共查询到20条相似文献,搜索用时 36 毫秒
1.
Activity of Free and Clay-Bound Insecticidal Proteins from Bacillus thuringiensis subsp. israelensis against the Mosquito Culex pipiens 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Bacillus thuringiensis subsp. israelensis produces parasporal insecticidal crystal proteins (ICPs) that have larvicidal activity against some members of the order Diptera, such as blackflies and mosquitoes. Hydrolysis of the ICPs in the larval gut results in four major proteins with a molecular mass of 27, 65, 128, and 135 kDa. Toxicity is caused by synergistic interaction between the 25-kDa protein (proteolytic product of the 27-kDa protein) and one or more of the higher-molecular-mass proteins. Equilibrium adsorption of the proteins on the clay minerals montmorillonite and kaolinite, which are homoionic to various cations, was rapid (<30 min for maximal adsorption), increased with protein concentration and then reached a plateau (68 to 96% of the proteins was adsorbed), was significantly lower on kaolinite than on montmorillonite, and was not significantly affected by the valence of the cation to which the clays were homoionic. Binding of the toxins decreased as the pH was increased from 6 to 11, and there was 35 to 66% more binding in phosphate buffer at pH 6 than in distilled water at pH 6 or 7.2. Only 2 to 12% of the adsorbed proteins was desorbed by two washes with water; additional washings desorbed no more toxins, indicating that they were tightly bound. Formation of clay-toxin complexes did not alter the structure of the proteins, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the equilibrium supernatants and desorption washes and by dot blot enzyme-linked immunosorbent assay of the complexes, which was confirmed by enhanced chemiluminescence Western blot analysis. Free and clay-bound toxins resulted in 85 to 100% mortality of the mosquito Culex pipiens. Persistence of the bound toxins in nonsterile water after 45 days was significantly greater (mortality of 63% ± 12.7%) than that of the free toxins (mortality of 25% ± 12.5%). 相似文献
2.
The equilibrium adsorption and binding of the delta-endotoxin proteins, i.e., the protoxins (Mr=132 kDa) and toxins (Mr=66 kDa), fromBacillus thuringiensis subsp.kurstaki were greater on montmorillonite than on kaolinite (five-fold more protoxin and three-fold more toxin were adsorbed on montmorillonite). Approximately two- to three-fold more toxin than protoxin was adsorbed on these clay minerals. Maximum adsorption occurred within 30 min (the shortest interval measured), and adsorption was not significantly affected by temperatures between 7° and 50°C. The proteins were more easily desorbed from kaolinite than from montmorillonite; they could not be desorbed from montmorillonite with water or 0.2% Na2CO3, but they could be removed with Tris-SDS (sodium dodecyl sulfate) buffer. Adsorption was higher at low pH and decreased as the pH increased. Adsorption on kaolinite was also dependent on the ionic nature of the buffers. The molecular mass of the proteins was unaltered after adsorption on montmorillonite, as shown by SDS-PAGE (polyacrylamide gel electrophoresis) of the desorbed proteins; no significant modifications occurred in their structure as the result of binding on the clay, as indicated by infrared analysis; and there was no significant expansion of the clay by the proteins, as shown by x-ray diffraction analysis. The bound proteins appeared to retain their insecticidal activity against the third instar larvae ofTrichoplusia ni. 相似文献
3.
The clay minerals montmorillonite and kaolinite protected bacteria, including actinomycetes, and filamentous fungi from the inhibitory effects of cadmium (Cd). Montmorillonite provided greater protection than did equivalent concentrations of kaolinite. The protective ability of the clays was correlated with their cation exchange capacity (CEC). The greater the CEC, the greater the absorbancy of exogenous Cd by the exchange complex and the greater the protection. The greater protection afforded by montmorillonite, as compared to kaolinite, was correlated with its higher CEC. Clays homoionic to Cd did not protect against exogenous Cd, as the exchange complex was already saturated with Cd. Montmorillonite homoionic to Cd was more detrimental to microbial growth than was kaolinite homoionic to Cd, as more Cd was present on and apparently desorbed from the montmorillonite. 相似文献
4.
Adsorption of reovirus to clay minerals: effects of cation-exchange capacity, cation saturation, and surface area 总被引:1,自引:0,他引:1
The adsorption of reovirus to clay minerals has been reported by several investigators, but the mechanisms defining this association have been studied only minimally. The purpose of this investigation was to elucidate the mechanisms involved with this interaction. More reovirus type 3 was adsorbed, in both distilled and synthetic estuarine water, by low concentrations of montmorillonite than by comparable concentrations of kaolinite containing a mixed complement of cations on the exchange complex. Adsorption to the clays was essentially immediate and was correlated with the cation-exchange capacity of the clays, indicating that adsorption was primarily to negatively charged sites on the clays. Adsorption was greater with low concentrations of clays in estuarine water than in distilled water, as the higher ionic strength of the estuarine water reduced the electrokinetic potential of both clay and virus particles. The addition of cations (as chloride salts) to distilled water enhanced adsorption, with divalent cations being more effective than monovalent cations and 10(-2) M resulting in more adsorption than 10(-3) M. Potassium ions suppressed reovirus adsorption to montmorillonite, probably by collapsing the clay lattices and preventing the expression of the interlayer-derived cation-exchange capacity. More virus was adsorbed by montmorillonite made homoionic to various mono-, di-, and trivalent cations (except by montmorillonite homoionic to potassium) than by comparable concentrations of kaolinite homoionic to the same cations. The sequence of the amount of adsorption to homoionic montmorillonite was Al greater than Ca greater than Mg greater than Na greater than K; the sequence of adsorption to kaolinite was Na greater than Al greater than Ca greater than Mg greater than K. The constant partition-type adsorption isotherms obtained when the clay concentration was maintained constant and the virus concentration was varied indicated that a fixed proportion of the added virus population was adsorbed, regardless of the concentration of infectious particles. A heterogeneity within the reovirus population was indicated. 相似文献
5.
Adsorption of reovirus to clay minerals: effects of cation-exchange capacity, cation saturation, and surface area. 总被引:10,自引:7,他引:3
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The adsorption of reovirus to clay minerals has been reported by several investigators, but the mechanisms defining this association have been studied only minimally. The purpose of this investigation was to elucidate the mechanisms involved with this interaction. More reovirus type 3 was adsorbed, in both distilled and synthetic estuarine water, by low concentrations of montmorillonite than by comparable concentrations of kaolinite containing a mixed complement of cations on the exchange complex. Adsorption to the clays was essentially immediate and was correlated with the cation-exchange capacity of the clays, indicating that adsorption was primarily to negatively charged sites on the clays. Adsorption was greater with low concentrations of clays in estuarine water than in distilled water, as the higher ionic strength of the estuarine water reduced the electrokinetic potential of both clay and virus particles. The addition of cations (as chloride salts) to distilled water enhanced adsorption, with divalent cations being more effective than monovalent cations and 10(-2) M resulting in more adsorption than 10(-3) M. Potassium ions suppressed reovirus adsorption to montmorillonite, probably by collapsing the clay lattices and preventing the expression of the interlayer-derived cation-exchange capacity. More virus was adsorbed by montmorillonite made homoionic to various mono-, di-, and trivalent cations (except by montmorillonite homoionic to potassium) than by comparable concentrations of kaolinite homoionic to the same cations. The sequence of the amount of adsorption to homoionic montmorillonite was Al greater than Ca greater than Mg greater than Na greater than K; the sequence of adsorption to kaolinite was Na greater than Al greater than Ca greater than Mg greater than K. The constant partition-type adsorption isotherms obtained when the clay concentration was maintained constant and the virus concentration was varied indicated that a fixed proportion of the added virus population was adsorbed, regardless of the concentration of infectious particles. A heterogeneity within the reovirus population was indicated. 相似文献
6.
Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact. 相似文献
7.
Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact. 相似文献
8.
Insecticidal Activity of the Toxins from Bacillus thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure and Soil Clays 总被引:6,自引:0,他引:6
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The release of transgenic plants and microorganisms expressing truncated genes from various subspecies of Bacillus thuringiensis that encode active insecticidal toxins rather than inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clays and other soil particles. Toxins from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis, either free or adsorbed at equilibrium or bound on pure clay minerals (montmorillonite or kaolinite) or on the clay size fraction of soil, were toxic to larvae of the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata), respectively. The 50% lethal concentrations (LC(inf50)) of free toxins from B. thuringiensis subsp. kurstaki were higher than those of both bound and adsorbed complexes of these toxins with clays, indicating that adsorption and binding of these toxins on clays increase their toxicity in diet bioassays. The LC(inf50) of the toxin from B. thuringiensis subsp. tenebrionis that was either free or adsorbed on montmorillonite were comparable, whereas the toxin bound on this clay had higher LC(inf50) and the toxin bound on kaolinite had lower LC(inf50) than when adsorbed on this clay. Results obtained with the clay size fraction separated from unamended soil or soil amended with montmorillonite or kaolinite were similar to those obtained with the respective pure clay minerals. Therefore, insecticidal activity of these toxins is retained and sometimes enhanced by adsorption and binding on clays. 相似文献
9.
Adsorption/desorption of toluene on montmorillonite, illite, and kaolinite was studied using the batch equilibrium method. The isotherms measured fit the Freundlich equation (r2 >0.95). Montmorillonite adsorbed more toluene than illite or kaolinite; the adsorption of toluene on illite and kaolinite was not significantly different. Adsorption of toluene by montmorillonite showed an exponential increase as the ratio of toluene to clay was increased from 5 to 100. The rate studies showed that 62% of the adsorption was completed within 6 h. A rapid desorption was observed initially, followed by slow desorption after 1 h. The desorption rate decreased as the time of adsorption was increased. Almost all of the adsorbed toluene was extracted with water from the clay when the adsorption time was 0.1 h, but only 61% of the toluene could be desorbed when the adsorption time was 24 h. 相似文献
10.
研究了Bt库斯塔克亚种(kurstaki)毒素(65 kDa)在高岭土、针铁矿和氧化硅表面的吸附和解吸特性.结果表明:在磷酸盐缓冲体系(pH 8)中,3种矿物的等温吸附曲线均符合Langmuir方程(R2>0.9661),它们对Bt毒素的吸附顺序为:针铁矿﹥高岭土﹥二氧化硅.矿物对Bt毒素的吸附1 h就基本达到了吸附平衡.在pH 6~8范围内,针铁矿、高岭土和二氧化硅对Bt毒素的吸附量随pH值的升高而降低.10 ℃~50 ℃范围内,针铁矿和氧化硅对Bt毒素吸附量随温度升高有所下降(8.39%和47.06%),高岭土对Bt毒素吸附则略有升高(5.91%).红外光谱分析显示,Bt毒素被矿物吸附后结构仅有微小变化.被矿物吸附的Bt毒素不易被去离子水解吸,水洗3次总解吸率为28.48%~42.04%. 相似文献
11.
Guno Haskå 《Microbial ecology》1974,1(1):234-245
Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their
ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not
the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption
of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on
kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed
bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase. 相似文献
12.
Guno Haskå 《Microbial ecology》1975,1(1):234-245
Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase. 相似文献
13.
The adsorption of the enzyme glucose oxidase (EC 1.1.3.4) to clays followed the pattern described for other proteins as being pH dependent. Maximum adsorption occurred at or below the isoelectric point of the enzyme. The amount of enzyme adsorbed to clay was influenced by the type of clay used, and also the saturating cations. Initially adsorbed enzyme showed low specific activities, and as amounts of enzyme adsorbed approached maximum stauration of clay, specific activities increased approaching that determined for free enzyme. The adsorption of glucose oxidase involved a temperature-independent cation-exchange mechanism, and enzyme adsorbed to surfaces of clay could be desorbed in active form by elevation of pH of suspending solution. This was followed by a slower temperature-dependent fixation, probably by hydrogen bonding, which resulted in protein being irreversibly adsorbed to clay surfaces. It is proposed that on adsorption of glucose oxidase to clay surfaces unravelling of the protein structure occurred, which allowed penetration of protein into the interlamellar spaces of montmorillonite. This proposal was based on the observed expansion of montmorillonite to 23 A, and the decreases in amount of a second-protein lysozyme adsorbed with extended incubation times of glucose oxidase - clay complexes at pH 4.5. 相似文献
14.
Dot Blot Enzyme-Linked Immunosorbent Assay for Monitoring the Fate of Insecticidal Toxins from Bacillus thuringiensis in Soil 总被引:10,自引:0,他引:10
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The release of transgenic plants and microorganisms expressing truncated genes from Bacillus thuringiensis that code for active insecticidal toxins rather than for the inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clay minerals and other soil particles. To monitor the fate of these toxins in soil, a dot blot enzyme-linked immunosorbent assay (ELISA) that detects free and particle-bound toxins from B. thuringiensis subsp. kurstaki and subsp. tenebrionis was developed. The lower limit of detection of the toxins, either free or adsorbed or bound on the clay minerals montmorillonite (M) or kaolinite (K) or on the clay-particle-size fraction separated from soil (by sedimentation according to Stokes' Law), was approximately 3 ng. Antibodies (Ab) to the toxins from B. thuringiensis subsp. kurstaki and from B. thuringiensis subsp. thuringiensis were raised in goats and rabbits, respectively, and each Ab was rendered specific by adsorption onto CNBr-activated Sepharose coupled with the other toxin. The preadsorbed Ab were specific for the toxins from both subspecies, both free and bound on M, K, or the clay-particle-size fraction of soil. The toxins that were added to sterile and nonsterile soil amended with M or K or not amended were detected on the clay-particle-size fraction of the soil after various periods of incubation by the dot blot ELISA. No toxins were detected on the silt- and sand-particle-size fractions. Each dot blot, containing various amounts of toxins and/or clays, was applied to a polyvinylidene difluoride membrane in a dot blot vacuum system. The toxins were still detectable on the clay-particle-size fraction of nonsterile soil after 40 days. This agreed with preliminary results of other studies in this laboratory that when these toxins bind on clay minerals, they become resistant to utilization by microorganisms. 相似文献
15.
To investigate the specificity of Bacillus thuringiensis var. kurstaki strain HD1 insecticidal crystal proteins (ICP), we used membrane preparations obtained from the midgut of Heliothis virescens larvae to perform separate ligand-blot experiments with the three activated CryIA toxins. The CryIA(a) and the CryIA(b) toxins bind the same 170-kDa protein, but most likely at two different binding sites. The CryIA(c) toxin binds two proteins of molecular masses 140 kDa and 120 kDa. We also demonstrate that the binding proteins for each of the B. thuringiensis toxins are not part of a covalent complex. Although the 170-kDa protein is a glycoprotein, endoglycosidase treatment does not prevent the binding of the CryIA(a) or CryIA(b) toxin. This indicates that the sugars are not important for the binding of these toxins. A model for a protein complex binding the B. thuringiensis HD1 ICPs is presented. Our results support the idea that binding proteins on membranes of the gut epithelial cells of H. virescens larvea are important for the specificity of the bacterial toxins. 相似文献
16.
Xueyong Zhou Jianbao Gao Shouwen Chen Bujiang Wang 《Biocontrol Science and Technology》2008,18(10):1051-1061
The adsorption, desorption and anti-ultraviolet light characteristics of the protoxin from Bacillus thuringiensis strain WG-001 on montmorillonite, kaolinite, zinc oxide and rectorite were studied. The protoxin was easily adsorbed onto minerals and the adsorption reached equilibrium within 0.5–1.0 h (except for rectorite). The adsorption isotherms of protoxin at different concentrations in sodium carbonate buffer (pH 9) followed the Langmuir (R 2 >0.97) and Freundlich (R 2 >0.95) equations. The maximum amounts of protoxin adsorbed were in the order: montmorillonite>rectorite>znic oxide>kaolinite. In the range of pH from 9 to 11 (carbonate buffer), the protoxin adsorbed decreased with increasing pH. The adsorption was not significantly affected by the temperature between 5 and 45°C. Both free and adsorbed protoxin were toxic to larvae of Heliothis armigera. The LC50 value of free and adsorbed protoxin on montmorillonite, rectorite, zinc oxide and kaolinite were 14±1.16, 1.76±0.31, 2.94±0.71, 4.78±2.08 and 1.91±0.91 µg mL?1, respectively. After 1 h of ultraviolet irradiation, the LC50 of the above samples increased by 41.4, 19.3, 16.3, 125.9 and 62.3%, respectively. The desorption of adsorbed protoxin in water ranged from 30.1 to 64.9% and from 18.5 to 48.7% in carbonate buffer. 相似文献
17.
D I Oltean A K Pullikuth H K Lee S S Gill 《Applied and environmental microbiology》1999,65(11):4760-4766
Although extensively studied, the mechanism of action of insecticidal Bacillus thuringiensis Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (Heliothis virescens) was partially purified, and its corresponding cDNA was cloned. The cDNA encodes a protein with a putative glycosyl phosphatidylinositol anchor and a polythreonine stretch clustered near the C terminus with predicted O-glycosylation. Partial purification of the 170-kDa aminopeptidase also resulted in isolation of a 130-kDa protein that was immunologically identical to the 170-kDa protein, and the two proteins had identical N termini. These proteins were glycosylated, as suggested by soybean agglutinin lectin blot results. Cry1Ac toxin affinity data for the two proteins indicated that the 130-kDa protein had a higher affinity than the 170-kDa protein. The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins. 相似文献
18.
Partial Purification and Characterization of Bacillus thuringiensis Cry1A Toxin Receptor A from Heliothis virescens and Cloning of the Corresponding cDNA 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Daniela I. Oltean Ashok K. Pullikuth Hyun-Ku Lee Sarjeet S. Gill 《Applied microbiology》1999,65(11):4760-4766
Although extensively studied, the mechanism of action of insecticidal Bacillus thuringiensis Cry toxins remains elusive and requires further elucidation. Toxin receptors in the brush border membrane demand particular attention as they presumably initiate the cascade of events leading to insect mortality after toxin activation. The 170-kDa Cry1Ac toxin-binding aminopeptidase from the tobacco budworm (Heliothis virescens) was partially purified, and its corresponding cDNA was cloned. The cDNA encodes a protein with a putative glycosyl phosphatidylinositol anchor and a polythreonine stretch clustered near the C terminus with predicted O-glycosylation. Partial purification of the 170-kDa aminopeptidase also resulted in isolation of a 130-kDa protein that was immunologically identical to the 170-kDa protein, and the two proteins had identical N termini. These proteins were glycosylated, as suggested by soybean agglutinin lectin blot results. Cry1Ac toxin affinity data for the two proteins indicated that the 130-kDa protein had a higher affinity than the 170-kDa protein. The data suggest that posttranslational modifications can have a significant effect on Cry1A toxin interactions with specific insect midgut proteins. 相似文献
19.
Protein chromatography on adsorbents with hydrophobic and ionic groups. Chromatography of dodecyl sulphate-solubilized proteins of the human erythrocyte membrane on N-(3-carboxypropionyl)aminodecyl-sepharose.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Human erythrocyte 'ghosts' were solubilized in 0.5% (w/v) sodium dodecyl sulphate at pH 4.0(I = 0.012 mol/I). At a loading of 1-2 mg of protein/ml of column volume, all of membrane proteins were adsorbed to a column of CPAD [N-(3-carboxypropionyl)-aminodecyl]-Sepharose at pH 4.0 (I = 0-012 mol/1) and room temperature (22 degrees C). Many proteins were subsequently desorbed by raising the pH or by including sodium dodecyl sulphate continuously in the eluting buffer. Experiments with a series of adsorbents homologous with CPAD-Sepharose, in which the length of the hydrocarbon chain was varied, provided strong evidence of hydrophobic interactions, in addition to ionic interactions, in the binding of these proteins to CPAD-Sepharose. Elution with increasing-pH gradients at different concentrations of sodium dodecyl sulphate showed that glycophorin (the major sialoglycoprotein) was eluted in the void volume, at recoveries close to 100%, when the detergent concentration was greater than or equal to 0.3% (w/v). Protein E, the major protein, was desorbed late in the pH gradient even at a high (0.5%, w/v) concentration of the detergent, and was always incompletely desorbed, the maximum recovery recorded being 40%. Spectrin (the high-molecular-weight polypeptide pair) did not behave in a well-defined manner, and was found widely distributed among the effluent fractions under all the conditions that were tested. 相似文献
20.
Oestergaard J Ehlers RU Martínez-Ramírez AC Real MD 《Applied and environmental microbiology》2007,73(11):3623-3629
Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa. 相似文献