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1.
Reduction kinetics of the ferredoxin-ferredoxin-NADP+ reductase complex: a laser flash photolysis study 总被引:1,自引:0,他引:1
The kinetics of reduction of spinach ferredoxin (Fd), ferredoxin-NADP+ reductase (FNR), and the Fd-FNR complex have been investigated by the laser flash photolysis technique. 5-Deazariboflavin semiquinone (5-dRf), generated in situ by laser flash photolysis under anaerobic conditions, rapidly reduced both oxidized Fd (Fdox) (k = 2 X 10(8) M-1 s-1) and oxidized FNR (FNRox) (K = 6.3 X 10(8) M-1 s-1) at low ionic strength (10 mM) at pH 7.0, leading to the formation of reduced Fd (Fdred) and FNR semiquinone (FNR.), respectively. At higher ionic strengths (310 and 460 mM), the rate constant for the reduction of the free Fdox increased about 3-fold (k = 6.7 X 10(8) M-1 s-1 at 310 mM and 6.4 X 10(8) M-1 s-1 at 460 mM). No change in the second-order rate constant for reduction of the free FNRox was observed at high ionic strength. At low ionic strength (10 mM), 5-dRf. reacted only with the FAD center of the preformed 1:1 Fdox-FNRox complex (k = 5.6 X 10(8) M-1 s-1), leading to the formation of FNR.. No direct reduction of Fdox in the complex was observed. No change in the kinetics occurred in the presence of excess NADP+. The second-order rate constant for reduction of Fdox by 5-dRf. in the presence of a stoichiometric amount of fully reduced FNR at low ionic strength was 7 X 10(6) M-1 s-1, i.e., about one-thirtieth the rate constant for reduction of free Fdox.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
Complex formation and the kinetics of electron transfer between ferredoxin-NADP+ reductase (FNR) and two structurally homologous acidic 4Fe-4S high-potential ferredoxins (HiPIP's) from Ectothiorhodospira halophila (HP1 and HP2) and two structurally homologous cytochromes c2 from Paracoccus denitrificans and Rhodospirillum rubrum (PC2, and RC2, respectively) have been investigated by gel filtration and laser flash photolysis techniques. Gel filtration studies indicated that complex formation occurred between FNRox and HP1ox or HP2ox at low ionic strength (10 mM) and that the complexes were completely dissociated at high ionic strength (310 mM). Laser flash photolysis using lumiflavin as the reductant demonstrated that both free HP1ox and HP2ox reacted primarily with the anionic form of fully reduced lumiflavin (LFH-), whereas FNR was unreactive. Second-order rate constants of 1 X 10(6) and 0.8 X 10(6) M-1 s-1 were obtained for these reactions at 10 mM ionic strength. Increasing the ionic strength to 310 mM resulted in an approximately 1.5-fold increase in the rate constant. Inclusion of stoichiometric amounts of FNRox into the reaction mixture at low ionic strength led to a 2.5-fold increase in the rate constants. The reaction of 5-deazariboflavin semiquinone (5-dRf.) with the oxidized HiPIP's was also investigated by laser flash photolysis. Second-order rate constants of 3.0 X 10(8) M-1 s-1 (HP1) and 2.5 X 10(8) M-1 s-1 (HP2) were obtained for the free proteins at 10 mM ionic strength. Under the same conditions, 5-dRf. reacted with free FNRox, resulting in the formation of the neutral protein-bound semiquinone (FNR.), with a second-order rate constant of 6 X 10(8) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
V M Dvorkin 《Biulleten' eksperimental'no? biologii i meditsiny》1983,96(11):44-46
Gangliosides in aqueous media of low ionic strength (2-5 mM NaCl) and in concentrations over the critical ones (10(-5) M) form micellas which do not differ from liposomes as regards the chromatographic behavior on Sepharose 4R, with a molecular weight of greater than or equal to 10(7) dalton. In aqueous media of a higher ionic strength (greater than or equal to 20 mM NaCl), gangliosides form micellas which are eluted during chromatography in far later fractions than liposomes 70-80 nm in diameter, with a molecular weight of (1-5) X 10(5) dalton. It is assumed that the conclusions about ganglioside incorporation into the liposomal membrane made on the basis of their peaks coincidence are correct, provided that ganglioside-containing liposomes are obtained and chromatographed under high ionic strength (greater than or equal to 20 mM NaCl). 相似文献
4.
A comparative study was made of gamma-inactivation of subtilisin-72 solutions in 5 X 10(-3) M acetate buffer and 0.1 M NaCl in the presence and absence of Ca2+ ions. It was shown that the acetate buffer had a protective action, and the influence of Ca2+ ions depended on the ionic strength of the solution. In general, Ca2+ ions exerted a stabilizing effect irrespective of the subtilisin concentration in the acetate buffer, but this effect competed with the destabilizing influence of the ionic strength increased by Ca2+ ions. 相似文献
5.
Influence of Ca2+ on the structure of reptilase-derived and thrombin-derived fibrin gels. 总被引:1,自引:0,他引:1
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The effects of Ca2+ ion on the structure of thrombin-derived and reptilase-derived fibrin gels formed at various ionic strengths were studied turbidimetrically. For both enzymes clotting times were shorter, final gel turbidities were higher and fibre mass/length ratios were increased as the ionic strength was lowered. The addition of 5 mM-Ca2+ augmented each of these effects for any given ionic strength. In the thrombin system, Ca2+ increased the final gel turbidity from 0.04 to 0.26 A632.8 at ionic strength 0.15. Under identical conditions in the reptilase system, the final gel turbidity increased from 0.03 A632.8 in the absence of Ca2+ to 0.345 A632.8 in the presence of 5 mM-Ca2+. In the thrombin system, fibre mass/length ratios increased from 0.4 X 10(12) to 6.9 X 10(12) Da/cm in the absence of Ca2+, and from 4.4 X 10(12) to 7.9 X 10(12) Da/cm in the presence of Ca2+, as the ionic strengths were decreased from 0.15 to 0.08 and to 0.11 respectively. In the reptilase system, the mass/length ratios increased from 0.9 X 10(12) to 5.8 X 10(12) Da/cm in the absence of Ca2+, and from 4.8 X 10(12) to 8.7 X 10(12) Da/cm in the presence of Ca2+, as the ionic strengths were decreased from 0.15 to 0.08 and to 0.10 respectively. At ionic strengths below 0.10, the presence of 5 mM-Ca2+ caused precipitation and macroscopic aggregation of fibrinogen upon the addition of either enzyme. In the presence of 5 mM-Ca2+, the fibres composing thrombin-induced and reptilase-induced gels were virtually identical. 相似文献
6.
T B Hakvoort K M Sinjorgo B F Van Gelder A O Muijsers 《Journal of inorganic biochemistry》1985,23(3-4):381-388
The aggregation state of two types of bovine heart cytochrome c oxidase preparations in the presence of laurylmaltoside was investigated by high performance liquid chromatography in two buffers of ionic strengths of 388 mM and 45 mM, respectively. At high ionic strength, it was found that the Fowler cytochrome c oxidase preparation was monomeric (Mr = 2 X 10(5)), while monomers and dimers (2 X aa3, Mr = 4 X 10(5)) could be isolated from the Yonetani preparation. Under these conditions there was no rapid equilibrium between the two forms. Covalent cytochrome c oxidase-cytochrome c complexes were largely dimeric, and addition of ascorbate and cytochrome c to the oxidase also promoted dimerization. At low ionic strength (I = 45 mM) in the presence of laurylmaltoside the oxidase and the covalent complex with cytochrome c were largely monomeric. In the steady-state oxidation of ferrous horse heart cytochrome c, the monomeric enzyme displayed biphasic kinetics at I = 45 mM. This suggests that the presence of high- and low-affinity reactions is an intrinsic property of the cytochrome c oxidase monomer. 相似文献
7.
C T Przysiecki A K Bhattacharyya G Tollin M A Cusanovich 《The Journal of biological chemistry》1985,260(3):1452-1458
The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1). 相似文献
8.
NAD glycohydrolase from Neurospora crassa conidia has been immobilized by hydrophobic interaction on Sepharose 4B beads coated with propyl residues through CNBr activation. The bond resulted stable under a wide range of conditions (ionic strength, temperature, pH). As a result of immobilization the pH optimum for catalytic activity shifted by about 0.2 pH unit in the acidic direction, to lie between 7.5 and 7.3. The stability of the enzymatic activity was largely enhanced by effect of immobilization but the Km value towards NAD+ was increased compared with that of the free enzyme (1 X 10(-3) and 2 X 10(-4) M respectively). 相似文献
9.
The kinetics and mechanism of the reduction of oxidized cytochrome c by ascorbate has been investigated in potassium nitrate, potassium 4-morpholineethanesulfonate (KMes), potassium sulfate and potassium ascorbate media. The results are consistent with simple second order electron transfer from ascorbate dianion to cytochrome c and do not support electron transfer from an ascorbate dianion bound to the protein of the cytochrome as recently proposed by Myer and Kumar. A rate constant of 8 X 10(5) M-1 X s-1 (25 degrees C, ionic strength, 0.1) was found for the electron-transfer step. This rate constant is essentially independent of the specific ions used in controlling ionic strength. 相似文献
10.
Comprehensive binding studies using direct and indirect methods yield stoichiometry and affinities for the binding of Mg X ADP and uncomplexed ADP to the active site of myosin subfragment-1. Additionally, the binding parameters for Mg2+ in the ternary complex protein X Mg X ADP are presented for the first time. The indirect method makes use of reactivity changes of the critical thiol-1 and thiol-2 groups, which occur upon the binding of the ligand at the active site. The affinity constants derived by this method are corroborated by two independent direct methods, equilibrium dialysis and centrifugation transport. For Mg2+, ADP and Mg X ADP just one mole of ligand binds/mole subfragment-1. The affinity of Mg X ADP at low ionic strength is 2.1 X 10(6) M-1 and only five-times lower in the absence of Mg2+. In the ternary complex Mg2+ has a low affinity of 4.1 X 10(4) M-1. At high ionic strength the uncomplexed ADP binds with a 43-times-lower affinity than Mg X ADP, whose affinity is 6.9 X 10(5) M-1. In this case Mg2+ interacts in the ternary complex with the higher affinity of 3.2 X 10(5) M-1, implying that at high salt concentration it plays a more prominent role in anchoring ADP at the active site. 相似文献
11.
E Buszman T Wilczok B Witman G Siebert 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1977,358(7):819-824
Colchicine (7.5 X 10-6M or 3.3 X 10-5M) was incubated at pH 7.0, 10.0, or 12.0 with high-molecular DNA from salmon sperm (7 X 10-5M or 3.5 X 10-5M DNA-P) at 40 degrees C. Interaction was monitored by UV spectrophotometry; Amax/Amin ratios, difference and additive spectra, and the quantity of colchicine bound to DNA were presented as functions of time, ionic strength and DNA concentration. Using NMR techniques, delta deltav 1/2/deltac values, chemical shifts and half-line widths of colchicine protons were analyzed as a function of the DNA/colchicine ratio, thus proving the interaction between alkaloid and DNA. An intercalation of the tropolone moiety of colchicine between the nitrogenous bases of DNA is suggested. 相似文献
12.
Binding of spermidine to transfer ribonucleic acid 总被引:1,自引:0,他引:1
The binding of spermidine to yeast tRNAPhe and Escherichia coli tRNAGlu2 at low and high ionic strength was studied by equilibrium dialysis. Once corrected for the expected Donnan effect, the binding at low ionic strength obeys the simple relationship of equivalent binding sites, and cooperative binding of spermidine to tRNA could not be detected. At low ionic strength (0.013 M Na+ ion), tRNAPhe (yeast) has 13.9 +/- 2.3 strong spermidine binding sites per molecule with Kd = 1.39 X 10(-6) M and a few weak spermidine binding sites which were inaccessible to experimentation; tRNAGlu2 (E. coli) has 14.8 +/- 1.6 strong spermidine binding sites and 4.0 +/- 0.1 weak spermidine binding sites with Kd = 1.4 X 10(-6) M and Kd = 1.23 X 10(-4) M, respectively. At high ionic strength (0.12 M monovalent cation) and 0.01 M Mg2+, tRNAPhe (yeast) has approximately 13 strong spermidine binding sites with an apparent Kd = 3.4 X 10(-3) M while the dimeric complex tRNAPhe X tRNAGlu2 has 10.4 +/- 1.2 strong spermidine binding sites per monomer with an apparent Kd = 2.0 X 10(-3) M. In the presence of increasing Na+ ion or K+ ion concentration, spermidine binding data do not fit a model for competitive binding to tRNA by monovalent cations. Rather, analysis of binding data by the Debye-Hückel approximation results in a good fit of experimental data, indicating that monovalent cations form a counterion atmosphere about tRNA, thus decreasing electrostatic interactions. On the basis of equilibrium binding analyses, it is proposed that the binding of spermidine to tRNA occurs predominantly by electrostatic forces. 相似文献
13.
Guanine aminohydrolase (E.C. 3.5.4.3) has been purified 11-fold from the supernatant fraction of guinea-pig liver homogenates in 0.25 M sucrose (centrifuged at 50,000 X g) through thermic denaturation at 60 degrees C and ammonium sulphate fractionation (30--60% saturation). The enzyme in the homogenates and purified preparations exhibited two Km values. In both preparations four enzymatic electrophoretic bands have been detected. Purified guanine aminohydrolase is chromatographically resolved on DEAE-sephadex in three components whose active forms appeared separately on their pherograms. The enzymatic form eluted at lower ionic strength has the least anodic mobility, is inhibited by guanine (4 X 10(-5) M) and presents only one Km value (1.5 X 10(-5) M). The enzymatic form eluted at greater ionic strength exhibits the highest anodic mobility, is also inhibited by guanine (7 X 10(-5) M) and its Km value seems to be 6.3 X 10(-6) M. Molecular weight of enzymatics forms determined by Sephadex G-200 chromatography, is 120,000 +/- 5,000. The preceding results, correlated with the chromatographic homogeneity of guanine aminohydrolase, purified in Sephadex G-100, suggests that the four molecular forms of the native enzyme may be considered as isozymes. 相似文献
14.
D A Abbott D E Schlarman P H Patrick D M Tal W H Elliott 《Analytical biochemistry》1985,146(2):437-441
The mobilities of coenzyme A and coenzyme A derivatives of cholate, chenodeoxycholate, deoxycholate, lithocholate, and their 5 alpha analogs were studied in reversed-phase high-performance liquid chromatography. With a C18 Radial-PAK A cartridge (10-micron particles) and a solvent mixture of 2-propanol/10 mM phosphate buffer (pH 7.0, 140:360), separation of the chenodeoxycholyl and deoxycholyl coenzyme A derivatives was not observed. An increase in ionic strength of the buffer to 50 mM afforded separation, which was markedly augmented with a C18 Radial-PAK A cartridge with 5-micron particles. Lowering the pH of the buffer to 5.5 did not materially change the separations regardless of the ionic strength. Quantitation was carried out to a lower level of 8.5 X 10(-12) mol. 相似文献
15.
H Suzuki W F Stafford H S Slayter J C Seidel 《The Journal of biological chemistry》1985,260(27):14810-14817
Gizzard heavy meromyosin (HMM) sediments in the ultracentrifuge as a single peak, whose sedimentation coefficient (S20,w) decreases from 9 to 7.5 S upon increasing the NaCl concentration from 0.02 to 0.3 M. This decrease is accompanied by a parallel increase in Mg2+-ATPase activity, suggesting that both changes have a common molecular basis. Phosphorylation decreases S20,w and increases ATPase activity, while ATP increases S20,w. Sedimentation equilibrium studies indicate that HMM undergoes no detectable aggregation at 0.02 or 0.4 M NaCl, remaining monomeric with a molecular weight of 3.4 X 10(5). In contrast, S20,w of subfragment 1 does not change with changes in ionic strength, and its ATPase activity does not decrease at low ionic strengths. Electron micrographs of samples of HMM prepared at low ionic strength show that up to half of the molecules are flexed, i.e. the heads are bent at the neck and project back toward the tail, while the remaining molecules have either one or both of the heads pointing away from the tail. In samples prepared at high ionic strength only about 10% of the molecules are flexed. There is a linear relationship between the fraction of flexed molecules and S20,w, with no significant bending or folding of the tail and no detectable change in the shape of the heads. This correlation suggests that the changes in ATPase activity and S20,w may be a result of the reorientation of the heads. 相似文献
16.
1. Double-stranded f2 sus11 or Qbeta RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaCl/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of double-stranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S1 from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermal-transition profile of double-stranded RNA (with a Tm of 89 degrees C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-stranded RNA degradation by ribonucleases specific for single-stranded RNA. 相似文献
17.
The spin-label electron paramagnetic resonance (EPR) technique has been used to study the interaction between human hemoglobin and erythrocyte membranes as a function of temperature and ionic strength. We show, for the first time, experimental evidence for the existence of the interaction at physiological pH, ionic strength and temperature. In addition to the pH dependence that we have previously reported, the interactions are also temperature and ionic strength dependent. Using a simple two-state equilibrium model to analyze the EPR data, we obtain an equilibrium dissociation constant of about 8.1 +/- 5.6 X 10(-5) M for hemoglobin-membrane systems in 5 mM phosphate with 150 mM NaCl at pH 7.4 and 37 degrees C. 相似文献
18.
W H Koppenol F Levine T L Hatmaker J Epp J D Rush 《Archives of biochemistry and biophysics》1986,251(2):594-599
Complexes of manganese, copper, cobalt, and iron with a variety of aminopolycarboxylates at concentrations from 2 X 10(-7) to 3 X 10(-6) M were tested for superoxide dismutase activity with horse ferricytochrome c as the competing reagent for superoxide. In the presence of excess ligand only manganous nitrilotriacetate and manganous ethylenediaminediacetate showed activity with catalytic rate constants of 2.2 X 10(7) and 1.8 X 10(7) M-1 S-1, respectively, at pH 6, 22 +/- 1 degree C, and 10 mM ionic strength. These rate constants decrease considerably at higher pH. Manganous N-hydroxyethylethylenediaminetriacetate is oxidized by superoxide, but does not appear to have catalytic activity. From the experimental conditions under which the two complexes mentioned above exhibit catalysis, and the inactivity of other metal chelates, it is concluded that an open coordination site is essential but not sufficient to catalyze the dismutation reaction. 相似文献
19.
《The Journal of cell biology》1983,96(6):1761-1765
Tomato activation inhibiting protein (AIP) is a molecule of an apparent molecular weight of 72,000 that co-purifies with tomato actin. In an assay system containing rabbit skeletal muscle F-actin and rabbit skeletal muscle myosin subfragment-1 (myosin S-1), tomato AIP dissociated the acto-S-1 complex in the absence of Mg+2ATP and inhibited the ability of F-actin to activate the low ionic strength Mg+2ATPase activity of myosin S-1. At a molar ratio of 5 actin to 1 AIP, a 50% inhibition of the actin-activated Mg+2ATPase activity of myosin S-1 was observed. The inhibition can be reversed by raising the calcium ion concentration to 1 X 10(-5) M. The AIP had no effect on the basal low ionic strength Mg+2ATPase activity of myosin S-1 in the absence of actin. The protein did not bind directly to actin nor did it cause depolymerization or aggregation of F-actin but appeared, instead, to interact with the actin binding site on myosin S-1. Since AIP is a potent, reversible inhibitor of the rabbit acto-S-1 ATPase activity, it is postulated that it may be responsible for the low levels of actin activation exhibited by tomato F-actin fractions containing the AIP. 相似文献
20.
Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies 总被引:2,自引:1,他引:1
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Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites. 相似文献