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1.
Iron-sulfur core extrusions from spinach [( 2Fe-2S]) and Clostridium pasteurianum (2[4Fe-4S]) ferredoxins in aqueous Triton X-100/urea containing excess benzenethiol yield quantitatively [FenSn(SPh)4]2- with n = 2 and n = 4, respectively. The iron-sulfur cluster can be separated from the corresponding apoprotein by rapid passage of the extrusion mixture over a small anaerobic column of Whatman DE-52 anion-exchange cellulose. Essentially quantitative recovery of [FenSn (SPh)4]2- is achieved in the eluate. The apoprotein remaining on the column can be eluted with 0.5 M NaCl. Most of the residual Triton X-100 and benzenethiol can be removed by passage of the apoprotein eluate over a small column of Bio-Beads SM-2, a hydrophobic polystyrene adsorbent. Apoprotein recovery is comparable to that obtained by other chromatographic methods. At least with spinach ferredoxin, the apoprotein prepared in this fashion can be reconstituted. The procedures developed in this work are potentially most applicable to selective removal of [2Fe-2S] and [4Fe-4S] centers from a multicenter enzyme without irreversible denaturation. 相似文献
2.
Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines. 总被引:3,自引:1,他引:2
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C. Sekharudu B. Ramakrishnan B. Huang R. T. Jiang C. M. Dupureur M. D. Tsai M. Sundaralingam 《Protein science : a publication of the Protein Society》1992,1(12):1585-1594
The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues. 相似文献
3.
Human plasma retinol-binding protein has been purified to homogeneity by a simple method that requires an ammonium sulfate fractionation, a hydrophobic interaction chromatography on phenyl-Sepharose, which dissociates the complex between retinol-binding protein and its carrier, transthyretin, and a gel filtration on Sephadex G-50. The yield of pure protein is comparable or higher than that obtained with the more complex procedures previously reported. 相似文献
4.
Summary The permeability and partition coefficients of tetraphenylarsonium (TPA) and several other organic cations were studied in the human erythrocyte using an ion-selective electrode. The permeability constant for the different cations could be explained quite well by differences in oil/water partition coefficients. No evidence for facilitated transport could be found. Binding of the organic ions occurred to both the cell membrane and to intracellular contents. Partitioning to the membrane remained relatively constant despite variation from ion intracellular binding with blood samples from different donors. TPA flux is stimulated by substoichiometric amounts of tetraphenylboron and other organic anions, suggesting an ion-pairing mechanism. 相似文献
5.
Liposome-mediated labeling of adrenocorticotropin fragments parallels their biological activity 总被引:2,自引:0,他引:2
To test our hypothesis that specific interactions of ACTH peptides with model lipid membranes reflect the biological importance of similar interactions on target cells, we investigated the liposome-mediated labeling of ACTH fragments with the extremely hydrophobic photolabel, 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine. Correlations were found between the labeling rates and the agonistic and antagonistic potencies of the peptides for in vitro steroidogenesis and inhibition of a synaptosomal protein kinase. A model for the cross-reactivity between ACTH and opioid peptides is discussed. 相似文献
6.
The native state of apomyoglobin described by proton NMR spectroscopy: interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS. 总被引:4,自引:3,他引:1
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Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His-64) in horse apomyoglobin. This residue was previously reported to titrate with a pKa below 5 and tentatively labeled as His-82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31:6481-6491). The packing of the side chains and the low pKa of His-64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter-amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin. 相似文献
7.
Neal C. Robinson 《Journal of bioenergetics and biomembranes》1993,25(2):153-163
Bovine cytochromec oxidase usually contains 3–4 mol of tightly bound cardiolipin per cytochromeaa
3 complex. At least two of these cardiolipins are required for full electron transport activity. Without the tightly bound cardiolipin, cytochromec oxidase has only 40–50% of its original activity when assayed in detergents that support activity, e.g., dodecyl maltoside. By measuring the restoration of electron transport activity, functional binding constants for cardiolipin and a number of cardiolipin analogues have been evaluated (K
d,app=1 µM for cardiolipin). These binding constants agree reasonably well with direct measurement of the binding using [14C]-acetyl-cardiolipin (K
d
<0.1 µM) when the enzyme is solubilized with Triton X-100. These data are discussed in relationship to the wealth of data that is known about the association of cardiolipin with cytochromec oxidase and the other mitochrondrial electron transport complexes and transporters. 相似文献
8.
Leckband D Chen YL Israelachvili J Wickman HH Fletcher M Zimmerman R 《Biotechnology and bioengineering》1993,42(2):167-177
The adhesion forces between various surfaces were measured using the "surface forces apparatus" technique. This technique allows for the thickness of surface layers and the adhesion force between them to be directly measured in controlled vapor or liquid environments. Three types of biological surfaces were prepared by depositing various lipid-protein monolayers (with thicknesses ranging from 1 to 4 nm) on the inert, molecularly smooth mica surface: (i) hydrophobic lipid monolayers; (ii) amphiphilic polyelectrolyte surfaces of adsorbed polylysine; and (iii) deposited bacterial S-layer proteins. The adhesion, swelling, and wetting properties of these surfaces was measured as a function of relative humidity and time. Initial adhesion is due mainly to the van der Waals forces arising from nonpolar (hydrophobic) contacts. Following adhesive contact, significant molecular rearrangements can occur which alter their hydrophobic-hydrophilic balance and increase their adhesion with time. Increased adhesion is generally enhanced by (i) increased relative humidity (or degree of hydration); (ii) increased contact time; and (iii) increased rates of separation. The results are likely to be applicable to the adhesion of many other biosurfaces, and show that the hydrophobicity of a lipid or protein surface is not an intrinsic property of that surface but depends on its environment (e.g., on whether it is in aqueous solution or exposed to the atmosphere), and on the relative humidity of the atmosphere. It also depends on whether the surface is in adhesive contact with another surface and-when considering dynamic (nonequilibrium) conditions-on the time and previous history of its interaction with that surface. (c) 1993 John Wiley & Sons, Inc. 相似文献
9.
Tore Eid 《Biotechnic & histochemistry》1993,68(4):189-192
Hydrophobic adhesive tape was used to produce miniature wells on microscope slides for staining several sections of tissue with minimal amounts of cytochemical reagents. The wells could be tailored to individual specifications and the method allowed coverslips to be mounted close to the sections using either aqueous or xylene based mounting media. This method was especially useful for multiple immunolabelling of serial semithin cryosections. 相似文献
10.
Frost damage to potatoes, grapevine and citrus plants was assessed following treatment with either an acrylic polymer (Antistress?) or with a hydrophobic particle film (CM‐96–018). In large freezing tests, the application of the hydrophobic particle film consistently led to less damage whilst the acrylic polymer led to the same amount or more damage when compared to control plants. Detailed examination of the freezing of leaves of all three species using infrared thermal imaging revealed that the hydrophobic particle film delayed the entry of ice from a frozen water droplet containing ice nucleating active bacteria and in some cases for the complete duration of the frost test. In contrast, the acrylic polymer was only able to influence the time of ice nucleation of the leaves of citrus plants. It was concluded that the hydrophobic particle film shows considerable promise as a frost protectant applied to susceptible crops just prior to a freezing event. 相似文献