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1.
Anthroylcholine was utilized as an extrinsic fluorescent probe in rapid kinetic studies of calcium dissociation from calmodulin (koff = 10 S?1) and the calmodulin-troponin I complex (koff = 6 S?1). At concentrations lower than 70 μM, the mechanism of dye binding agreed with the simple kinetic scheme in which the dye binds exclusively to the respective calcium complexes of calmodulin and calmodulin-troponin I. The sensitivity of anthroylcholine also made possible the estimation of values for the association (1.0 ± 0.8) × 108M?1 S?1) and dissociation rate constants (2 ± 170 S?1) for troponin I binding to the calcium4-calmodulin complex.  相似文献   

2.
Previous proton nuclear magnetic resonance (nmr) studies have indicated that inositol hexaphosphate (IHP) can stabilize hemoglobin (Hb) Kansas in a deoxy-like quaternary structure even when fully liganded with carbon monoxide (CO) (S. Ogawa, A. Mayer, and R. G. Shulman, 1972, Biochem. Biophys. Res. Commun., 49, 1485–1491). In the present report we have investigated both CO binding at equilibrium and the CO binding and release kinetics to determine if Hb Kansas + IHP is devoid of cooperativity, as would be suggested by the nmr studies just quoted. The equilibrium measurements show that Hb Kansas + IHP has a very low affinity for CO (P12 = 1.2 mm Hg and Keq = 5.4 × 105M?1) and almost no cooperativity (n = 1.1) at pH 7, 25 °C. The CO “on” and “off” kinetics also show no evidence for cooperativity. In addition, the equilibrium constant estimated from the kinetic rate constants (Keq = 5.2 × 105M?1 with kon = 1.03 × 105M?1 · S? and koff = 0.198 S?1) is in excellent agreement with the equilibrium constant determined directly. Thus, both kinetic and equilibrium measurements allow us to conclude that CO binding to Hb Kansas + IHP occurs without significant cooperativity.  相似文献   

3.
NADPH-cytochrome c reductase has been isolated from a top-fermenting ale yeast, Saccharomyces cerevisiae (Narragansett strain), after ca. a 240-fold purification over the initial extract of an acetone powder, with a final specific activity (at pH 7.6, 30 °C) of ca. 150 μmol cytochrome c reduced min?1mg?1 protein. The preparation appears to be homogeneous by the criteria of: sedimentation velocity; electrophoresis on cellulose acetate in buffers above neutrality; and by polyacrylamide gel electrophoresis. Although the reductase appeared to partially separate into species “A” and “B” on DEAE-cellulose at pH 8.8, the two species have proven to be indistinguishable electrophoretically (above pH 8) and by sedimentation. By sedimentation equilibrium at 20 °C, a molecular weight of ca. 6.8 (± 0.4) × 104 was obtained with use of a V?20 ° = 0.741 calculated from its amino acid composition. After disruption in 4 m guanidinium chloride- 10 mm dithioerythritol- 1 mm EDTA, pH 6.4 at 20 °C, an M?r of 3.4 (± 0.1) × 104 resulted, which points to a subunit structure of two polypeptide chains per mole. Confirmatory evidence of the two-subunit structure with similar, if not identical, polypeptide chains was obtained by polyacrylamide gel electrophoresis in dodecyl-sulfate, after disruption in 4 m urea and 2% sodium dodecyl sulfate, and yielded a subunit molecular weight of ca. 4 × 104. Sulfhydryl group titration with 4,4′-dithiodipyridine under acidic conditions revealed one sulfhydryl group per monomer, which apparently is necessary for the catalytic reduction of cytochrome c. NADPH, as well as FAD, protects this-SH group from reaction with 5,5′-dithiobis (2-nitrobenzoate). The visible absorption spectrum of the oxidized enzyme (as prepared) has absorption maxima at 383 and 455 nm, typical of a flavoprotein. Flavin analysis (after dissociation by thermal denaturation of the “A” protein) conducted fluorometrically, revealed the presence of 2.0 mol of FAD per 70,000 g, in confirmation of the deduced subunit structure. The identity of the FAD dissociated from either “A” or “B” protein was confirmed by recombination with apo-d-amino acid oxidase and by thin-layer chromatography. A kinetic approach was used to estimate the dissociation constant for either FAD or FMN (which also yields a catalytically active enzyme) to the apoprotein reductase at 30 °C and pH 7.6 (0.05 m phosphate) and yielded values of 4.7 × 10?8m for FAD and 4.4 × 10?8m for FMN.  相似文献   

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Kinetic parameters for high affinity [HA] uptake in vitro in synaptosomes from different mouse brain regions were investigated. Vmax was highest in the striatum [200 pmol.· mg protein?1 · 4 min?1], followed by the cortex [111 pmol · mg protein?1 · 4 min?1], hippocampus [63 pmol · mg protein?1 · 4 min?1], midbrain [21 pmol · mg protein?1 · 4 min?1] and, lowest, medulla oblongata [5 pmol · mg protein?1 · 4 min?1]. Km was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between Vmax for synaptosomal HA uptake of Ch in vitro and apparent turnover of ACh in vivo was found between the brain regions. Administration of oxotremorine [1 mg·kg?1 i.p.] decreased Vmax for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.  相似文献   

7.
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   

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2-deoxyglucose uptake rates at low sugar concentrations (less than 500 μM) appeared to be lower than those predicted by the Michaelis-Menten model which correctly described higher concentrations. This phenomenon which we will call concentration-dependent transport lag, was also observed for L-glucose uptake which suggest that this phenomenon is carrier-independent. A model involving the perimembrane space is developed which, for L-glucose, gives k1 = 0.931 ± 0.072 × 10?6 l. mg protein?1. minute?1, k2 = 2.97 ± 0.19 × 10?7 l. mg protein?1. minute?1 and So = 88,8 ± 4,3 μM; where k1 is the diffusion constant in the cell membrane, k2 is the diffusion constant in the perimembrane space and So the sugar concentration required in the external medium in order to provide an équivalent sugar concentration in the transport carrier area.  相似文献   

11.
Relaxation measurements on the kinetics of the double helix to coil transition for the self-complementary ribo-oligonucleotide A7U7 are reported over a concentration range of 6.9 μM to 19.6 μM in single strand in 1 M NaCl. The rate constants for helix formation are about 2 × 106 M?1 s?1 and decrease with increasing temperature yielding an activation enthalpy of ?6 kcalmole. The rate constants for helix dissociation range from 3 to 250 s?1 and increase with increasing temperature yielding an activation enthalpy of +45 kcalmole. The kinetic data reported here for 1 M NaCl is compared with previously published results obtained at lower salt concentrations. These data are discussed in terms of the quantitative effect of ionic strength on the kinetics of helix-coil transitions in oligo- and polynucleotides.  相似文献   

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Brinolase, a thrombolytic fungal protease capable of forming vasoactive kinins, has been shown to hydrolyze kinins after their formation. Using synthetic bradykinin as a substrate, the kinetics and mechanism of hydrolysis have been elucidated, evidently explaining the apparently low kinin formation in vivo, Bradykinin hydrolysis proceeded rapidly in vitro with a pH optimum of 7.0–7.5, and a half-life of 5.1 min, using 250 ng/ml bradykinin and 50 μg/ml brinolase. The Km was 3.2×10?6 M and the Vmax was 4.6 × 10?8 mol/liter/min, using 5 μg/ml brinolase. Two-dimensional paper fractionation of the brinolase-bradykinin digest revealed the presence of free arginine amongst the five peptide fragment spots.  相似文献   

14.
In the determination of the rates of oxygen consumption with a Clark-type oxygen electrode, and experimental error is caused by finite response time of the oxygen electrode for a rapid oxidation reaction. A theoretical equation between the observed pseudo first-order rate constant (kobs) and the true rate constant (k)
1kobs=1k+T
where T is a time constant for a first-order response of the oxygen electrode, was derived and found to hold up to k = 23 min?1 in oxidation of hydroquinone at pH 7.60–8.63.  相似文献   

15.
The magnesium ion-dependent equilibrium of vacant ribosome couples with their subunits
70 S?k?1k150 S+30S
has been studied quantitatively with a novel equilibrium displacement labeling method which is more sensitive and precise than light-scattering. At a concentration of 10?7m, tight couples (ribosomes most active in protein synthesis) dissociate between 1 and 3 mm-Mg2+ at 37 °C with a 50% point at 1.9 mm. The corresponding association constants Ka′ are 5.1 × 105m?1 (1 mm-Mg2+), 3.5 × 107m?1 (2 mm), and 1.2 × 109m?1 (3 mm), about five orders of magnitude higher than the Ka′ value of loose couples studied by Spirin et al. (1971) and Zitomer & Flaks (1972).In this range of Mg2+ concentrations (37 °C, 50 mm-NH4+) the rate constants depend exponentially and in opposite ways on the Mg2+ concentration: k1 = 2.2 × 10?3s?1, k?1 = 7.7 × 104m?1s?1 (2mm-Mg2+); k1 = 1.5 × 10?4s?1, k?1 = 1.7 × 107m?1s?1 (5 mm-Mg2+). Under physiological conditions (Mg2+ ~- 4 mm, ribosome concn ~- 10?7m), the equilibrium strongly favors association and the rate of exchange is slow (t12 ~- 10 min). In the range of dissociation (2 mm-Mg2+), association of subunits proceeds without measurable entropy change and hence ΔGO = ΔHO. The negative enthalpy change of ΔHO = ? 10 kcal suggests that association of subunits involves a shape change.Below a critical Mg2+ concentration (~- 2 mm), the 50 S subunits are converted irreversibly into the b-form responsible for the transition to loose couples. The results are compatible with two classes of binding sites, one class binding Mg2+ non-co-operatively and contributing to the free energy of association by reduction of electrostatic repulsion, and another class probably consisting of hydrogen bonds between components at opposite interfaces whose critical spatial alignment rapidly denatures in the absence of stabilizing magnesium ions.  相似文献   

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Hybrids were constructed between E. coli K12 chl? mutants defective in nitrate respiration and an F′ plasmid carrying nitrogen fixation genes from K. pneumoniae. Examination of these hybrids showed that expression of nifKp+ genes does not require a functional nitrate respiratory system, but that nitrate reductase and nitrogenase do share some Mo-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the Mo-X cofactor encoded by chl genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity.  相似文献   

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The kinetics of methemoglobin reduction by Fe(EDTA)2? have been studied and found to follow a second order rate law with k = 29.0 M?1 s?1 [25°C, μ = 0.2 M, pH 7.0 (phosphate)], ΔH3 = 5.5 ± 0.7 kcal/mol, and ΔS2= ?33 ± 2 e.u.. The electrostatics-corrected self-exchange rate constant (k11corr) for hemoglobin based on the Fe(EDTA)2? cross-reaction is 2.79×10?3M?1 s?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)2? and Fe(CDTA)2?/?. The k11corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.  相似文献   

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