to orient the prolate ellipsoidal-shaped cells which possess a highly ordered internal structure, consisting of concentric, nearly cylindrical membranes. The field-oriented suspension of cells exhibits a highly dichroic EPR signal for the iron-semiquinone complex, showing that the iron possesses a low-symmetry ligand field and exists in a preferred orientation within the native reaction-center membrane complex. The EPR spectrum is analyzed utilizing a spin hamiltonian formalism to extract physical information describing the electronic structure of the iron and the nature of its interaction with the semiquinones. Exact numerical solutions and analytical expressions for the transition frequencies and intensities derived from a perturbation theory expansion are presented, and a computer-simulated spectrum is given. It has been found that, for a model which assumes no preferred orientation within the plane of the membranes, the orientation of the Fe2+ ligand axis of largest zero-field splitting (Z, the principal magnetic axis) is titled 64±6° from the membrane normal. The ligand field for Fe2+ has low symmetry, with zero-field splitting parameters of |D1|=7.0±1.3 cm?1 and |E1|=1.7±0.5 cm?1 and |E1D1|=0.26 for the redox state Q1?Fe2+Q2?. The rhombic character of the ligand field is increased in the redox state Q1Fe2+Q?2, where 0.33>|E2D2|>0.26. This indicates that the redox state of the quinones can influence the ligand field symmetry and splitting of the Fe2+. There exists an electron-spin exchange interaction between Fe2+ and Q?1 and Q?2, having magnitudes |J1|=0.12±0.03 cm?1 and |J2|?0.06 cm?1, respectively. Such weak interactions indicate that a proper electronic picture of the complex is as a pair of immobilized semiquinone radicals having very little orbital overlap (probably fostered by superexchange) with the Fe2+ orbitals. The exchange interaction is analyzed by comparison with model systems of paramagnetic metals and free radicals to indicate an absence of direct coordination between Fe2+ and Q?1 and Q?2. Selective line-broadening of some of the EPR transitions, involving Q? coupling to the magnetic sublevels of the Fe2+ ground state, is interpreted as arising from an electron-electron dipolar interaction. Analysis of this line-broadening indicates a distance of 6.2–7.8 ? between Fe2+ and Q?1, thus placing Q1 outside the immediate coordination shell of Fe2+.  相似文献   

17.
Salivary glands of Chironomus thummi larvae were incubated in media composed of those NaClKCl, MgCl2NaClorMgCl2KCl combinations and at concentrations which they tolerated without visible damage. Resulting changes in puffing activity were recorded for three chromosomal segments. Within certain combinatorial ranges NaClKCl, MgCl2NaClandMgCl2KCl induced puffs in the three segments. Each inducing range is depicted as a ‘puff-inducing field’ for extracellular ion concentrations (IFe) in a two-dimensional lattice. The IFes are coherent, distinctly delineated and highly overlapping. At most places a transition from 0 to 100 % puff induction (induction of respective puff in each nucleus) depends on changes in media composition of 10–20 mM. Ion sensitivities of the three chromosomal segments were computed for NaCl/KCl/MgCl2 combinations and were found to conform to actual puff-inducing capacities of selected NaCl/KCl/MgCl2 media.  相似文献   

18.
Reversible flbrinogen polymer formation was examined at pH 6.6 and Γ/2 0.3. The equilibrium fraction of fibrinogen present as polymer, (Pmf)e, was determined by gel filtration for fibrinogen concentrations, FO, from 48 to 166 μm. Using FO in molarity, the experimental relation is ln [FO(Pmf)e] = 3.53 ln[FO(1 ? (Pmf)e)] + 23.73. This relation and attendant confidence limits are examined assuming, during filtration, that the original polymer population is either stable or selected polymer species dissociate to monomer. The possibility that all polymers are open is excluded since the calculated microscopic association constant would then increase with FO. Acceptable models are based on the assumptions that polymers are open, with association constant Ka, until restricted by closure, with association constant Kr, at an integral degree of polymerization, n. Values are selected on the basis that interaction parameters are independent of FO and that the required molar decrease in free energy is a minimum. Assuming polymer stability, the experimental relation at 273 °K gives n = 4, KrKa = 1.2 m, and Ka = 736 m?1. Temperature dependence gives ΔH= ?16.9 kcal/mol and ΔSOa = ?48.8 e.u. KrKa indicates a relation between changes in entropy. The probability is >0.90 that KrKa ? 56 m, which indicates a greater loss of degrees of freedom on closure than on association. Conclusions are not altered by the assumption that only the closed polymer species is stable. As ionic strength is decreased at pH 6.6, Ka increases. The clotting time of an otherwise constant system decreases as system Pmf is increased.  相似文献   

19.
Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (ka) for complex formation (ka = 54,000 s?1m? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k?a = 6.2 s?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio ka?a (0.9 × 104m?1) was in reasonable agreement with value of 1.1 ± 0.1 × 104m?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln(kaT) and ln(kaT) vs 1T were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.  相似文献   

20.
If the bicyclic peptide ring proposed by Gross etal. (1,2) does in fact exist in nisin and related antibiotics, then the unusual β-methyllanthionine component must be significantly distorted from its conformation in the free state, as determined by x-ray structure analysis. The torsion angles about the SCβ bonds are 50–100° from the torsion angles in models of the sulfur-bridged peptide ring proposed for nisin. The chirality of the methylated β-carbon atom is (S). The conformation of the amino acid differs from that of meso-lanthionine only by a 180° rotation of a carboxyl group about the CαDCβ(CH3) bond.  相似文献   

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