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1.
Miyashita M Nakamori T Miyagawa H Akamatsu M Ueno T 《Bioscience, biotechnology, and biochemistry》2003,67(3):635-638
The effect of the host-specific phytotoxins, AM-toxins, on the photosynthetic activity of leaves from susceptible apple cultivars was investigated by using an oxygen electrode. The photosynthetic O2 evolution was inhibited by AM-toxin I in a host-specific manner. The inhibitory activity of several AM-toxin analogs against photosynthesis was also evaluated and the findings were correlated with their necrosis-inducing activity. 相似文献
2.
Tsutomu Higashijima Yasuyuki Shimohigashi Tetsuo Kato Nobuo Izumiya Tamio Ueno Tatsuo Miyazawa 《Biopolymers》1983,22(4):1167-1187
Cyclic tetradepsipeptides, AM-toxin I and II, are the host-specific phytotoxins of Alternaria mali. In order to elucidate conformation-toxicity relationships, we analyzed the 270-MHz proton nmr spectra of AM-toxins and hydrogenated analogs, (D -Ala2)AM-toxin I (toxic) and (L -Ala2)AM-toxin I (not toxic), in (C2H3)2SO. These cyclic tetradepsipeptides do not contain N-substituted amino acid residues, and all the peptide and ester groups have been found to be transoid. Two conformers with very unequal populations have been found for AM-toxin I and II; the Cβ?Cα? C?O conformations of the Dha2 residues are nonplanar S-trans in the major conformer and nonplanar S-cis in the minor conformer. Only one ring conformation has been found for each of (L -Ala2) and (D -Ala2)AM-toxin I. (L -Ala2)AM-toxin I takes a C4-type ring conformation; all the C?O groups and Cα-H bonds are oriented to the same side of the ring. (D -Ala2)AM-toxin I takes a new ring conformation; the side chain and C?O group of the L -Amp1 residue are oriented to the same side of the ring. This new conformation is also found for the major conformers of AM-toxin I and II and thus appears to be required for the toxicity. The ring conformations of Tyr(OCH3)1-bearing analog tetradepsipeptides have been found to be much the same as those of Amp1-bearing depsipeptides. Furthermore, on the basis of the two distinct conformations of (D -Ala2) and (L -Ala2)AM-toxin I, an empirical rule is proposed for the stable ring conformations of cyclic tetra-D ,L -peptides, not containing N-substituted amino acid residues. 相似文献
3.
Miyashita M Nakamori T Murai T Yonemoto T Miyagawa H Akamatsu M Ueno T 《Zeitschrift für Naturforschung. C, Journal of biosciences》2001,56(11-12):1029-1037
AM-toxins are host-specific phytotoxins of the Alternaria alternata apple pathotype, which induce necrosis on apple leaves. In this study, we developed a new assay to measure the necrotic activity of AM-toxin analogs using cultured leaves from meristem cells. This method was not only more sensitive to AM-toxin I, but also more reliable than the previous one that used tree leaves due to the homogeneous nature of cultured leaves and to the method of application of toxins. Using this assay method we investigated a structure-activity relationship of AM-toxin analogs synthesized in this study. Most residues and the macrocyclic ring structure were strictly recognized by AM-toxin putative receptor, whereas the L-Ala binding subsite of the receptor allowed for side chain structures with various stereoelectronic properties. These findings are important for designing ligands for further experimental probing of the nature of the receptor. 相似文献
4.
Johnson RD Johnson L Itoh Y Kodama M Otani H Kohmoto K 《Molecular plant-microbe interactions : MPMI》2000,13(7):742-753
Afternaria afternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. afternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction. 相似文献
5.
We produced mutants resistant to Alternaria blotch disease in several cultivars of apple (Malus × domestica Borkh.) by irradiation with X- or γ-rays. An efficient in vitro assay method was established using chemically-synthesized AM-toxin I of Alternaria alternata (Fr.) Keissler to screen for mutants resistant to Alternaria blotch disease. The frequency of necrotic lesions was investigated by applying various concentrations of AM-toxin I to leaf discs of the first, third, and fifth leaves from the shoot apex of several apple cultivars, including Jonathan, Fuji, Oorin, and Indo. In vitro-grown apple shoots of susceptible cultivars were then treated with various doses of X- or γ-ray irradiation. Several mutants resistant to AM-toxin I were obtained by combining the techniques for tissue culture of apple shoots with the AM-toxin I screening method. Following a repeat second screening test with AM-toxin I, mutant plants were sprayed with a spore suspension of A. alternata and found resistant to be the fungal pathogen. These mutants showed normal phenotypic appearance, and so far, no difference has been observed between the original plants and mutants except for the susceptibility to Alternaria blotch. 相似文献
6.
In order to explore the route for the preparation of cyclodepsipeptide by cyclization through an ester bond formation, two analogs of AM-toxin II, cyclotetradepsipeptide, were synthesized. As a preliminary experiment, synthesis of [L-Phe3, L-Ser(Bzl)4]-AM-toxin II, containing L-Phe and L-Ser(Bzl) in place of L-App (2-amino-5-phenyl-pentanoic acid) and delta Ala (alpha, beta-dehydroalanine), respectively, was attempted. Cyclization of H-L-Hmb-L-Phe-L-Ser(Bzl)-L-Ala-OH in CH2Cl2 at 10 mM concentration using water-soluble carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) successfully gave a cyclic monomer in 16% yield. Cyclization of H-L-Hmb-L-App-L-Ser(Bzl)-L-Ala-OH under the same conditions also afforded a cyclic monomer, [L-Ser(Bzl)4]AM-toxin II, in 19% yield. Analytical parameters of these cyclic monomers obtained were identical to those of the authentic samples obtained by cyclization through a peptide bond formation. 相似文献
7.
Bunzo Matsuura Maoqing Dong Laurence J Miller 《The Journal of biological chemistry》2002,277(12):9834-9839
The predicted second extracellular loop domain of the motilin receptor is of particular interest because it is a region that is quite distinct from the analogous regions in other family members that are most closely related and because the initial report of the photoaffinity labeling of a domain of this receptor included this region (Coulie, B. J., Matsuura, B., Dong, M., Hadac, E. M., Pinon, D. I., Feighner, S. D., Howard, A. D., and Miller, L. J. (2001) J. Biol. Chem. 276, 35518-35522). In the current work, motilin receptor constructs were prepared that included sequential deletions ranging from single residues to twelve amino acid segments throughout this 67 amino acid domain. Each construct was expressed in COS cells and characterized for motilin radioligand binding and motilin-stimulated intracellular calcium responses. The only segments that had negative impact on motilin binding and biological activity included deletion constructs DeltaCys(235), Delta179-182, and Delta241-246. Cys(235) is likely involved in the highly conserved and functionally important disulfide bond linking the first and second loops of G protein-coupled receptors. Alanine replacements for each of the amino acid residues in the other two segments revealed that the perimembranous residues at both ends of this loop, Val(179) and Leu(245) and Arg(246), were responsible for the negative impact on motilin binding and biological activity. Of note, these mutants responded normally to the non-peptidyl agonist, erythromycin. These data support important functional roles for both amino-terminal and carboxyl-terminal perimembranous regions of the second loop for responses to the natural agonist peptide, while supporting independent determinants for action of a non-peptidyl agonist ligand. 相似文献
8.
K A Jacobson M S Verlander R P Rosenkranz K L Melmon M Goodman 《International journal of peptide and protein research》1983,22(3):284-304
A series of monodisperse oligopeptide conjugates related to the catecholamine, isoproterenol, has been synthesized. The peptide carrier molecules used were synthesized by stepwise and fragment condensation techniques and ranged in size from a single, blocked amino acid derivative to isomeric pentapeptides. The amino acid compositions and sequences of the carriers were chosen so as to provide specific information concerning the effects of molecular weight, hydrophilic/hydrophobic balance, charge, etc., on the biological activity of the final conjugates. The common point of attachment for the drug in all carriers was a p-aminophenylalanine residue. The peptide-catecholamine conjugates were prepared via the attachment of carboxyl-containing catecholamine congeners, to the peptide carriers by techniques described previously. The conjugates were purified rigorously by chromatographic techniques and characterized by high-field n.m.r. spectroscopy. 相似文献
9.
M R Sairam 《International journal of peptide and protein research》1979,14(2):153-160
The subunits of ovine lutropin prepared by acid dissociation and salt precipitation were characterized by end group analysis, tryptic peptide mapping, SDS gel electrophoresis and biological activity. No evidence of internal peptide cleavage was found in the alpha subunit. The subunits possessed low activity. The alpha and beta subunits recombined effectively to generate a complex that had full receptor binding activity and in vitro biological activity. The recombinants of subunits prepared by countercurrent distribution showed only 50% activity in both assays. The salt precipitation method alpha subunit could be completely reduced and reoxidized in the absence of denaturants. The reoxidized alpha subunit combines with the native beta subunit generating full activity. However, this recombined hormone tends to lose activity with time, suggesting that the reoxidation may not fully restore the native structur of the reduced alpha subunit. The native lutropin alpha subunit effectively combined with follitropin beta subunit generating complete follitropin activity. 相似文献
10.
The glucagon receptor antagonist BI-32169, recently isolated from Streptomyces sp., was described as a bicyclic peptide, although its primary structure comprises conserved elements of class I and class II lasso peptides. Tandem mass spectrometric and nuclear magnetic resonance spectroscopic studies revealed that BI-32169 is a lasso-structured peptide constituting the new class III of lasso peptides. The determined lasso fold opens new avenues to improve the promising biological activity of BI-32169. 相似文献
11.
J Stagsted W A Baase A Goldstein L Olsson 《The Journal of biological chemistry》1991,266(20):12844-12847
It was recently shown that a 25-residue peptide, Dk-(61-85), derived from the alpha 1 domain of a murine major histocompatibility class I molecule (H-2Dk), affects insulin receptor functions (Hansen, T., Stagsted, J., Pedersen, L., Roth, R. A., Goldstein, A., and Olsson, L. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3123-3126; Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now report that this peptide can reversibly assume a biologically active or inactive state as measured in the rat adipocyte glucose uptake assay, implying that the peptide has at least two interconvertible conformations. The peptide has an ordered conformation in 0.1 M HCl or 0.1 M NaCl stock solution as shown by circular dichroism, but has a disordered molecular structure and is inactive when dissolved in H2O. The biologically active peptide forms liquid crystals at the stock solution concentration (1 mM), so the CD spectra do not provide information on the secondary structure. Under all conditions tested, biological activity (measured after transfer to assay buffer) is associated with an ordered conformation in stock solution. Biological activity and an ordered conformation of the peptide in H2O stock solution can be induced by increasing ionic strength (greater than 100 mM NaCl for maximal effect) or increasing pH (greater than 5 for maximal effect). The induction rate of the ordered conformation is slow with a half-maximal value obtained after approximately 20 min. Both biological activity and the ordered structure are lost upon heating of stock solution to 90 degrees C or upon transfer to assay buffer. A similar correlation of ordered structure with biological activity was observed with two truncated peptides derived from Dk-(61-85). It is inferred from these results that the Dk-(61-85) peptide and related peptides only affect insulin-stimulated glucose uptake in rat adipocytes if they have assumed an ordered conformation in stock solution prior to transfer to assay buffer and exposure to cells. 相似文献
12.
Modulation of plasminogen activation and type IV collagenase activity by a synthetic peptide derived from the laminin A chain. 总被引:3,自引:0,他引:3
Laminin is a large multidomain glycoprotein with diverse biological activities which include stimulation of neurite outgrowth, enhancement of tumor metastasis, and promotion of cell growth, adhesion, and differentiation. A 19 amino acid synthetic peptide derived from the E8 fragment of the laminin A chain (Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp -Arg- NH2) was identified which promotes metastasis and stimulates collagenase IV activity in the culture medium of B16 melanoma cells (Kanemoto et al., 1990). We report that this peptide, here designated LamA2091-2108, is also a potent stimulator of tissue plasminogen activator (t-PA)-catalyzed plasminogen activation, resulting in a 22-fold increase in the kcat/Km of the activation reaction. The activity of purified type I and type IV collagenase was inhibited by LamA2091-2108 with IC50 values of 3 and 43 microM, respectively. These data support an alternative mechanism for the appearance of collagenase activity in the culture media of melanoma cells, namely, that the peptide stimulates plasminogen activation, subsequently generating collagenase activity. 相似文献
13.
利用噬菌体展示技术筛选特异性人源抗ICAM-1单链抗体(Anti-human ICAM-1 scFv)并进行生物学活性鉴定。应用Tomlinson I+J噬菌体抗体库,以P1抗原肽为包被抗原,经过4轮“吸附-洗脱-扩增”进行亲和富集筛选。以PCR反应、ELISA抗原交叉反应和Dot blotting实验进行阳性克隆的鉴定。scFv经原核表达和分离纯化后,以Western blotting实验、竞争ELISA实验和细胞黏附抑制实验对其生物学活性进行初步鉴定。Tomlinson I+J噬菌体抗体库经4轮亲和富集筛选,利用ELISA方法成功筛出4株阳性克隆。通过PCR鉴定反应、ELISA抗原交叉反应和Dot blotting实验,最终获得了1株既能与P1抗原肽特异结合又能与人ICAM-1抗原特异结合的阳性克隆J-A1。对scFv进行原核表达和亲和层析后获得了高纯度的目的蛋白。竞争ELISA实验和细胞黏附抑制实验证实纯化的scFv具有良好的亲和活性和抗细胞黏附活性。文中成功利用噬菌体展示技术筛选到特异性人源抗ICAM-1 scFv,为进一步探索该抗体在炎症相关性疾病治疗中的应用奠定了基础。 相似文献
14.
D H Silverman J M Krueger M L Karnovsky 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(6):2195-2201
Two radiolabeled (125I) muramyl peptide derivatives of high specific activity were prepared: a tripeptide with an iodinated C-terminal tyrosine methyl ester (Ligand I), and a muramyl tripeptide with a C-terminal lysine derivatized with Bolton-Hunter reagent (Ligand II). These were used to characterize binding of muramyl peptides to monolayers of murine macrophages. Saturable high-affinity binding to resident, caseinate-elicited, and Listeria-activated peritoneal cells was observed with both radioligands. Binding affinities varied with the state of activation of the macrophages, and KD values ranged from 48 +/- 33 pM (for resident macrophages, Ligand I) to 1020 +/- 90 pM (for activated macrophages, Ligand II). Specific binding sites were also found on a macrophage-derived cell line. The ability of several unlabeled muramyl peptides to compete with Ligands I and II for their binding sites was tested. Competition was stereospecific and correlated with known biological activities of these compounds (i.e., immunoadjuvanticity, pyrogenicity, and somnogenicity). The sites identified here for Ligands I and II may mediate some of the effects that muramyl peptides have previously been demonstrated to have on macrophages. 相似文献
15.
Relaxin is a member of the insulin superfamily and has many biological actions including angiogenesis and collagen degradation. It is a 6 kDa peptide hormone consisting of two peptide chains (A and B) tethered by two disulphide bonds. Past structure-function relationship studies have shown the key receptor binding site of relaxin to be principally situated within the B-chain alpha-helix. Molecular dynamic simulations were performed to aid the design of conformationally constrained relaxin B-chain analogues that possess alpha-helical structure and relaxin-like activity. Restraints included disulphide bonds, both single and double, and lactam bonds. Each peptide was prepared by solid phase synthesis and, following purification, subjected to detailed conformational analysis by circular dichroism spectroscopy. Of 15 prepared relaxin B-chain mimetics, one was able to mimic the secondary structure of the native ligand as indicated by biomolecular recognition/interaction analysis using surface enhanced laser desorption ionization mass spectroscopy together with a relaxin antibody. However, none of the mimetics possess characteristic relaxin-like biological activity which strongly indicates that the pharmacophore comprises additional structural elements other than the relaxin B-chain alpha-helix. These findings will assist in the design and preparation of novel relaxin agonists and antagonists. 相似文献
16.
Wang YJ Zhao Y Meredith J Phillips JE Theilmann DA Brock HW 《Archives of insect biochemistry and physiology》2000,45(3):129-138
Ion transport peptide (ITP) stimulates Cl(-) transport (measured as short-circuit current, I(sc)) and fluid reabsorption in Schistocerca gregaria ilea. We report that Drosophila Kc1 cells transfected with preproITP cDNA secrete a peptide (KcITP(75)) that, while cleaved correctly at the N-terminus, had reduced (10-fold) stimulatory activity on ileal I(sc) compared to both native ITP (ScgITP) and synthetic ITP (synITP). We provide evidence that the reduced activity of KcITP(75) is due to incomplete processing of the C-terminal sequence LGKK (KcITP(75)) to L-amide. In support of this, in vitro amidation of glycine extended ITP (i.e., KcITP(73) ending in LG) but not KcITP(75) (ending in LGKK) significantly increased specific activity in the bioassay. Further evidence for C-terminus involvement includes complete loss of stimulation by truncated mutants (e.g., KcITP(71) which lacks LGKK) and a mutant in which alanine is substituted for the terminal glycine in KcITP(73). Moreover a natural homologue (KcITP-L, which differs only in the C-terminal sequence) expressed by Kc1 cells does not stimulate ileal I(sc). Rather KcITP-L acts as a weak ITP antagonist, as does the truncated mutant KcITP(71). KcITP(70) has no antagonistic effect. A short synthetic peptide fragment of the C-terminus (VEIL-amide) does not stimulate ileal I(sc), indicating that other regions of ITP are also essential to biological activity. Arch. 相似文献
17.
Szekat C Jack RW Skutlarek D Färber H Bierbaum G 《Applied and environmental microbiology》2003,69(7):3777-3783
The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity. 相似文献
18.
Ishino T Urbina C Bhattacharya M Panarello D Chaiken I 《The Journal of biological chemistry》2005,280(24):22951-22961
The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor alpha (IL5Ralpha) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N- and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556-568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5Ralpha interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide.receptor complex is comparable with that of the cytokine.receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5Ralpha, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its binding footprint on IL5Ralpha by alanine substitution of Asp(55), Asp(56), Glu(58), Lys(186), Arg(188), and Arg(297) of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5Ralpha, in particular Asp(55), Arg(188), and Arg(297) in the D1, D2, and D3 domains, respectively. This footprint represents a significant subset of that for IL5 binding. The fact that AF17121 mimics the receptor binding capability of IL5 but antagonizes biological function evokes several models for how IL5 induces activation of the multisubunit receptor system. 相似文献
19.
A K Koul G F Wasserman P K Warme 《Biochemical and biophysical research communications》1979,89(4):1253-1259
New semi-synthetic peptide analogs of horse heart cytochrome have been prepared by replacing tyrosine at position 67 by phenylalanine and L-parafluorophenylalanine. The former has 56% biological activity in the succinate oxidase system whereas the parafluorophenylalanine analog is totally inactive. The tyrosine at position 74 has also been replaced by leucine. It shows a high level of biological activity (58%), which demonstrates that the presence of an aromatic residue at this position is not required for electron transfer. 相似文献
20.
Construction of an Expression System for Site-Directed
Mutagenesis of the Lantibiotic
Mersacidin 总被引:4,自引:2,他引:2
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Christiane Szekat Ralph W. Jack Dirk Skutlarek Harald Frber Gabriele Bierbaum 《Applied microbiology》2003,69(7):3777-3783
The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity. 相似文献