共查询到20条相似文献,搜索用时 31 毫秒
1.
M.F. Budyka A.M. Khenkin A.A. Shteinman 《Biochemical and biophysical research communications》1981,101(2):615-622
Oxygenation of heme-mercaptide as well as spectroscopic characteristics of the dioxygen complex formed have been studied. Absorption and magnetic circular dichroism spectra of the O2 complex support the retention of mercaptide in the heme fifth position. A release of in the decomposition of the oxygenated complex and an independent formation of the latter from hemine-dimercaptide and together with electron paramagnetic resonance and Mössbauer data support the O2 presence in the heme coordination sphere. The similarity of optical and magnetic circular dichroism spectra and the closeness of the ratio for oxy-heme-mercaptide and oxycytochrome P450 unequivocally confirm the presence of an axial cystein mercaptide ligand in oxycytochrome P450. 相似文献
2.
The binding characteristics of the β-adrenergic antagonist, [3H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity () but low capacity ( membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both β-antagonists and β-agonists, competition at the low-affinity sites occurred only with β-antagonists and was non-stereoselective. Thus, the β-agonist (?)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity () binding sites with a binding capacity of membrane protein (corresponding to sites per cell), and independent kinetic analysis provided a value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a subtype, yielding values in good agreement with the and the values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of β-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [3H]dihydroalprenolol binding sites defined as the binding displaceable by (?)-isoproterenol represent the physiologically relevant β-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (?)-norepinephrine showed that the inhibitory effect of the of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the in the adrenergic control of lipolysis in hamster white adipocytes. 相似文献
3.
The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO3? or (OHCH2CH2)2N+C(CH2OH)3 HCl (BisTris+HCl). A small kinetic hydrogen-deuterium isotope effect () was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase. 相似文献
4.
Zoltán Spolarics Jozsef Mandl Raymund Machovich Patrick Lambin Tamás Garzó Ferenc Antoni István Horváth 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,845(3):389-395
125I-labelled complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent values calculated from displacement experiments were 7.9 · 10?8 M for and 8.5 · 10?8 M for . Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. itself bound also to hepatocytes, but the affinity of the complexes was higher than that of the inhibitor alone, and was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their complexes. The complex competed with the complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for and antithrombin III-proteinase complexes are different. 相似文献
5.
Determination of delta psi, delta pH and the proton electrochemical gradient in isolated cholinergic synaptic vesicles 总被引:1,自引:0,他引:1
The electrical potential (Δψ) of intact cholinergic synaptic vesicles was measured in the presence and absence of the proton translocator carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), and the results were utilized to calculate the vesicular proton chemical gradient (ΔpH) and proton electrochemical potential . At external pH = 7.4 the vesicles maintain a proton electrochemical gradient of (positive inside) which is composed of (negative inside) and (acidic inside). The proton chemical gradient (ΔpH) increases as a function of pHout whereas the vesicular electrical potential (Δψ) is only slightly affected by the external pH. Consequently, is larger at basic external pH values ( at pHout = 9.0) and smaller at acidic external pH values ( at (pHout = 5.6). The possible physiological role of the electrochemical potentials in maintaining high concentrations of acetylcholine within the cholinergic synaptic vesicle is discussed. 相似文献
6.
According to previous authors, cytochrome , when extracted from bovine liver by a detergent method, is called cytochrome . On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome .Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, , decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome , whereas does not change for cytochrome and cytochrome . This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome , because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature. 相似文献
7.
The reduction-oxidation reactions of NADH and cytochrome to incipient oxygen insufficiency caused by nitrogen ventilation or hemorrhagic hypotension were examined in the exposed cerebral cortex of the cat. A comparison of the onset of redox changes with each procedure shows that cytochrome reduction precedes the reduction of mitochondrial NAD. This constitutes evidence that, in the living brain, NADH maintains its resting oxidation state at lower cellular oxygen tensions than cytochrome does, consistent with the differences in oxygen affinity these respiratory chain components exhibit during oxygen titration . 相似文献
8.
Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation with , where ko is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, kn is the rate constant in a protium oxide-deuterium oxide mixture, and is the isotopic fractionation factor. 相似文献
9.
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 () and dissociation () shows a nearly identical 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
10.
Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N4-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C3), -A-U-G-A- (mutant C4) and -A-U-A-A- (mutant ) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C3), 3.2 (C4) and 0.3 (). The finding that mutant C3 no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C4 RNA bound more efficiently than wild type, and that mutant RNA showed substantial ribosome binding capacity whereas the single mutant C3 did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon. 相似文献
11.
Jean-Louis Dasseux Jean-François Faucon Michael Lafleur Michel Pezolet Jean Dufourcq 《生物化学与生物物理学报:生物膜》1984,775(1):37-50
Perturbations induced by melittin on the thermotropism of dimyristoyl-, dipalmitoyl-, distearoylphosphatidylcholine and natural sphingomyelin are investigated and rationalized from data obtained by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy. Depending on the technique and / or experimental conditions used, the observed effects differ at the same lipid to protein molar ratio, due to partial binding of melittin. The binding is more efficient for tetrameric than for monomeric melittin, but in both cases its affinity is weaker for phosphatidylcholine dispersions in the gel phase than for sonicated vesicles. For temperatures efficient binding occurs whatever the initial state of the lipids is. One can summarize the effects induced by melittin on the transition temperature as follows: (i) No upward shift is observed on synthetic phosphatidylcholines when lipid degradation is avoided. This is achieved by using highly purified melittin, phospholipase inhibitors, and / or non-hydrolysable lipids. (ii) Melittin monomer does not change . (iii) When melittin tetramer is stabilized, it decreases by 10–15 deg. C. The transition broadens, and is finally abolished for . Very similar results are found for natural sphingomyelin. Fluorescence polarization indicates similar changes in order and dynamics of the acyl chains for all lipid studied. For , fluorescence and Raman show that melittin decreases the amount of CH2 groups in ‘trans’ conformation and the intermolecular order of the chains. According to fluorescence data, there is an increase of the rigid-body orientational order at , while from Raman the positional intermolecular order decreases without significant change in the CH2 groups ‘trans’/‘gauche’ ratio. 相似文献
12.
Cytochrome was extracted and purified from beef liver by a detergent method (cytochrome ). The hydrophilic moiety which carries the heme group (cytochrome ) was prepared by trypsin action upon pure cytochrome .Single-shelled lecithin liposomes form complexes with cytochromes up to a molar ratio of one protein for 35 phospholipids. The lipid-protein complexes were isolated by gel filtration on Sepharose 4B. They are hollow vesicles in which [3H]-glucose can be trapped. Their diameter is greater than that of the initial liposomes.Cytochrome does not interact with the vesicles. These results show that the hydrophobic tail is necessary for the binding and that the hydrophilic part of the protein is located on the outer face of the vesicles. This asymmetry is also proved by the action of reducing agents.Experiments with saturated phosphatidylcholines show that the protein interacts with the lipids both below the transition temperature . i.e. when the aliphatic chains are in a crystalline state, and above , when the alipathic chain are in a fluid state.1H NMR spectra show that even at the maximum cytochrome concentration the presence of the proteins does not markedly change the dynamics to the phospholipid molecules. An asymmetric single-shelled vesicle structure is proposed for the complex. 相似文献
13.
Shakunthala Narasimhulu 《生物化学与生物物理学报:生物膜》1979,556(3):457-468
The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant () and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome binding reaction and in the temperature dependency of the . 相似文献
14.
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
15.
(i) It is proved that only four independent constants can ever be obtained by extrapolation procedures applied to non-hyperbolic steady-state or binding data, (ii) Analysis of the algebraic graphs , , and ( is shown to require a knowledge of the sign of six curve shape determinants. In each case, the sign is a necessary and sufficient condition for a specific curve shape feature, (iii) The precise graphical effect of positive and negative co-operativity then requires the definition of two reference curves, the osculating hyperbola at zero substrate concentration, OH(0), and the osculating hyperbola at infinite substrate concentration OH(∞). These are better first order approximations than the Hill equation, (iv) Rules for determining unambiguously the sign of initial, final and overall co-operativity coefficients by inspection of non-hyperbolic binding curves are then possible, (v) These rules require that saturation data for: be fitted by computer for low concentrations to the hyperbola: while regression of high substrate concentration data is to: . Comparisons of the best fit pseudo-kinetic constants then gives the type of co-operativity present in an unambiguous way with no assumptions as to molecular mechanism, (vi) These rules are then applied to the MWC and KNF allosteric models of ligand binding and the constraints necessary for specific curve shape effects are given, (vii) The graphical expression of positive or negative final co-operativity depends only on events at high substrate concentration but overall and initial co-operativities produce specific geometric effects depending upon the difference between behaviour of saturation data at both extremes of concentration, (viii) This apparent anomaly is explained by a discussion of the relationships between the osculating hyperbolae, the theoretical parent hyperbola and the Hill plot asymptotes. 相似文献
16.
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
17.
Winston F. Moo-Penn Danny L. Jue Mary H. Johnson Melisenda J. McDonald Susan M. Turci Tsu-Bi Shih Richard T. Jones Bradford L. Therrell Arthur Arnone 《Journal of molecular biology》1984,180(4):1119-1140
Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated α-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity ( and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect ( and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 (l′ = 5.6 × 106m?1s?1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding (l′ = 2.6 × 106m?1s?1), whereas Hb Wayne Asp showed heterogeneous binding (l′ = 6.1 × 106 and 2.6 × 106m?1s?1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function. 相似文献
18.
19.
Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous from methanol to caused an inhibition of calcium uptake and an enhancement of ATPase activity. The treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of vesicles was about twice that of control vesicles. With increasing number () of carbon atoms of the , the maximum increment of ATPase activity increased, and both the alcohol concentration () required to inhibit calcium uptake by 50% and the alcohol concentration () required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows. The ratio, to , was constant for all values. The apparent free energy of binding of the methylene groups of to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of in octanol and water (?670 cal/mole). The effects of on membrane vesicles are discussed on the basis of these data. 相似文献
20.
The oxidation of unsaturated fatty acid micelles by the superoxide free radical (), 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 () and lipid hydroperoxide initiates a chain oxidation process in the micelles. While tetranitromethane, which reacts rapidly with (), 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