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1.
One of the side chains of Orn residues in gramicidin S (GS) was connected with alanine (AGS), sarcosine (SGS), or histidine (HGS) residue, aiming at developing membrane-active artificial enzymes by virtue of the membrane-associating property of GS. The conformation of the GS analogs was similar to that of GS. However, the affinity of GS and its analogs for dipalmitoylphosphatidylcholine (DPPC) vesicles decreased in the order of GS greater than SGS greater than HGS congruent to AGS. The addition of GS analogs at 10 microM to DPPC vesicles decreased the membrane fluidity, indicating that GS analogs did not disrupt the vesicular structure of DPPC vesicles. On the other hand, GS analogs enhanced carboxyfluorescein-leakage from DPPC vesicles. It was therefore considered that the GS analogs induced the phase-separation of the lipid bilayer membrane. Hydrolytic reactions of HGS in the presence of DPPC vesicles were studied using N-methylindoxyl alkanoate as substrate. HGS reacted only with N-methylindoxyl hexanoate below the phase-transition temperature of the membrane. The substrate specificity of HGS was ascribed to the condensation of HGS in the neighbourhood of the substrate in the lipid bilayer membrane due to the phase-separation below the phase-transition temperature of the membrane.  相似文献   

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To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (Kb). The Kb1 and Kb2 values for endothermic two-step binding processes corresponded to high and low binding affinities (Kb1 ≥ 100 Kb2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.  相似文献   

4.
Potassium and sodium chlorides, sulfates, acetates and phosphates activated the lytic action of gramicidin S and its derivatives on protoplasts of M. lysodeikticus. The derivatives used were positively charged and neutral by the free amino groups in the ornithine moieties. The salts had no effect on lysis of the bacillar protoplasts by gramicidin S and its positively charged derivatives. The lytic effect of the neutral derivative on the bacillar protoplasts markedly increased in the presence of the salts, activation of the lysis by the phosphates being more pronounced than that by the other salts. Increased membrane activity of gramicidin S in the presence of the salts was not connected with association of the substance molecules in solution. Probably it was due to increased destruction of the membranes at the account of activated detergent effect of the antibiotic and its derivatives.  相似文献   

5.
Dipeptides L-phenylalanyl-proline, D-phenylalanyl-proline, prolyl-valine, valyl-lysine, lysyl-leucine and leucyl-phenylalanine, derived from the sequence of gramicidin S, are substrates of the gramicidin S synthetase. When any of these dipeptides are used to replace the two corresponding amino acids in the reaction assay, cyclodecapeptide antibiotic synthesis occurs, and requires the whole multienzyme system. Active esters, like the thiophenyl and p-nitrophenyl esters of D-phenylalanyl-proline are unable to promote gramicidin S biosynthesis with the gramicidin S synthetase system or with the heavy enzyme alone.  相似文献   

6.
The S100 proteins are a unique class of EF-hand Ca2+ binding proteins distributed in a cell-specific, tissue-specific, and cell cycle-specific manner in humans and other vertebrates. These proteins are distinguished by their distinctive homodimeric structure, both intracellular and extracellular functions, and the ability to bind transition metals at the dimer interface. Here we summarize current knowledge of S100 protein binding of Zn2+, Cu2+ and Mn2+ ions, focusing on binding affinities, conformational changes that arise from metal binding, and the roles of transition metal binding in S100 protein function.  相似文献   

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Conformation of gramicidin S   总被引:3,自引:0,他引:3  
A molecular conformation of Gramicidin S was derived on the basis of conformational calculations taking into account the available experimental data. The conformation is characterized by a dyad axis which relates the two chemically equivalent halves of the molecule and contains four hydrogen bonds; other structural features agree with experimental results. X Ray Crystallographic evidences for the relative position of the Ornithine residues is also reported which supports an important feature of the structure of Gramicidin S.  相似文献   

9.
The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern-Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids.  相似文献   

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Cell-free synthesis of gramicidin S   总被引:6,自引:0,他引:6  
S Tomino  M Yamada  H Itoh 《Biochemistry》1967,6(8):2552-2560
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13.
We determined changes in the volume and adiabatic compressibility of large multi- and unilamellar vesicles composed of dimyristoylphosphatidylcholine containing various concentrations of the antimicrobial peptide gramicidin S (GS) by applying densitometry and sound velocimetry. Gramicidin S incorporation was found to progressively decrease the phase transition temperature of DMPC vesicles as well as to decrease the degree of cooperativity of the main phase transition and to increase the volume compressibility of the vesicles. GS probably enhanced thermal fluctuations at the region of main phase transition and provide more freedom of rotational movement for the phospholipid hydrocarbon chains. The ability of GS to increase the membrane compressibility and to decrease the phase transition temperature is evidence for regions of distorted membrane structure around incorporated gramicidin S molecules. At relatively high GS concentration (10 mol%), more significant changes of specific volume and compressibility appear. This might suggest changes in the integrity of the lipid bilayer upon interaction with high concentrations of GS.  相似文献   

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The acid-base transformations of the gramicidin S molecule in water were studied. The protonization constants of the antibiotic amino group were calculated by the data of the potentiometric titration and the antibiotic distribution in the system of chloroform-water: K1 1.55 X 10(10), K2 1.38 X 10(6), the logarithm of the distribution coefficient of gramicidin S in the system of chloroform-water (1:1) lg alpha G 4.10. By the same data the constants of water solubility of gramicidin S base (1.02 X 10(7) mol/l), gramicidin S monohydrate (1.06 X 10(-4) mol/l) and gramicidin S dihydrochloride (2.08 X 10(-4) mol/l) were calculated.  相似文献   

16.
A complex of the electron-rich ion Cu(I) with the flavoquinone analogue 10-methylisoalloxazine has been synthesized and characterized by x-ray methods. The complex is unstable to oxygen. It is black-green in color, in contrast with the bright yellow, orange, or orange-brown crystalline complexes of 10-methylisoalloxazine or riboflavin with Cu(II), Ag(I), and Pb(II). These results are indicative of strong perturbation of the flavin electronic structure by the Cu(I) ion and suggest that this complex is a reasonable model for incipient transfer of an electron from a reduced metal to flavoquinone. the crystal structure is orthorhombic, Pna2-1, with unit cell constants a = 31.24(1) (figures in parentheses are estimated standard deviations), b = 12.862(4), c = 6.239(2) A, Pobs = 1.76 g per cm-3 and Pcalc = 1.77 g per cm-3 for Z = 4 and asymmetric formula CuClO4-2(C11H8N4O2). HCOOH. The final R factor based on 1250 counter-measured data is 8.8%. The 2 independent 10-methylisoalloxazine molecules, A and B, bind strongly to the cuprous ion throug N(5) of each flavin. The copper is approximately linearly coordinated with an N-Cu-N angle of 153(1) degrees, and Cu-N(5) distances of 1.94(2) A and 1.92(2) A. The next nearest atoms to Cu are the O(4) oxygens of each flavin, forming weak bonds with distances Cu-O(4) = 2.27(2) A and 2.21(2) A for molecules A and B. The dihedral angle between the 2 10-methylisoalloxazine molecules is 65.4 degrees.  相似文献   

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Production of gramicidin S in batch and continuous culture   总被引:2,自引:0,他引:2  
A mathematical model for the production of gramicidin S in batch and continuous culture is proposed. It is based on the division of the age of a cell into two phases—an immature and a mature one. A nongrowth associated product, such as an antibiotic, is assumed to be produced when the organism is in the older of these two phases, the mature state. The parameters describing the model were evaluated from batch and single stage transient continuous culture of Bacillus brevis, which produces the antibiotic gramicidin S. The predictive value of the model was studied in steady-state single stage continuous culture and in a transient two stage system. Good agreement between the theoretical curves and the experimental results was found in the transient response of both the first and second stage systems, although at high dilution rates (0.34 hr?1) in the first stage, deviations from the predicted response were observed in the second stage. These may have been due to chemostat instability at dilution rates close to washout, lags in cell growth, and a metabolic lag on going from stage one to stage two.  相似文献   

19.
The effect of the interaction of gramicidin (GA) with lysophosphatidylcholine (LPC) on the change in lipid structure upon heat incubation was revealed by differential scanning calorimetry (DSC) and fluorescence spectroscopy. DSC showed a large endothermic transition in both pure LPC micelles and GA-containing LPC micelles after prolonged heat incubation at 70 degrees C. To elucidate this behavior, fluorescence spectra of 1-anilinonaphthalene-8-sulfonate embedded in LPC micelles were measured. About 40% of the resultant LPC micelles was found to be transformed into the interdigitated gel structures after prolonged heat incubation. On the other hand, intrinsic fluorescence spectra of GA-containing LPC micelles caused a blue-shift of the emission maxima with incubation time, suggesting that tryptophans near the C-terminus of GA moved into a more apolar environment. In addition, GA-containing LPC micelles caused quenching of fluorescence with incubation time, due to the interaction between GA molecules. To determine the location of GA in LPC membranes, surface pressure was measured using the mixed monolayers composed of GA and LPC. The result suggests that GA molecule is localized by interdigitating the C-terminal part of adjacent to acyl chain of LPC.  相似文献   

20.
The biosynthesis of gramicidin S in a cell-free system   总被引:3,自引:0,他引:3       下载免费PDF全文
1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of 14C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [14C]-leucine, -proline and -phenylalanine over a period of 4hr. With [14C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [14C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.  相似文献   

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