共查询到20条相似文献,搜索用时 15 毫秒
1.
The interrelation was studied between the phototransient absorbing maximally at 412 nm (M412) and light-induced proton release under steady-state conditions in aqueous suspensions of ‘purple membrane’ derived from Halobacterium halobium. The decay of M412 was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M412 which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M412 was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M412 without affecting the level of M412 which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca2+ and their antagonists La3+ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed. 相似文献
2.
Peter Scherrer Lester Packer Stanley Seltzer 《Archives of biochemistry and biophysics》1981,212(2):589-601
Bacteriorhodopsin in the purple membrane of Halobacterium halobium is coupled to a photocycle that results in the release and uptake of protons. The role of tyrosyl residues in the photocycle of bacteriorhodopsin has been investigated by the technique of chemical modifications of these residues by iodination and nitration. The studies indicate that modification of a tyrosyl residue accelerates M412 formation, whereas modification of another type of tyrosine residue(s) accessible from the cytoplasmic surface of the purple membrane inhibits M412 decay. The results support the hypothesis that a reversible deprotonation of tyrosine residues prior to and after M412 formation in the photocycle are steps in the light-driven pathway of H+ translocation by bacteriorhodopsin. 相似文献
3.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(4):283-288
AbstractThe effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respetively) to Leuproteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21°C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from ~6.9, in the free Leu-proteinase, to ~5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c - Ka = 2.2 × 1011 M-1, δG°= - 64kJ/mol, δH° = + 5.9kJ/mol, and δS° = + 240J/molK; Leu-proteinase:BBI - Ka = 3.2 × 1010 M-1, δG° = - 59kJ/mol, δH°= + 8.8kJ/mol, and δS° = + 230J/molK; and Leu-proteinase:F-C - Ka = 1.1 × 106 M-1, δG°= - 34kJ/mol, δH° = + 18J/mol, and δS° = + 180J/molK (values of Ka, δG° and δS° were obtained at 21.0°C; values of δH° were temperature-independent over the range explored, i.e. between 10.0°C and 40.0°C). F-T does not inhibit Leu-proteinase up to an inhibitor concentration of 1.0 × 10-3 M, suggesting that the upper limit of Ka is 1 × 102 M-1. Considering the known molecular models, the observed binding behaviour of eglin c, BBI, F-C and F-T to Leu-proteinase has been related to the inferred stereochemistry of the enzyme/inhibitor contact region 相似文献
4.
Large Scale Global Structural Changes of the Purple Membrane during the Photocycle 总被引:2,自引:2,他引:0
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Both the solution and the oriented film absorption and circular dichroic spectra of the bacteriorhodopsin (bR568) and M412 intermediate of the purple membrane photocycle were compared over the wavelength region 800-183 nm to assess structural changes during this photocycle. The main findings are (a) loss of the excitonic interaction among the chromophoric retinal transitions indicating disordering of the retinal orientations in the membrane and distortions of the membrane hexagonal crystal lattice, (b) structural change of the chromophoric retinal, (c) changes in the key interactions between the retinal and specific groups in the local environment of the apoprotein, (d) significant changes of the tertiary structure of the bR with negligible secondary structure involvement, and (e) a net tilting of the rodlike segments of the bR polypeptides away from the membrane normal. These findings are in accord with large scale global structural changes of the membrane during the photocycle and with structural metastability of the bR molecules. An important implication of these changes is the possibility of transmembrane retinal-regulated pulsating channels during the photocycle. The significance of this possibility in respect to models for the proton translocation function of this membrane is discussed. 相似文献
5.
Angelo Filosa A. A. Ismail A. M. English 《Journal of biological inorganic chemistry》1999,4(6):717-726
Fourier transform infrared (FTIR) spectroscopy is used to compare the thermally induced conformational changes in horse,
bovine and tuna ferricytochromes c in 50 mM phosphate/0.2 M KCl. Thermal titration in D2O at pD 7.0 of the amide II intensity of the buried peptide NH protons reveals tertiary structural transitions at 54 °C in horse and at 57 °C in bovine c. These transitions, which occur well before loss of secondary structure, are associated with the alkaline isomerization involving
Met80 heme-ligand exchange. In tuna c, the amide-II-monitored alkaline isomerization occurs at 35 °C, followed by a second amide II transition at 50 °C revealing
a hitherto unreported conformational change in this cytochrome. Amide II transitions at 50 °C (tuna) and 54 °C (horse) are
also observed during the thermal titration of the CN–-ligated cytochromes (where CN– displaces the Met80 ligand), but a well-defined 35 °C amide II transition is absent from the titration curve of the CN–adduct of tuna c. The different mechanisms suggested by the FTIR data for the alkaline isomerization of tuna and the mammalian cytochromes c are discussed. After the alkaline isomerization, loss of secondary structure and protein aggregation occur within a 5 °C
range with T
m values at 74 °C (bovine c), 70 °C (horse c) and 65 °C (tuna c), as monitored by changes in the amide I′ bands. The FTIR spectra were also used to compare the secondary structures of the
ferricytochromes c at 25 °C. Curve fitting of the amide I (H2O) and amide I′ (D2O) bands reveals essentially identical secondary structure in horse and bovine c, whereas splitting of the α-helical absorption of tuna c indicates the presence of less-stable helical structures. CN– adduct formation results in no FTIR-detectable changes in the secondary structures of either tuna or horse c, indicating that Met80 ligation does not influence the secondary structural elements in these cytochromes. The data provided
here demonstrate for the first time that the selective thermal titration of the amide II intensity of buried peptide NH protons in D2O is a powerful tool in protein conformational analysis.
Received: 1 April 1999 / Accepted: 24 August 1999 相似文献
6.
《Molecular membrane biology》2013,30(6):197-206
AbstractFTIR and cryomicroscopy have been used to study mouse embryonic fibroblast cells (3T3) during freezing in the absence and presence of DMSO and glycerol. The results show that cell volume changes as observed by cryomicroscopy typically end at temperatures above ?15°C, whereas membrane phase changes may continue until temperatures as low as ?30°C. This implies that cellular dehydration precedes dehydration of the bound water surrounding the phospholipid head groups. Both DMSO and glycerol increase the membrane hydraulic permeability at subzero temperature and reduce the activation energy for water transport. Cryoprotective agents facilitate dehydration to continue at low subzero temperatures thereby decreasing the incidence of intracellular ice formation. The increased subzero membrane hydraulic permeability likely plays an important role in the cryoprotective action of DMSO and glycerol. In the presence of DMSO water permeability was found to be greater compared to that in the presence of glycerol. Two temperature regimes were identified in an Arrhenius plot of the membrane hydraulic permeability. The activation energy for water transport at temperature ranging from 0 to ?10°C was found to be greater than that below ?10°C. The non-linear Arrhenius behavior of Lp has been implemented in the water transport model to simulate cell volume changes during freezing. At a cooling rate of 1°C min-1, ~5% of the initial osmotically active water volume is trapped inside the cells at ?30°C. 相似文献
7.
An in vitro system for the uptake of 125l-vitellogenin (VG) or vitellin into isolated follicles of the tobacco hornworm, Manduca sexta, is described. After incubation with 125l-VG, follicles were disrupted and the internal yolk contents separated from the follicle membranes. The results showed that 125l-VG was associated principally with the membranes (92%) after incubation at 4°C. However, at 27°C, 125l-VG was mainly in the yolk (92%). Furthermore, trypsin treatment removed approximately 70% of VG bound to the follicles at 4°C. Labeled VG was shown to bind to sonicated follicle membranes with high specificity and affinity (KD ? 1.3 × 10?8 M). This binding was sensitive to pH and calcium concentration. The total binding sites were estimated at 4 × 1014 sites/g of membrane protein. Competition studies showed that binding of 125l-VG to follicle membranes was blocked by excess unlabeled vitellin and deglycosylated vitellogenin but not by lipophorin (the major hemolymph lipoprotein), microvitellogenin, a female-specific protein (Mr ~ 31,000) found in both hemolymph and eggs, and the smaller vitellogenin subunit, apovitellogenin-II (Mr ~ 45,000). These results suggest that selective uptake of M. sexta VG from the hemolymph involves binding to specific receptors located on the follicle membranes. 相似文献
8.
The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (TL and TH) were found to be independent of the external K+, Ca2+, or Mg2+ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (TM) was markedly influenced by the external Ca2+ concentration. Ca2+ increased TM from 12°C (no Ca2+ added) to 22°C (10 mM Ca2+), i.e., growth temperature. K+ reversed this Ca2+ effect, downshifting TM from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in TM, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment. 相似文献
9.
《FEBS letters》1986,203(1):36-40
Signal II of plant photosynthesis, which is thought to be due to a plastosemiquinone cation radical, has been studied by EPR at 9 and 35 GHz in non-oriented and partly oriented PS II particles. The spectra measured of the oriented particles at 35 GHz show that the molecular Z-axis, which is the axis perpendicular to the plane of the radical, makes an angle of 60° with the membrane normal. All spectra could be computer-simulated with one set of parameters. This set is essentially the same as that given earlier on the basis of EPR spectroscopy on non-oriented membranes [(1985) Biochim. Biophys. Acta 809, 421-428], except that the bond bending of the hydroxyl group on ring position 1 is found to be 60°, resulting in a somewhat smaller isotropie hyperfine splitting of the hydroxyl proton.Signal IIEPROrientationHyperfine coupling 相似文献
10.
The mammalian neurofilament triplet proteins (210, 160 and 68 × 103Mr proteins) are resolved by anion exchange chromatography in the presence of urea. Upon dialysis against physiological buffers at 37 °C only the 68 × 103Mr protein shows self-assembly into morphologically normal intermediate-sized filaments. Addition of 210 × 103Mr protein to 68 × 103Mr protein leads to shorter filaments, which upon embedding reveal a rough surface and whisker-like protrusions that are not present on the smooth surface of filaments assembled from 68 × 103Mr protein alone. Certain emerging principles of neurofilament structure are discussed, emphasizing a possible relation between neurofilaments and other intermediate-sized filaments. 相似文献
11.
Abstract: δ-Opioids mobilize Ca2+ from intracellular stores in undifferentiated NG108-15 cells, but the mechanism involved remains unclear. Therefore, we examined the effect of [d -Pen2,5]enkephalin on inositol 1,4,5-trisphosphate formation in these cells. [d -Pen2,5]enkephalin caused a dose-dependent (EC50 = 3.1 nM) increase in inositol 1,4,5-trisphosphate formation (measured using a specific radioreceptor mass assay), which peaked (25.7 ± 1.2 pmol/mg of protein with 1 µM, n = 9) at 30 s and returned to basal levels (10.6 ± 0.9 pmol/mg of protein, n = 9) within 4–5 min. This response was fully naloxone (1 µM) reversible and pertussis toxin (100 ng/ml for 24 h) sensitive. Preincubation with Ni2+ (2.5 mM) or nifedipine (1 µM) had no effect on the [d -Pen2,5]enkephalin (1 µM)-induced inositol 1,4,5-trisphosphate response, and K+ (80 mM) was unable to stimulate inositol 1,4,5-trisphosphate formation, indicating Ca2+ influx-induced activation of phospholipase C is not involved. Preincubation with the protein kinase C inhibitor Ro 31-8220 (1 µM) enhanced, whereas acute exposure to phorbol 12,13-dibutyrate (1 µM) abolished, the [d -Pen2,5]enkephalin (0.1 µM)-induced inositol 1,4,5-trisphosphate response, suggesting protein kinase C exerts an autoinhibitory feedback action. [d -Pen2,5]Enkephalin also dose-dependently (EC50 = 2.8 nM) increased the intracellular [Ca2+], which was maximal (24 nM increase with 1 µM, n = 5) at 30 s. This close temporal and dose-response relationship strongly suggests that δ-opioid receptor-mediated increases in intracellular [Ca2+] results from inositol 1,4,5-trisphosphate-induced Ca2+ release from intracellular stores, in undifferentiated NG108-15 cells. 相似文献
12.
Thermodynamics of the B to Z transition in poly(dGdC) 总被引:1,自引:0,他引:1
The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl2, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔHcal = 2.02 kcal (mol base pair)?1 and a cooperative unit of 150 base pairs (bp). The tm of this transition is independent of both polynucleotide and Mg2+ concentrations. A second transition, with ΔHcal = 2.90 cal (mol bp)?1, follows the B to Z conversion, the tm of which is dependent upon both the polynucleotide and the Mg2+ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔHcal = 12.4 kcal (mol bp)?1 and a cooperative unit of 290 bp. In 5 mM MgCl2, a single transition is seen centered at 75.5°C, characterized by ΔHcal = 2.82 kcal (mol bp)?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔHcal = 10.0 kcal (mol bp)?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions. 相似文献
13.
Reiner Dolle 《Physiologia plantarum》1988,73(1):7-14
Chlorophyll-free plasma membranes of the unicellular green alga Chlamydomonas reinhardtii Dangeard were purified from a microsomal fraction using an aqueous polymer two-phase system of 6.5% (w/w) dextran T500, 6·5% (w/w) polyethylene glycol 3350, 60 mM NaCI, 0 33 M sucrose and 5 mM potassium phosphate (pH 7·8). The plasma membrane fraction contained only 2·4% of the microsomal membrane protein. Specific activity of the plasma membrane marker enzyme, K*, Mg2+-ATPase (EC 3.6.1.3). was enriched 9-fold over the microsomal fraction, and 22% of total activity was recovered in the upper, polyethylene glycol-rich phase. Contamination from intracellular membranes was minimal. K*, Mg2+-ATPase showed a pH optimum at about 6·5, and addition of 0·05% (w/v) Triton X-100 stimulated the activity 3-fold. [3H]-Nimodipinc was employed to characterize 1,4-dihydropyridine-specific membrane receptors. Two apparent binding sites with different affinities to nimodipine were found in the crude microsomal fraction. The separation of plasma membranes from intracellular membranes revealed that one binding site with higher affinity (KD= 9 nM) was located on the plasma membrane and a second binding site with lower affinity (KD= 36 nM) on an intracellular membrane The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments were comparable to those determined by equilibrium experiments. The maximum number of binding sites of the plasma membrane fraction and the intracellular membrane fraction was Bmax= 440 and 470 fmol (mg protein)-1, respectively. [3H]-Nimodipinc binding was inhibited by (±) verapamil and stimulated by D-cis-diltiazem in both fractions. Moreover, ethyle-neglycol-bis(2-aminoethylcther)-N, N'-tetraacctic acid (EGTA) inhibited [3H]-nimo-dipinc binding in the plasma membrane fraction but not in the intracellular membrane fraction This effect was cancelled by the addition of CaCl2. 相似文献
14.
K. Rosenheck M. Brith-Lindner P. Lindner A. Zakaria S. R. Caplan 《European biophysics journal : EBJ》1978,4(4):301-313
Pronase treatment of aqueous suspensions of purple membrane fragments from H. halobium leads to the cleavage of bacteriorhodopsin. The protein fragments remaining in the membrane after treatment with relatively small concentrations of enzyme (2% w/w) in normal daylight range in molecular weight from 20,000-21,000 daltons, indicating that cleavage occurs mainly near the extremities of the protein chain. At higher enzyme concentrations the relative amounts of protein fragments having smaller molecular weight increase. Generally, the relative loss of retinal chromophore is larger than that of protein and thus the retinal binding site seems to be located near one of the chain ends that is cleaved off by enzyme.Irradiation with white light during the time of proteolysis (at both low and high enzyme concentrations) results in extensive cleavage, so that under certain conditions no high molecular weight components can be detected in SDS-polyacrylamide gels. It, therefore, appears that parts of the bacteriorhodopsin chain become more exposed to enzyme digestion when the purple membrane is illuminated.Enzyme treated aqueous purple membrane fragment suspensions still show photocycle activity. The main consequence of proteolysis is a pronounced appearance of biphasicity in the decay of M412 and the regeneration of bR570. Simultaneously the yield of O660 is reduced. As with untreated purple membrane, the correlation between the rates of decay of M412 and regeneration of bR570 is greatest when the yield of O660 is lowest. 相似文献
15.
The combined action of electric field (105–107 V · m?1) and light (380–580 nm, 80 W · m?2) activating the photoenergetic reaction of bacteriorhodopsin (BR) in dry films of purple membranes from Halobacterium halobium was studied. A new stimulating effect of the field on the BR412 intermediate accumulation in the normal photochromic cycle of BR570 has been observed. The formation of the product BR412 is supposed to be accompanied by specific rearrangements of certain charged, polar and polarizable groups in the BR pigment-protein matrix. Such an intrinsic polarization could be promoted by an external electric field, the displacement vector of those groups being oriented in the direction of the field. The dielectric polarization properties of the purple membranes have been demonstrated by electret-thermal analysis. 相似文献
16.
《Redox report : communications in free radical research》2013,18(4):173-178
AbstractThe reaction of human myeloperoxidase (MPO) with hypochlorous acid (HOCl) was investigated by conventional stopped-flow spectroscopy at pH 5, 7, and 9. In the reaction of MPO with HOCl, compound I is formed. Its formation is strongly dependent on pH. HOCl (rather than OCl-) reacts with the unprotonated enzyme in its ferric state. Apparent second-order rate constants were determined to be 8.1×107 M-1s-1 (pH 5), 2.0×108 M-1s-1 (pH 7) and 2.0×106 M-1s-1 (pH 9) at 15°C. Furthermore, the kinetics and spectra of the reactions of halides and thiocyanate and of physiologically relevant one-electron donors (ascorbate, nitrite, tyrosine and hydrogen peroxide) with this compound I were investigated using the sequential-mixing technique. The results show conclusively that the redox intermediates formed upon addition of either hydrogen peroxide or hypochlorous acid to native MPO exhibit the same spectral features and reactivities and thus are identical. In stopped-flow investigations, the MPO/HOCl system has some advantage since: (i) in contrast to H2O2, HOCl cannot function as a one-electron donor of compound I; and (ii) MPO can easily be prevented from cycling by addition of methionine as HOCl scavenger. As a consequence, the observed absorbance changes are bigger and errors in data analysis are smaller. 相似文献
17.
Evidence that the intermediate electron acceptor,A2, in Photosystem I is a bound iron-sulfur protein
Absorption changes accompanying the formation of light-induced P-700+ were investigated in a highly enriched Photosystem I preparation where an intermediate electron acceptor preceding P-430 could be detected. In an enriched Photosystem I particle, light-induced reversible absorption changes observed at 700 nm in the presence of dithionite resembled those previously seen at 703 nm and 820 nm [9], thus indicating the presence of a backreaction between P-700+ and A?2. After this same Photosystem I particle was treated to denature the bound iron-sulfur centers, the photochemical changes that could be attributed to P-700 A2 were completely lost. These results provide evidence that the intermediate electron acceptor, A2, is a bound iron-sulfur protein. Additional studies in the 400–500 nm region with Photosystem I particles prepared by sonication indicate that the spectrum of A2 is different from that of P-430. 相似文献
18.
Chlorogenic acid, 3’-O-caffeoyl D-quinic acid, is an inherent ligand present inHelianthus annuus L. The effect of pH on chlorogenic acid binding to helianthinin suggests that maximum binding occurs at pH 6.0. The protein-polyphenol
complex precipitates as a function of time. The association constant of the binding of chlorogenic acid to helianthinin, determined
by equilibrium dialysis, at 31°C has a value of 3.5 ± 0.1 × 104M−-1 resulting in a ΔG value of − 6.32 ± 0.12 kcal /mol. The association constantK
ais 1.0 ± 0.1 × 104M−1 as determined by ultraviolet difference spectral titration at 25°C with ΔG° of -5.46 ± 0.06 kcal/mol. From fluorescence spectral
titration at 28°C, theK
avalue is 1.38 ± 0.1 × 1 0 4M−1 resulting in a ΔG of − 5.70 ± 0.05 kcal/mol. The total number of binding sites on the protein are 420 ± 50 as calculated
from equilibrium dialysis. Microcalorimetric data of the ligand-protein interaction at 23°C suggests mainly two classes of
binding. The thermal denaturation temperature,T
mof the protein decreases from 76°C to 72°C at 1 × 10−3M chlorogenic acid concentration upon complexation. This suggests that the complexation destabilizes the protein. The effect
of temperature onK
aof chlorogenic acid shows a nonlinear increase from 10.2°C to 45°C. Chemical modification of both lysyl and tryptophanyl residues
of the protein decreases the strength of binding of chlorogenic acid. Lysine, tryptophan and tyrosine of protein are shown
to be present at the binding site. Based on the above data, it is suggested that charge-transfer complexation and entropically
driven hydrophobic interaction are the predominant forces that are responsible for binding of chlorogenic acid to the multisubunit
protein, helianthinin.
Publication No. 324. 相似文献
19.
Proton uptake mechanism of bacteriorhodopsin as determined by time-resolved stroboscopic-FTIR-spectroscopy 总被引:5,自引:5,他引:0
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Bacteriorhodopsin's proton uptake reaction mechanism in the M to BR reaction pathway was investigated by time-resolved FTIR spectroscopy under physiological conditions (293 K, pH 6.5, 1 M KCl). The time resolution of a conventional fast-scan FTIR spectrometer was improved from 10 ms to 100 μs, using the stroboscopic FTIR technique. Simultaneously, absorbance changes at 11 wavelengths in the visible between 410 and 680 nm were recorded. Global fit analysis with sums of exponentials of both the infrared and visible absorbance changes yields four apparent rate constants, k7 = 0.3 ms, k4 = 2.3 ms, k3 = 6.9 ms, k6 = 30 ms, for the M to BR reaction pathway. Although the rise of the N and O intermediates is dominated by the same apparent rate constant (k4), protein reactions can be attributed to either the N or the O intermediate by comparison of data sets taken at 273 and 293 K. Conceptionally, the Schiff base has to be oriented in its deprotonated state from the proton donor (asp 85) to the proton acceptor (asp 96) in the M1 to M2 transition. However, experimentally two different M intermediates are not resolved, and M2 and N are merged. From the results the following conclusions are drawn: (a) the main structural change of the protein backbone, indicated by amide I, amide II difference bands, takes place in the M to N (conceptionally M2) transition. This reaction is proposed to be involved in the “reset switch” of the pump, (b) In the M to N (conceptionally M2) transition, most likely, asp-85's carbonyl frequency shifts from 1,762 to 1,753 cm-1 and persists in O. Protonation of asp-85 explains the red-shift of the absorbance maximum in O. (c) The catalytic proton uptake binding site asp-96 is deprotonated in the M to N transition and is reprotonated in O. 相似文献
20.
A method for the separation of the outer membrane (OM) from the cytoplasmic membrane (CM) of Acinetobacter calcoaceticus 69/V grown on different carbon sources is described. The contamination of the OM with CM was less than 10%. Independent of the carbon source, five protein bands with apparent molecular weights of 47 000, 33000, 21 000, 19 000 and 12 000 were found by solubilization at 37°C and six bands at 100°C (apparent Mr 53 000, 47 000, 38 000, 26 000, 21000, 12000). Three proteins were modifiable by heat. With the periodic acid-Schiff procedure the bands with apparent Mr of 33 000 and 12 000 were made visible. After growth on d,l-carnitine an additional two non-heat-modifiable protein bands with apparent Mr between 40 000 and 45 000 were detected. By cultivation on acetate and peptone as carbon source one additional band (Mr 15 000) from OM of cells could be found. 相似文献