共查询到20条相似文献,搜索用时 0 毫秒
1.
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
2.
Cell-free synthesis of gramicidin S 总被引:6,自引:0,他引:6
3.
4.
5.
Mode of antibacterial action by gramicidin S 总被引:1,自引:0,他引:1
To elucidate the mode of antibacterial action by gramicidin S (GS), a detailed experiment on GS distribution on bacteria cells was carried out. 14C-Labeled gramicidin S ([14C]GS) was incubated with cells of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and the amount of [14C]GS adsorbed on the cells was measured. Adsorption on B. subtilis cells was observed from 1 microgram/ml of [14C]GS. As the concentration of [14C]GS increased, the amount adsorbed on B. subtilis increased discontinuously, producing a curve which had three plateaus. On the other hand, [14C]GS was not easily adsorbed on E. coli cells at lower concentrations, but the amount adsorbed increased above 6 micrograms/ml, and the cells were temporarily saturated with GS at 10 micrograms/ml, which is the minimum inhibitory concentration for E. coli. The amount of [14C]GS adsorbed on the protoplast membrane of B. subtilis was the same as that of natural cells. However, the amount of [14C]GS adsorbed on the cell wall dropped to about 20% of that of natural bacteria. These facts indicate that GS is adsorbed on the cell membrane of bacteria particularly. The uptake of amino acid or glucose in B. subtilis was inhibited by GS. Therefore, it is concluded that GS damages the phospholipid bilayer of the cell membrane by adsorption, and prevents the functioning of the cell membrane. The amount of [14C]GS adsorbed on the spheroplast membrane of E. coli increased remarkably as compared with natural cells, even at a lower concentration of GS. The poor GS adsorption on E. coli cells may be due to the permeability barrier of the E. coli cell wall. 相似文献
6.
7.
The ability of gramicidin to induce bilayer formation in lysophosphatidylcholine (LPC) systems was investigated as a function of the conformation of the peptide. The conformation was varied by using different solvents to cosolubilize gramicidin and lipid. Using circular dichroism (CD), it was found that when codissolved in trifluoroethanol (TFE), after drying and subsequent hydration, gramicidin is mainly present in the single-stranded beta 6.3-helical configuration, whereas when using chloroform/methanol or ethanol as the solvent, it is proposed that the dominant conformation of gramicidin in the membrane is that of the double-stranded antiparallel dimer. Employing 31P NMR, the stoichiometry for bilayer formation was found to be 6 to 7 lipid molecules per gramicidin monomer, when samples were prepared from TFE, whereas a stoichiometry of 4 was found when chloroform/methanol or ethanol was the solvent. Upon heating the latter samples, a conversion was observed in the CD pattern toward that indicative of the beta 6.3-helical configuration. This change was accompanied by an increase in the extent of bilayer formation. Next, it was investigated whether the conformation of gramicidin and its ability to induce bilayer formation were dependent on the lipid acyl chain length. CD measurements of samples prepared from TFE indicated that gramicidin, independent of acyl chain length, was present in the beta 6.3-helical configuration but the intensity of the ellipticities at 218 nm increased with the length of the acyl chain. The extent of bilayer formation in these samples was found to be largely chain length independent.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
9.
10.
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
11.
Proteinchemical and kinetic features of gramicidin S synthetase 总被引:1,自引:0,他引:1
J Vater W Schlumbohm J Salnikow K D Irrgang M Miklus T Choli H Kleinkauf 《Biological chemistry Hoppe-Seyler》1989,370(9):1013-1018
The amino-acid compositions of both enzymes of gramicidin S synthetase were determined. These proteins contain a high number of acidic amino-acid residues. Phenylalanine racemase, the light enzyme, was sequenced from the N-terminus until position 10. The kinetics of the thioester formation reactions were studied. The half-life times of these processes under substrate saturation conditions were found in the range between seconds and a few minutes. The valine activation at the heavy enzyme was detected as one of the rate-limiting steps of the biosynthesis of gramicidin S. 相似文献
12.
Gramicidin S (GS) is a cyclic decapeptide antibiotic active against both Gram‐positive and Gram‐negative bacteria as well as against several pathogenic fungi. However, clinical application of GS is limited because of GS hemolytic activity. The large number of GS analogues with potentially attenuated hemolytic activity has been developed over the last two decades. For all new GS derivatives, the antimicrobial test is accompanied with the hemolytic activity assay. At the same time, neither GS nor its analogues were tested against other blood cells. In the present work, the effects of GS on platelets and platelet aggregates have been studied. GS interaction with platelets is concentration dependent and leads either to platelet swelling or platelet shape change. Effect of GS on platelets is independent of platelet aggregation mechanism. GS induces disaggregation of platelet aggregates formed in the presence of aggregation agonists. The rate of the GS interaction with platelet membranes depends on membrane lipid mobility and significantly increases with temperature. The interaction of GS with the platelet membranes depends strongly on the state of the membrane lipids. Factors affecting the membrane lipids (temperature, lipid peroxidation and ionising irradiation) modify GS interaction with platelets. Our results show that GS is active not only against erythrocytes but also against other blood cells (platelets). The estimated numbers of GS molecules per 1 µm2 of a blood cell required to induce erythrocyte hemolysis and disaggregation of platelet aggregates are comparable. This must be considered when developing new antimicrobial GS analogues with improved hemolytic properties. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
13.
14.
Chu H. Tzeng Kathleen D. Thrasher John P. Montgomery Bruce K. Hamilton Daniel I. C. Wang 《Biotechnology and bioengineering》1975,17(1):143-152
A high productivity tank fermentation for gramicidin S synthetases has been developed to supply biocatalyst for a preparative-scale ATP-driven cell-free enzymatic synthesis employing the polypeptide antibiotic, gramicidin S, as a model product. A rich, complex medium supports rapid and dense growth of the enzyme-producing microorganism, Bacillus brevis ATCC 9999, accompanied by the appearance of excellentenzyme activities. Under conditions used, the two enzyme fractions of the gramicidin S synthesizing system, as well as the total enzymatic activity for synthesis of gramicidin S, all reach their maxima simultaneously at the point where growth enters the stationary phase. Successful batch enzyme fermentations have been performed at the bench (14 liter) and pilot (180 liter)scales. 相似文献
15.
16.
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
17.
18.
C. C. Matteo M. Glade A. Tanaka J. Piret A. L. Demain 《Biotechnology and bioengineering》1975,17(1):129-142
Rapid and extensive growth of Bacillus brevis ATCC 9999 was obtained in a complex medium containing yeast extract and peptone. Gramicidin S (GS) production in this medium reached 2.5 g/liter and 0.25 g/g dry cell weight. GS synthetase I production was also high in this complex medium. Chemically defined media were also developed for this strain. In a glycerol-ammonium sulfate-Tris-salts medium, the culture grew about 40% as well (rate and extent) as in complex medium. Although GS production was low (0.23 g GS/liter), peak specific activity of GS synthetase I was as high as on complex medium. Nutritional experiments showed that growth was stimulated by glutamine, methionine, proline, arginine, and histidine. Addition of these amino acids almost doubled the rate and extent of growth and GS production on a volumetric basis. However the increase in GS was due merely to the increased cell density; GS synthetase I specific activity was in fact decreased by the supplement. Complex medium is better than defined medium for GS and GS synthetase production due to increased cell density and a slower rate of synthetase disappearance. 相似文献
19.
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
20.
T Katsu H Kobayashi T Hirota Y Fujita K Sato U Nagai 《Biochimica et biophysica acta》1987,899(2):159-170
The previous study of the action of gramicidin S on bacteria (Katsu, T., Kobayashi, H. and Fujita, Y. (1986) Biochim. Biophys. Acta 860, 608-619) prompted us to investigate further the structure-activity relationship of the gramicidin S analogues on membrane permeability. Two types of the gramicidin S analogues were used in the present study: (1) cyclo(-X-D-Leu-D-Lys-D-Leu-L-Pro-)2, where X = Gly, D-Leu and D-cyclohexylalanine (D-cHxAla); (2) N,N'-diacetyl derivative of gramicidin S (diacetyl-gramicidin S) which lacks a cationic moiety of gramicidin S. All the analogues have a beta-sheet conformation as gramicidin S. The following cellular systems were used: Staphylococcus aureus as Gram-positive bacteria, Escherichia coli as Gram-negative bacteria, human erythrocytes, rat liver mitochondria and artificial liposomal membranes. It was found that gramicidin S and one of the type 1 analogues having X = D-cHxAla induced the efflux of K+ through the cytoplasmic membrane of all types of the cells. In addition, these two peptides had the ability to lower the phase transition temperature of dipalmitoylphosphatidylcholine. Accordingly, it was concluded that, if peptides can expand greatly the membrane structure of neutral lipids which constitute main parts of the biological membrane, they can stimulate the permeability of cells without any selectivity. The action of the type 2 peptide, diacetyl-gramicidin S, was strongly cell dependent. Although this peptide stimulated the efflux of K+ from mitochondria, it did not do so efficiently, if at all, from S. aureus, E. coli and erythrocytes. In experiments using liposomes, diacetyl-gramicidin S increased markedly the permeability of liposomes composed of egg phosphatidylcholine. The presence of egg phosphatidylethanolamine or cholesterol reduced its activity. These results on liposomes explained well the low sensitivity of diacetyl-gramicidin S against E. coli and erythrocytes in terms of lipid constituents of the membranes. The mechanism of action of diacetyl-gramicidin S was discussed from the formation of a boundary lipid induced by this peptide. 相似文献