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1.
A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.  相似文献   

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Chitin is one of the most abundant biopolymers widely distributed in the marine and terrestrial environments. Chitinase enzyme has received increased attention due to its wide range of biotechnological applications, especially in agriculture for biocontrol of phytopathogenic fungi and harmful insects. In the present study, 58 bacterial isolates were screened for chitinolytic activity and on the basis of chitin hydrolysis zone 6 isolates were selected for chitinase production in broth media. Based on enzyme production, two most potent isolates identified as Aeromonas hydrophila HS4 and Aeromonas punctata HS6 were selected for further study. The effects of media composition and various fermentation conditions for optimization of chitinase production were studied. The maximum chitinase production was obtained at 37 °C and pH 8.0 after 24–48 h of incubation by HS4; and at 37 °C and pH 7 after 48 h incubation by HS6. Among the substrates colloidal chitin was the best for both the strains. Regarding carbon sources, starch (1%) was the best for both strains; while malt and yeast extract (1%) was found as the best nitrogen source for HS4 and HS6, respectively. Out of metal ions Mn2+ and Cu2+ enhanced enzyme production in the case of HS6. However, Co2+ was the most appropriate for HS4.  相似文献   

4.
Teng  Tieshan  Yu  Junping  Yang  Hang  Wei  Hongping 《中国病毒学》2015,30(1):76-79
<正>Dear Editor,Bacteriophages are powerful tools for investigating and manipulating their hosts(Fernandes et al.,2014).This holds particularly true for mycobacteriophages,which have facilitated the development of mycobacterial genetic systems and have generated tools for the clinical  相似文献   

5.
Using v-abl probes, we have identified and cloned a novel fes/fps-homologous human cDNA, which we have designated FER (pronounced "fair"). This apparently full-length cDNA of 3.0 kilobases has an open reading frame of 2,466 base pairs and the capacity to encode a protein of 94,000 molecular weight. The cDNA contains regions homologous to the highly conserved tyrosine protein kinase domain of other oncogenes and growth factor receptors but lacks a clear transmembrane region, indicating that it encodes a tyrosine kinase of the nonreceptor type. The deduced amino acid sequence of FER resembles that of c-fes/fps. Our data indicate that the protein product of FER, p94FER, corresponds to a previously reported cellular phosphoprotein, NCP94, detected with a v-fps-specific antipeptide antiserum.  相似文献   

6.
A Saito  K Ozaki  T Fujiwara  Y Nakamura  A Tanigami 《Gene》1999,239(2):325-331
Using differential display technique, we have isolated a novel human gene expressed specifically in the lung. Two forms of the gene, designated TSA1902, were transcribed by alternate mRNA splicing. The transcribed mRNAs, termed TSA1902-L and TSA1902-S, putatively encode proteins of 368 and 315 amino acids, respectively, which show high similarity to human chitotriosidase protein. The N-terminal region of TSA1902-L protein contains the conserved active site residues (DXXDXDXE) of the catalytic center of various chitinases which are essential for chitinase activity. The deduced protein sequence of TSA1902-S, however, does not possess this active site, with the N-terminal 54 amino acids present in TSA1902-L protein having been deleted. Both proteins lacked the secretory sequence of N-termini and, judging from the hydropathy profile, may be soluble proteins in the cytoplasm. Chromosomal mapping by radiation hybrid analysis localized this gene to the chromosome 1p13.1-p21.3.  相似文献   

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以西瓜尖镰孢菌诱导、提纯的豇豆抗真菌 I类几丁质酶 N端前 1 0个氨基酸序列测定的基础上 ,设计合成了引物 ,运用 PCR等分子生物学技术 ,从豇豆基因组中分离克隆了该特异几丁质酶成熟蛋白基因 ,测定分析了其全序列。该新基因全长 894bp,无内含子 ;具 Aat I、Aat II、Bgl I、Dpn I、Dpn II、Eco R II、Hae I、Hae II、Hae III、Hinf I、Hpa II、Mae II、Mae III、Nba I、Oxa I和 Sst IV酶切位点 43个 ;豇豆、Vigna unguiculata、菜豆、豌豆、烟草、小麦、水稻的同源性依次递减。扩增克隆了菜豆几丁质酶信号肽基因 ,并将其与豇豆几丁质酶成熟蛋白基因连接 ,再与 p BI1 2 1重组 ,成功构建了特异几丁质酶基因的植物表达载体 ,为进一步培育抗真菌病转基因西瓜新品种打下了坚实基础。  相似文献   

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Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences.  相似文献   

11.
Zhou  S.N.  Yang  C.Y.  Lu  Y.J.  Huang  L.  Cai  C.H.  Lin  Y.C. 《World journal of microbiology & biotechnology》1999,15(6):745-746
A chitinase was separated from the culture broth of Vibrio sp. 11211 isolated from sediment from the South China Sea. The chitinase was purified 18.3-fold with 33% recovery by ammonium sulphate precipitation and chromatography. The subunit molecular weight of the enzyme was estimated by SDS-PAGE to be about 30kDa. The enzyme showed optimum pH at 6.5 and optimum temperature at 50°C, and was stable in the pH range of 4 to 9 and at the temperature below 40°C.  相似文献   

12.
Chitinases are listed as one class of pathogenesis-related proteins, and they have become a popular research topic because of their resistance to plant-pathogenic diseases. A chitinase with antifungal activity was isolated from the Canadian cranberry beans (Phaseolus vulgaris). The procedure included extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, CM-Sephadex C-50, and Sephadex G-75. There was an almost 108-fold increase in specific activity of the purified chitinase compared with that of the crude extract. The enzyme exhibited a molecular mass of 30.6 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and non-reducing conditions, indicating that it was a monomeric protein. The pI was determined to be 7.6 by isoelectric-focusing electrophoresis. The optimum pH and the optimum temperature for activity towards N-acetyl-glucosamine was 5.4 and 40–55°C, respectively. It exerted a potent inhibitory action toward fungal species including Botrytis cinerea, Physalospora piricola, Fusarium oxysporum, and Pythium aphanidermatum. The chitinase was thermostable up to 58°C in both enzymatic reaction and antifungal activity. The present findings demonstrated a thermostable chitinase from cranberry beans with potentially exploitable significance.  相似文献   

13.
Amino acid sequence of chitinase from Streptomyces erythraeus   总被引:2,自引:0,他引:2  
The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.  相似文献   

14.
Chitinases are ubiquitous chitin-fragmenting hydrolases. Recently we discovered the first human chitinase, named chitotriosidase, that is specifically expressed by phagocytes. We here report the identification, purification, and subsequent cloning of a second mammalian chitinase. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and man the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Like chitotriosidase, AMCase is synthesized as a 50-kDa protein containing a 39-kDa N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain. In contrast to chitotriosidase, the enzyme is extremely acid stable and shows a distinct second pH optimum around pH 2. AMCase is capable of cleaving artificial chitin-like substrates as well as crab shell chitin and chitin as present in the fungal cell wall. Our study has revealed the existence of a chitinolytic enzyme in the gastrointestinal tract and lung that may play a role in digestion and/or defense.  相似文献   

15.
75-kDa chitinase, which showed potential as a biocontrol agent against Japanese pine sawyer, was characterized after purification from the integument of the fifth instar larvae of Bombyx mori by chromatography on diethylaminoethyl (DEAE)-Toyoperal 650 (M), hydroxylapatite, and Fractogel EMD DEAE 650 (M) columns. The optimum pH was 6.0 toward N-acetylchitopentaose (GlcNAc5) and 10 toward glycolchitin. The optimum temperature was 60 degrees C toward GlcNAc5 and 25 degrees C toward glycolchitn. The enzyme was stable at pH 7-10 and below 40 degrees C. Kinetic analysis and reaction-pattern analysis using glycolchitin and N-acetylchitooligosacchraides as substrates indicated that 75-kDa chitinase is an endo- or random-type hydrolytic enzyme to produce the beta anomeric product and that it prefers the longer N-acetylchitooligosaccharides, suggesting, together with the N-terminal amino acid sequence, that the 75-kDa chitinase belongs to family 18 of glycosyl hydrolases.  相似文献   

16.
The isolation of bombesin-related peptides in chicken proventriculus was monitored by radioimmunoassay using a C-terminal specific bombesin antibody. Two peptides were identified, one corresponded to the 27-residue, chicken gastrin-releasing peptide (GRP-27) previously identified; the other corresponded to its C-terminal hexapeptide. Chicken GRP-27 stimulated pancreatic and gastric acid secretion in anaesthetized turkeys, but the hexapeptide was inactive. No evidence could be found to suggest that the hexapeptide was an artifact of degradation generated during extraction or isolation. It is proposed that the hexapeptide is produced either by chymotryptic-like cleavage of GRP-27 or by trypsin-like cleavage followed by two cycles of dipeptidylaminopeptidase cleavage. This type of biosynthetic processing may be more common than formerly supposed.  相似文献   

17.
Family 18 chitinases play key roles in the life cycles of a variety of organisms ranging from bacteria to man. Very recently it has been shown that one of the mammalian chitinases is highly overexpressed in the asthmatic lung and contributes to the pathogenic process through recruitment of inflammatory cells. Although several potent natural product chitinase inhibitors have been identified, their chemotherapeutic potential or their use as cell biological tools is limited due to their size, complex chemistry, and limited availability. We describe a virtual screening-based approach to identification of a novel, purine-based, chitinase inhibitor. This inhibitor acts in the low micromolar (Ki=2.8+/-0.2 microM) range in a competitive mode. Dissection of the binding mode by x-ray crystallography reveals that the compound, which consists of two linked caffeine moieties, binds in the active site through extensive and not previously observed stacking interactions with conserved, solvent exposed tryptophans. Such exposed aromatics are also present in the structures of many other carbohydrate processing enzymes. The compound exhibits favorable chemical properties and is likely to be useful as a general scaffold for development of pan-family 18 chitinase inhibitors.  相似文献   

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In plants, various chitinases have been identified and categorized into several groups based on the analysis of their sequences and domains. We have isolated SafchiA, a novel class of chitinase from saffron (Crocus sativus L.). The cDNA encoding SafchiA is mainly expressed in roots and corms, and its expression is induced by elicitor treatment, methyl jasmonate, wounding, and by the fungi Fusarium oxysporum, Beauveria and Phoma sp., suggesting a defence role of the protein. Furthermore, in vitro assays with the recombinant native protein showed chitinolytic, and antifungal activity. The deduced protein shares high similarity with chitinases belonging to family 19 of glycosyl-hydrolases, although some changes in the enzyme active site are present. To explore the properties of SafchiA we have expressed recombinant SafchiA in Escherichia coli and generated four different mutants affected in residues involved in the catalytic activity. One glutamic acid essential for family 19 chitinases activity is not present in C. sativus chitinase suggesting that only one acidic residue is necessary for the enzyme activity, in a similar manner as family 18 glycosyl-hydrolases.  相似文献   

20.
Using a radioimmunoassay system employing an antiserum which recognises the common C-terminal tripeptide (YIL) of neurotensin (NT) and neuromedin N (NN), immunoreactivity was identified in extracts of brain (65.8 pmol/g), small intestine (44.2 pmol/g) and rectum (13.2 pmol/g) of the European common frog (Rana temporaria). No immunoreactivity was detected in extracts of stomach and skin. Reverse-phase HPLC analysis of each tissue extract resolved a single immunoreactive peptide with identical retention time in each case. The immunoreactive peptide was isolated by reverse-phase HPLC from brain extracts and an N-terminal pyroglutamyl residue was successfully removed enzymatically. The molecular mass of des(pyroglutamyl) frog NT, determined by plasma desorption mass spectroscopy, was 1440 Da. The primary structure of this peptide was determined by gas-phase sequencing and the calculated molecular mass, 1440.7 Da, was in close agreement with that derived by mass spectroscopy. The full primary structure of frog NT was established as: QSHISKARRPYIL. When compared with bovine NT, frog NT exhibits five amino acid substitutions in the N-terminal region, whereas the C-terminal hexapeptide sequence (RRPYIL), which mediates the classical biological effects of NT, is completely conserved. Amphibia thus possess a tridecapeptide NT which is analogous to that of higher vertebrates and considerable constraints on the primary structure of the C-terminal biologically-active core have existed for a vast evolutionary time span.  相似文献   

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