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排序方式: 共有116条查询结果,搜索用时 15 毫秒
1.
Binding of ions and hydrophobic probes to alpha-lactalbumin and kappa-casein as determined by analytical affinity chromatography 总被引:1,自引:0,他引:1
The technique of analytical affinity chromatography was extended to characterize binding of ions and hydrophobic probes to proteins. Using the immobilized protein mode of chromatography, alpha-lactalbumin and kappa-casein were covalently attached to 200-nm-pore-diameter controlled-pore glass beads and accommodated for high-performance liquid chromatography. The existence of a high affinity binding site (Kdiss = 0.16 microM) (site I) for calcium ion in alpha-lactalbumin was confirmed by chromatography of [45Ca2+]. In addition, chromatography of the hydrophobic probes, 1-(phenylamino)-8-naphthalene-sulfonate (ANS)2 and 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate (bis-ANS) indicated that Ca2+ bound to a second site (presumably the zinc site or site II) with weaker affinity. Dissociation constants obtained for apo-alpha-lactalbumin were about 80 microM for ANS and 4.7 microM for bis-ANS in the absence of sodium ion. Addition of Ca2+ initially caused a reduction in surface hydrophobicity (lowered affinity for the probe dyes) followed by an increase at higher Ca2+ concentrations (greater than 0.5 mM), suggesting that occupancy of site II restores an apo-like conformation to the protein. Moreover, the effect of Zn2+ was similar to that observed in the higher Ca2+ concentration range, whereas Na+ apparently bound to site I. A calcium binding site of moderate affinity also exists in kappa-casein (Kdiss = 15.6 microM). A cluster of negative charges, probably including the orthophosphate group, most likely comprise this binding site. By preventing self-association, analytical affinity chromatography permits microscale characterization of ligand equilibria in proteins that are unaffected by protein-protein interactions. 相似文献
2.
Sharon X. Chen Charles C. Hardin Harold E. Swaisgood 《Journal of Protein Chemistry》1993,12(5):613-625
Incubation of -lactoglobulin with immobilized trypsin at 5–10°C results in a time-dependent release of several fragments of the core domain in yields approaching 15%. Digests were fractionated by ion-exchange chromatography with a Mono Q HR5/5 column and analyzed after disulfide reduction by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Three fragments with approximate molecular weights of 13.8, 9.6, and 6.7 kD were identified. The fraction from ion-exchange chromatography yielding the 6.7 kD fraction after disulfide reduction was further characterized because it was most homogeneous and gave the highest yield. The C-terminal cleavage site of the 6.7 kD core fragment appeared to be Lys100 or Lys101 as determined by C-terminal amino acid analysis. The exact masses, after reduction with dithiothreitol, are 6195 and 6926 as determined by laser desorption mass spectrometry, corresponding to residues 48–101 and 41–100. Prior to reduction, -lactoglobulin C-terminal residues 149–162 are connected to these core domain fragments as shown by C-terminal analysis and mass spectrometry. Structural studies indicate that these 7.9 and 8.6 kD core domain fragments released by immobilized trypsin retain much of their native structure. CD spectra indicate the presence of antiparallel -sheet structure similar to the native protein but the -helix is lost. Spectra in the aromatic region indicate the existence of tertiary structure. Moreover, structural transitions in urea are completely reversible as measured by CD spectra, although the extrapolated G
D
H20
and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display apH-dependent binding to immobilizedtrans-retinal as does intact protein. A single endotherm is obtained for both core domain fragments and native protein upon differential scanning calorimetry, but again, the domain is less stable as indicated by a transition peak maxima of 56.9°C as compared with 81.1°C for native protein.Abbreviations used: CD, circular dichroism; CPG, controlled pore glass; DSC, differential scanning calorimetry; DTT, dithiothreitol; FPLC, fast flow liquid chromatography; HPLC, high-performance liquid chromatography; PITC, phenylisothiocyanate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEA, triethylamine; UV, ultraviolet. 相似文献
3.
The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation 总被引:48,自引:38,他引:10
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The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro. 相似文献
4.
Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions. 相似文献
5.
Megan A. Owen Ronald R. Swaisgood Laura McGeehan Xiaoping Zhou Donald G. Lindburg 《Ethology : formerly Zeitschrift fur Tierpsychologie》2013,119(10):869-880
Giant panda courtship behavior includes multimodal signaling assemblages consisting of olfactory, vocal, and postural elements. While signaling is generally conspicuous, successful copulation is inconsistently achieved in captivity, even when female behavioral and physiological data indicate that ovulation is imminent. We set out to characterize these complex patterns of social behavior by observing interactions between 26 unique pairs of giant pandas housed in adjoining pens throughout the females' reproductive cycle. We categorized social behaviors from a transactional perspective and examined social exchanges via analyses of the relative frequency of social behaviors, and via the sequential relationship between male and female social behavior. From non‐estrus to peak‐estrus, we found that the relative frequency of female affiliative and sexual behavior increased and that the relative frequency of ambivalent and aggressive behavior decreased. Male behavior was fairly constant, except for sexual behavior, which increased during peak‐estrus, when it was facilitated by female sexual behavior. Sequential analysis of social interactions showed that preceding behavior had a significant influence over the other panda's response behavior primarily during peak‐estrus, suggesting that pandas are most responsive to conspecific signaling during the peri‐ovulatory period. However, behavioral momentum was a dominant feature of the intra‐individual transitions. Females maintained sexual, ambivalent, and neutral behavior during interactions significantly more than would be expected by chance, with male behavior bearing little influence once the behavior was initiated. A similar pattern was also observed in males, who maintained affiliative, interested, and neutral behaviors. Overall, our data suggest that the multimodal signals used by giant pandas during courtship do not consistently evoke a discrete, immediate response from receivers. Instead, signals appear to advertise reproductive condition and influence potential mates over longer timeframes, suggesting the potential tonic role of communication. 相似文献
6.
Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli 总被引:1,自引:0,他引:1
Expression of fusion protein trypsin-streptavidin (TRYPSA)4 in Escherichia coli was evaluated and the protein purified. Protein expression was induced by 1 mM isopropylthio--D-galactoside (IPTG), and the enzyme activity was measured by the hydrolysis rate of p-toluenesulfonyl-l-arginine methyl ester (TAME). Expression of the fusion protein in the cell-free extract decreased with increased induction time; correspondingly, that in the inclusion bodies increased. The total expression in Luria–Bertani broth (LB) and Terrific Broth (TB) media reached the highest levels in 2 hr at 30°C. The optimum expression level was 35 and 48 U/L in LB and TB, respectively. Expression of the fusion protein was verified by Western Blot analysis using streptavidin antiserum, and the fusion protein was purified using a benzamidine Sepharose 6B affinity column at room temperature. The molecular size of the soluble purified fusion protein was determined by size-exclusion chromatography using Superose 12 FPLC. A molecular weight of 39–40 kDa was obtained, indicating that the soluble protein exists as a monomer; thus, the presence of the trypsin domain must prevent the streptavidin domain from tetramer formation. 相似文献
7.
Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores
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Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 104 B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads. 相似文献
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10.
William A Cafruny Richard G Duman Grace HW Wong Suleman Said Pam Ward-Demo Raymond RR Rowland Eric A Nelson 《Virology journal》2006,3(1):1-17