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1.
The goal of this study was to map an epitope on the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) at its C terminus, a region whose integrity is fundamental in maintaining the normal function of this molecule. Residues including the fourth alpha-helix (D, 103-116) were analyzed for their role in the interaction with antibodies (Abs) raised against the protein. Five peptides homologous to different segments of the C terminus of hGM-CSF were synthesized. Peptide-(102-121) included the same residues of the alpha-helix D and the next five amino acids toward the C terminus; peptide-[E108A]-(102-121) introduced the mutation E108A in order to verify the role of acidic residues; peptide-[C96A](93-110) encompassed the beta-sheet 2 and half of the alpha-helix D; peptide-[C121A]-(110-127) included the second half of the alpha-helix D and the C terminus of hGMCSF; peptide-(13-31)-Gly-Pro-Gly-(103-116) included both the alpha-helices A and D connected by the tripeptide Gly-Pro-Gly, which allows the original antiparallel orientation of the two alpha-helices to be maintained. Both anti-protein and anti-peptide-(102-121) antibodies, capable of neutralizing the stimulatory activity of hGMCSF in the bone marrow colony-forming assays, recognized a specific epitope in the C terminus of hGM-CSF. Molecular modeling estimated the surface accessibility of hGM-CSF and the stability of the synthetic peptides in aqueous solution. Altogether, our results showed that the immunogenic region includes part of the alpha-helix D and the residues 116-120, which are external to this helix and particularly exposed on the protein surface, confirming the feasible participation of this region in antibody binding. 相似文献
2.
We have been interested in partially folded proteins with marginal stability and activity, because they have a potential to be mature proteins by artificial evolution. A module is defined as a contiguous peptide chain forming a compact region in a globular protein. Modules may be used as building blocks to create partially folded proteins. Barnase, a ribonuclease consisting of 110 amino acids, has been divided into six modules (M1-M6), four peptide fragments, M12 (1-52), M123 (1-73), M1234 (1-88) and M12345 (1-98), have been constructed by progressive elongation of the modules from the N-terminus. Only M12345 (1-98) had a partially folded conformation, but it lacked detectable RNase activity. A mixture of M12345 (1-98) with M56 (89-110) showed weak but distinct RNase activity. Unfolded M12345 (1-96) was constructed by removal of two residues from the C-terminus of M12345 (1-98). The mixture of M12345 (1-96) with M56 (89-110) also showed RNase activity. Further, the interaction endowed M12345 (1-96) with conformational stability. We propose that N- and C-terminal fragments obtained by successive elongation of modules would interact to be a complex with marginal stability and activity, which would be used for creating a mature complex by artificial evolution. 相似文献
3.
Prion protein fragments that are extracted from the brains of patients with Gerstmann-Straussler-Scheinker disease are known to have stimulating action on circulating leukocytes. In particular, the amyloidogenic hydrophobic prion peptide HuPrP (113-127) AGAAAAGAVVGGLGG has been reported to be associated with significant cellular toxicity. In this paper we show that the self assembled form of HuPrP (113-127) and its valine rich domains viz. GAVVGGLG [HuPrP (119-126)] and VVGGLGG [HuPrP (121-127)] are toxic to peripheral lymphocytes. To explore the cytotoxic mechanism of these fragments, we studied 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) reduction, reactive oxygen species (ROS) generation, calcium influx and raft sequestration of' peptide treated lymphocytes. Langmuir monolayer studies on these peptides showed a maximum lipid perturbing property of HuPrP (121-127) as compared to the other two fragments. MTT reduction assays on lymphocytes treated with peptides indicated that the prion peptide fibrils are relatively more toxic than freshly solubilized peptide preparations. Lymphocytes treated with HuPrP (121-127), HuPrP (113-127) and HuPrP (119-126) fibrils underwent 60%, 30% and 40% cell death, respectively. Abeta(1-42), HuPrP (119-126) and HuPrP (121-127) fibrils caused 4 fold increases in intracellular ROS as compared with control cells. However, HuPrP (113-127) fibrils lacked such a significant ROS generating activity, indicating that a subtle difference in sequence leads to a difference in the toxic mechanism in the cell. HuPrP (119-126) and HuPrP (121-127) fibrils also produced maximum raft sequestration and calcium influx. Taken together, these data suggest that the assemblage of prion fragments has significant toxic activity on peripheral lymphocytes, a finding with implications for controlling reactive lymphocytes in prion infected subjects. 相似文献
4.
I Clark-Lewis C Schumacher M Baggiolini B Moser 《The Journal of biological chemistry》1991,266(34):23128-23134
Interleukin-8 (IL-8) is an inflammatory mediator that stimulates neutrophil migration and functional activation. Analogs of human IL-8 were chemically synthesized, purified, and compared with the full-length 72-residue synthetic IL-8 for their ability to stimulate neutrophil chemotaxis and exocytosis as measured by assaying for release of elastase, as well as their binding to specific receptors in competition assays. Analogs corresponding to the less abundant natural forms, 3-72, 4-72, and 77-residue IL-8, were evaluated and the 3-72 and 4-72 had 2-5-fold higher potencies, whereas the 77-residue IL-8 was 2-fold less potent. A major finding was that NH2-terminal residues 4, 5, and 6 were absolutely essential for IL-8 activity and receptor binding. Quantitative dissociation of elastase release and chemotaxis activity was detected with 5-72, which compared with 1-72, was 80-fold less potent in the elastase assay, but was only slightly less potent in stimulating chemotaxis. IL-8 6-72 lacked all the biological activities tested but had detectable receptor binding activity. The NH2-terminal peptide, AVLPRSAKEL, lacked activity and receptor binding, suggesting that the NH2-terminal region alone is not sufficient for function. Comparison of analogs shortened at the COOH terminus showed that potency was progressively reduced as the COOH-terminal residues were excluded. However activity was retained in an analog (1-51) with the entire COOH-terminal alpha helix and beta turn missing. A peptide corresponding to the COOH-terminal 22 residues, although inactive alone, synergized with the 1-51 analog in stimulating elastase release. The results suggest that the NH2-terminal residues 4, 5, and 6, which are disordered in the IL-8 solution structure, are directly involved in receptor binding, but the COOH-terminal alpha helix is probably important for stabilizing the three-dimensional structure. Other regions within residues 7-51 are also functionally important. 相似文献
5.
Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor. 总被引:7,自引:1,他引:6
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The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond. 相似文献
6.
We report the conformational and toxic properties of two novel fibril-forming prion amyloid sequences, GAVVGGLG (PrP(119-126)) and VVGGLGG (PrP(121-127)). The conformational preferences of these fragments were studied in differing microenvironments of TFE/water mixtures and SDS solution. Interestingly, with an increase in TFE concentration, PrP(119-126) showed a helical conformational propensity, whereas PrP(121-127) adopted a more random coil structure. In 5% SDS, PrP(119-126) showed more alpha-helical content than in TFE solution, and PrP(121-127) exhibited a predominantly random coil conformation. However, both peptides took a random coil conformation in water, and over time the random coil transformed into a beta-sheet structure with a significant percentage of helical conformation and beta-turn structure in PrP(119-126) and PrP(121-127), respectively, as observed with CD spectroscopy. The aged fibrils of PrP(119-126) were insoluble in SDS, and PrP(121-127) was extractable with SDS solution. These fibrils were characterized by transmission electron microscopy. Both PrP(119-126) and PrP(121-127) formed stable monolayer's consisting of multimeric assemblages at the air-water interface. Monomeric PrP(119-126) was more toxic to astrocytes than the control Abeta peptide; however, the fibrillar form of PrP(119-126) was less toxic to astrocytes. PrP(121-127) elicited moderate toxicity in both soluble and fibrillar forms on astrocytes. Furthermore, quenching experiments using acroyl-labeled PrP(119-126) and PrP(121-127) with eosin-labeled synaptosomal membrane revealed that these prion fragments bind to anion-exchange protein. The binding of PrP(119-126) and PrP(121-127) with a membrane microdomain (lipid raft) was also analyzed using pyrenated derivatives. We conclude that the formation of PrP(119-126) and PrP(121-127) fibrils is a concentration-dependent process that involves coil to sheet conversion with aging. PrP(119-126), the sequence with intrinsic helical propensity, is more toxic in monomer form, and the fibril formation in this case seems to be protective to cells. For PrP(121-127), the SDS-soluble fibrils are more cytotoxic, indicating that a higher order assemblage structure is required for cytotoxic activity of this peptide. 相似文献
7.
A linear analog of atrial natriuretic peptide (ANP) discriminates guanylate cyclase-coupled ANP receptors from non-coupled receptors 总被引:2,自引:0,他引:2
G M Olins D R Patton P R Bovy P P Mehta 《The Journal of biological chemistry》1988,263(22):10989-10993
Atrial natriuretic peptide (ANP) contains a disulfide which is generally considered to be required for biological activity. A truncated linear ANP analog, des-Cys105,Cys121-ANP-(104-126) (referred to as analog I), that lacks the 2 cysteine residues of the parent peptide was synthesized. In competition binding studies using rabbit lung membranes, ANP-(103-126) and analog I displaced bound 125I-ANP-(103-126) from specific ANP binding sites 100 and 73%, respectively. The concentrations of ANP-(103-126) and analog I that produced 50% inhibition of radioligand binding to the membranes were 0.26 +/- 0.07 and 0.31 +/- 0.09 nM, respectively. Radioiodinated ANP-(103-126) and analog I were chemically cross-linked to binding sites on rabbit lung membranes, and the labeled membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. 125I-Analog I specifically labeled a 65,000-dalton protein and a 135,000-dalton protein which, under reducing conditions, dissociated into 65,000-dalton subunits. In contrast, 125I-ANP-(103-126) labeled specifically a nonreducible 135,000-dalton protein, in addition to the 65,000-dalton species and the reducible 135,000-dalton species. ANP-(103-126) (100 nM) stimulated rabbit lung particulate guanylate cyclase activity, whereas analog I, at the same concentration, had no effect on cyclic GMP production and did not antagonize the effect of ANP-(103-126). From these observations, we conclude that analog I is a selective ligand which binds to approximately 73% of the total ANP binding sites present in rabbit lung membranes. Unlike ANP-(103-126), analog I does not bind to the remaining 27% of the binding sites and does not activate guanylate cyclase. Binding to the cyclase-linked ANP receptor correlates with the specific labeling by 125I-ANP-(103-126) of the nonreducible 135,000-dalton membrane protein. 相似文献
8.
Two regions of the adenovirus early region 1A proteins are required for transformation. 总被引:63,自引:36,他引:27
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Regions of the adenovirus type 5 early region 1A (E1A) proteins that are required for transformation were defined by using a series of deletion mutants. Deletion mutations collectively spanning the entire protein-coding region of E1A were constructed and assayed for their ability to cooperate with an activated ras oncogene to induce transformation in primary baby rat kidney cells. Two regions of E1A (amino acids 1 to 85 and 121 to 127) were found to be essential for transformation. Deletion of all or part of the region from amino acids 121 to 127 resulted in a total loss of transforming ability. An adjacent stretch of amino acids (residues 128 to 139), largely consisting of acidic residues, was found to be dispensable for transformation but appeared to influence the efficiency of transformation. Amino acids 1 to 85 made up a second region of the E1A protein that was essential for transformation. Deletion of all or part of this region resulted in a loss of the transforming activity. Even a mutation resulting in a single amino acid change at position 2 of the polypeptide chain was sufficient to eliminate transformation. Deletion of amino acids 86 to 120 or 128 to 289 did not eliminate transformation, although some mutations in these regions had lowered efficiencies of transformation. Foci induced by transformation-competent mutants could be expanded into cell lines that retained their transformed morphology and constitutively expressed the mutant E1A proteins. 相似文献
9.
Interaction of synthetic peptides of apolipoprotein C-II and lipoprotein lipase at monomolecular lipid films 总被引:2,自引:0,他引:2
R L Jackson A Balasubramaniam R F Murphy R A Demel 《Biochimica et biophysica acta》1986,875(2):203-210
The triacylglycerol hydrolyase and phospholipase A1 activities of bovine milk lipoprotein lipase toward long-chain fatty acyl ester substrates were investigated with monomolecular lipid films containing trioleoylglycerol and phosphatidylcholine. In a monolayer of egg phosphatidylcholine containing 3 mol% [14C]trioleoylglycerol, and in the presence of apolipoprotein C-II, a 79 amino acid activator protein for lipoprotein lipase, enzyme activity was maximal at a surface pressure of 21-22 mN X m-1 (37 mumol oleic acid released/h per mg enzyme); enzyme activity was enhanced 9-fold by apolipoprotein C-II. At surface pressures between 22 and 30 mN X m-1, lipoprotein lipase activity decreased over a broad range and was nearly zero at 30 mN X m-1. Apolipoprotein C-II and the synthetic fragments of the activator protein containing residues 56-79, 51-79 and 44-79 were equally effective at 20 mN X m-1 in enhancing lipoprotein lipase catalysis. However, at surface pressures between 25 and 29 mN X m-1, only apolipoprotein C-II and the phospholipid-associating fragment containing residues 44-79 enhanced enzyme catalysis. The effect of apolipoprotein C-II and synthetic peptides on the phospholipase A1 activity of lipoprotein lipase was examined in sphingomyelin:cholesterol (2:1) monolayers containing 5 mol% di[14C]myristoylphosphatidylcholine. At 22 mN X m-1, apolipoprotein C-II and the synthetic fragments containing residues 44-79 or 56-79 enhanced lipoprotein lipase activity (70-80 nmol/h per mg enzyme). In contrast to trioleoylglycerol hydrolysis, the synthetic fragments were not as effective as apolipoprotein C-II enhancing enzyme activity towards di[14C]myristoylphosphatidylcholine at higher surface pressures. We conclude that the minimal amino acid sequence of apolipoprotein C-II required for activation of lipoprotein lipase is dependent both on the lipid substrate and the packing density of the monolayer. 相似文献
10.
The Cbp3 protein of Saccharomyces cerevisiae is an enzyme-specific chaperone required for the assembly of ubiquinol-cytochrome c reductase of the mitochondrial respiratory chain. To gain preliminary insight into the role of Cbp3p during assembly, 29 independently isolated mutants were examined to define functional regions of the protein. Mutants were analyzed with respect to respiratory growth, ubiquinol-cytochrome c reductase assembly, and steady state amounts of enzyme subunits and Cbp3p. Three regions essential for Cbp3p activity were identified: regions 1 and 3 were required for Cbp3p function, while region 2 was necessary for protein stability. Mutation of Glu134 in region 1 (Cys124 through Ala140) impaired the ability of the Rieske FeS protein to assemble with the enzyme complex. Mutations targeted to region 3 (Gly223 through Asp229) primarily affected the 14 kDa subunit and cytochrome c(1) assembly. Gly223 was found especially sensitive to mutation and the introduction of charged residues at this site compromised Cbp3p functional activity. Region 2 (Leu167 through Pro175) overlapped the single hydrophobic domain of Cbp3p. Mutations within this area altered the association of Cbp3p with the mitochondrial membrane resulting in enhanced protein turnover. The role of the amino-terminus in Cbp3p activity was investigated using cbp3 deletion strains Delta12-23, Delta24-54, Delta56-96 and Delta12-96. All mutants were respiratory competent, indicating that residues 12-96 were not essential for Cbp3p function, stability or mitochondrial import. Analysis of carboxy-terminal deletion mutants demonstrated that the final 44 residues were not necessary for Cbp3p function; however, alterations in the secondary structure of the extreme carboxy-terminal 17 residues affected assembly protein activity. 相似文献
11.
Hasnain SS Murphy LM Strange RW Grossmann JG Clarke AR Jackson GS Collinge J 《Journal of molecular biology》2001,311(3):467-473
Here, we describe the structure of a C-terminal high-affinity copper-binding site within a truncated recombinant human PrP containing residues 91-231, which lacks the octapeptide repeat region. We show that at least two extra co-ordinating groups are involved in binding this copper(II) ion in conjunction with histidine residues 96 and 111 in a region of the molecule known to be critical in conferring strain type. In addition, using X-ray solution scattering, a low-resolution shape of PrP(91-231) is provided. The restored molecular envelope is consistent with the picture where the N-terminal segment, residues 91-120, extends out from the previously known globular domain containing residues 121-231. 相似文献
12.
M Sugimoto H Mohri R A McClintock Z M Ruggeri 《The Journal of biological chemistry》1991,266(27):18172-18178
We have used proteolytic fragments and overlapping synthetic peptides to define the domain of von Willebrand factor (vWF) that forms a complex with botrocetin and modulates binding to platelet glycoprotein (GP) Ib. Both functions were inhibited by the dimeric 116-kDa tryptic fragment and by its constituent 52/48-kDa subunit, comprising residues 449-728 of mature vWF, but not by the dimeric fragment III-T2 which lacks amino acid residues 512-673. Three synthetic peptides, representing discrete discontinuous sequences within the region lacking in fragment III-T2, inhibited vWF-botrocetin complex formation; they corresponded to residues 539-553, 569-583, and 629-643. The 116-kDa domain, with intact disulfide bonds, exhibited greater affinity for botrocetin than did the reduced and alkylated 52/48-kDa molecule, and both fragments had significantly greater affinity than any of the inhibitory peptides. Thus, conformational attributes, though not strictly required for the interaction, contribute to the optimal functional assembly of the botrocetin-binding site. Accordingly, 125I-labeled botrocetin bound to vWF and to the 116-kDa fragment immobilized onto nitrocellulose but not to equivalent amounts of the reduced and alkylated 52/48-kDa fragment; it also bound to the peptide 539-553, but only when the peptide was immobilized onto nitrocellulose at a much greater concentration than vWF or the proteolytic fragments. These studies demonstrate that vWF interaction with GP Ib may be modulated by botrocetin binding to a discontinuous site located within residues 539-643. The finding that single point mutations in Type IIB von Willebrand disease are located in the same region of the molecule supports the concept that this domain may contain regulatory elements that modulate vWF affinity for platelets at sites of vascular injury. 相似文献
13.
Because beta-amyloid precursor protein (APP) has the abilities both to interact with extracellular matrix and to inhibit gelatinase A activity, this molecule is assumed to play a regulatory role in the gelatinase A-catalyzed degradation of extracellular matrix. To determine a region of APP essential for the inhibitory activity, we prepared various derivatives of APP. Functional analyses of proteolytic fragments of soluble APP (sAPP) and glutathione S-transferase fusion proteins, which contain various COOH-terminal parts of sAPP, showed that a site containing residues 579-601 of APP(770) is essential for the inhibitory activity. Moreover, a synthetic decapeptide containing the ISYGNDALMP sequence corresponding to residues 586-595 of APP(770) had a gelatinase A inhibitory activity slightly higher than that of sAPP. Studies of deletion of the NH(2)- and COOH-terminal residues and alanine replacement of internal residues of the decapeptide further revealed that Tyr(588), Asp(591), and Leu(593) of APP mainly stabilize the interaction between gelatinase A and the inhibitor. We also found that the residues of Ile(586), Met(594), and Pro(595) modestly contribute to the inhibitory activity. The APP-derived decapeptide efficiently inhibited the activity of gelatinase A (IC(50) = 30 nm), whereas its inhibitory activity toward membrane type 1 matrix metalloproteinase was much weaker (IC(50) = 2 microm). The decapeptide had poor inhibitory activity toward gelatinase B, matrilysin, and stromelysin (IC(50) > 10 microm). The APP-derived inhibitor formed a complex with active gelatinase A but not with progelatinase A, and the complex formation was prevented completely by a hydroxamate-based synthetic inhibitor. Therefore, the decapeptide region of APP is likely an active site-directed inhibitor that has high selectivity toward gelatinase A. 相似文献
14.
To determine the amino acid residues required for the signal-transducing activity of the human c-Ha-Ras protein, we introduced point mutations at residues 45-54 near the 'effector region' (residues 32-40). We transfected PC12 cells with these mutant genes and also micro-injected the mutant proteins, bound with an unhydrolyzable GTP analog, into PC12 cells. Both procedures showed that Val45----Glu and Gly48----Cys mutations impaired the ability of the Ras protein to induce morphological change of PC12 cells. These mutations did not affect the guanine nucleotide-binding activity or GTPase activity in the absence or presence of bovine GTPase-activating protein (GAP). Therefore, the Val45 and Gly48 residues should be included by definition in the effector region responsible for the signal transduction, while only a subset of the effector-region residues is required for enhancement of the GTPase activity by GAP. 相似文献
15.
It has been hypothesized that resistance to nonenzymatic deamidation of asparagine and glutamine residues may be an important determinant of protein stability in vivo. As a test of this hypothesis, we analyzed the central region of old human lenses, which contain proteins such as gamma-S crystallin that were synthesized during the fetal-embryonic periods of development. Total protein from the fetal-embryonic region of old human lenses was digested with trypsin, followed by resolution of tryptic fragments containing amidated and deamidated forms using high pressure liquid chromatography-reverse phase chromatography together with synthetic peptide standards and mass spectral analysis. The results demonstrate no detectable deamidation of glutamine 92, glutamine 96, asparagine 143, and glutamine 170 from gamma-S crystallin from old human lenses, consistent with the hypothesis that very long-lived proteins can contain asparagine and glutamine residues that are extremely resistant to in vivo deamidation. 相似文献
16.
Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity 总被引:15,自引:0,他引:15
G Ju L Collins K L Kaffka W H Tsien R Chizzonite R Crowl R Bhatt P L Kilian 《The Journal of biological chemistry》1987,262(12):5723-5731
To locate functional domains of the interleukin-2 (IL-2) protein, a cDNA clone encoding biologically active human IL-2 was mutagenized using synthetic oligonucleotides to incorporate defined amino acid substitutions and deletions in the mature protein. The IL-2 analogs were then produced in Escherichia coli and assayed for the ability to induce proliferation of IL-2-dependent cells and the ability to compete for binding to the IL-2 receptor. Our analysis of over 50 different mutations demonstrated that the integrity of at least three regions of the IL-2 molecule is required for full biological activity: the NH2 terminus (residues 1-20), the COOH terminus (residues 121-133), and 2 of the 3 cysteine residues (58 and 105). Deletion of the NH2-terminal 20 amino acids or the COOH-terminal 10 amino acids resulted in the loss of greater than 99% of bioactivity and binding. Amino acid substitutions at specific positions in these regions also resulted in proteins which retained less than 1% activity. The NH2 terminus and an adjacent internal region were recognized by neutralizing anti-IL-2 antibodies. In combination with the results from epitope competition analysis with neutralizing antibodies, these data are consistent with the IL-2 protein being folded such that the NH2 terminus, the COOH terminus, and the internal 30- to 60-region are juxtaposed to form the binding site recognized by the IL-2 receptor. 相似文献
17.
Ovalbumin, one of the major proteins present in avian egg white, was proteolytically digested by trypsin and chymotrypsin and the peptide fragments were investigated for their antimicrobial activity. The antimicrobial peptides were isolated and characterized. From the tryptic digestion, the following five antimicrobial peptide fragments were obtained: SALAM (residues 36-40), SALAMVY (residues 36-42) YPILPEYLQ (residues 111-119), ELINSW (residues 143-148) and NVLQPSS (residues 159-165). Digestion of ovalbumin by chymotrypsin yielded the antimicrobial peptides AEERYPILPEYL (residues 127-138), GIIRN (residues 155-159) and TSSNVMEER (residues 268-276). The peptides were synthesized and found to exert antimicrobial activity. They were strongly active against Bacillus subtilis and to a lesser extent against the other bacterial strains examined. A weak fungicidal activity against Candida albicans was also shown by some peptides. Ovalbumin itself was not bactericidal against all the bacteria strains examined. Our results suggest that the food protein ovalbumin may supply the organism with antimicrobial peptides, supporting the immunodefences of the organism. 相似文献
18.
Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity. 总被引:5,自引:5,他引:0
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In vitro DNA binding results from a series of E1 proteins containing amino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Additional binding experiments with E1 proteins containing internal, in-frame insertions or deletions confirmed the importance of the region defined by truncated E1 proteins and also demonstrated that downstream sequences were not required for binding activity in the context of the full-length E1 protein. On the basis of mapping results from the E1 mutants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding. The E1(121-311) protein retained origin-specific DNA binding, confirming that this region was not only necessary but was also sufficient for origin recognition. In addition to origin binding, E1(121-311) bound E2 protein in a cold-sensitive manner. Therefore, DNA binding and E2 binding activities colocalize to a 191-amino-acid functional domain derived from the amino-terminal half of the E1 protein. Finally, three E1 proteins with mutations in this region all lacked DNA binding activity and were all defective for in vivo replication. Two of these E1 mutants retained E2 binding capability, demonstrating that origin recognition by E1 is critical for replication and cannot necessarily be rescued by an interaction with E2 protein. 相似文献
19.
20.
Mice expressing a C-terminal fragment of the prion protein instead of wild-type prion protein die from massive neuronal degeneration within weeks of birth. The C-terminal region of PrPc (PrP121-231) expressed in these mice has an intrinsic neurotoxicity to cultured neurones. Unlike PrPSc, which is not neurotoxic to neurones lacking PrPc expression, PrP121-231 was more neurotoxic to PrPc-deficient cells. Human mutations E200K and F198S were found to enhance toxicity of PrP121-231 to PrP-knockout neurones and E200K enhanced toxicity to wild-type neurones. The normal metabolic cleavage point of PrPc is approximately amino-acid residue 113. A fragment of PrPc corresponding to the whole C-terminus of PrPc (PrP113-231), which is eight amino acids longer than PrP121-231, lacked any toxicity. This suggests the first eight amino residues of PrP113-121 suppress toxicity of the toxic domain in PrP121-231. Addition to cultures of a peptide (PrP112-125) corresponding to this region, in parallel with PrP121-231, suppressed the toxicity of PrP121-231. These results suggest that the prion protein contains two domains that are toxic on their own but which neutralize each other's toxicity in the intact protein. Point mutations in the inherited forms of disease might have their effects by diminishing this inhibition. 相似文献