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1.
Legume seed lectins specific for N-acetyl-alpha-D-galactosaminyl end groups from Amphicarpaea bracteata, lima bean, Griffonia simplicifolia, Dolichos biflorus, and soybean were compared with respect to binding of several spin-labeled derivatives of D-galactosamine by electron spin resonance and precipitin inhibition analysis. Spin-label II [methyl 2-[[(2,2,5,5-tetramethyl-1-oxopyrrolidin-3-yl) carbonyl]amino]-2-deoxy-alpha-D-galactopyranoside], spin-label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6, 6-tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], and spin-label IV [1-[4-[[(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)amino]carbonyl]phenyl]-3-(2, 2,6-tetramethyl-1-oxypiperidin-4-yl)-2-thiourea] contain 2-N-(oxypiperidinyl) or 2-N-(oxypyrrolidinyl) substituents varying in length and polarity of the linker arm between the glycoside and nitroxide ring. Spin-labels II and III were found to bind very weakly to all the lectins tested (Kd greater than or equal to 1.0 mM). Spin-label IV, containing a planar, nonpolar 2-N-phenyl group, was bound very strongly (Kd = 0.1-0.4 mM) and was moderately immobilized (2T parallel = 48-56 G) by all lectins except that from D. biflorus. Notably, the affinity of spin-label IV to lima bean lectin was 18-fold greater than that for methyl N-acetyl-alpha-galactosaminide. These results suggest that when the bulky oxypiperidinyl moiety lies in a position close to the sugar ring, it interferes with binding; in the cases where a phenyl group spacer exists, the aromatic ring in some cases actually enhances binding.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
W M Rutherford W E Dick J F Cavins M A Dombrink-Kurtzman M E Slodki 《Biochemistry》1986,25(5):952-958
A new lectin from soybeans having specificity toward the extracellular 4-O-methyl-D-glucurono-L-rhamnans produced by certain strains of Rhizobium japonicum has been purified and characterized. Isolation was accomplished initially by isoelectric precipitation of contaminating globulins and subsequently by affinity chromatography on partially hydrolyzed glucuronorhamnan covalently coupled to amino-hexylagarose. Residual globulins were removed by adsorption of the lectin on concanavalin A-agarose and elution with methyl alpha-mannoside. The lectin is a glycoprotein (3-5% carbohydrate) with a molecular weight of approximately 175 000. It is a tetramer with subunit molecular weights of 45 000 when dissociated with sodium dodecyl sulfate. Reverse-phase high-pressure liquid chromatography analysis indicates the presence of two types of subunits, both having equivalent molecular weights. According to amino acid analyses, the lectin is rich in acidic but low in sulfur-containing amino acids. The carbohydrate portion of the lectin contains mannose; no hexosamines could be detected. Chemical modification of the lectin indicated that neither sulfhydryl groups nor amino groups participate in binding. Quantitative binding studies of the lectin with various carbohydrate haptens showed that specificity was directed toward 4-O-methyl-D-glucuronic acid, D-glucuronic acid, and their methyl glycosides with 4-O-methyl-D-glucuronic acid 3-4-fold more effective. In each instance, the methyl glycoside is a more effective hapten. 相似文献
3.
The ability of Pseudomonas aeruginosa PA-I lectin to bind the fluorescent hydrophobic probe, 2-(p-toluidinyl) naphthalene sulfonic acid (TNS), and adenine was examined by spectrofluorametry and equilibrium dialysis. Interaction of TNS with PA-I caused significant enhancement of TNS fluorescence. The Hill coefficient (3.8+/-0.3) and the dissociation constant (8.7+/-0.16 microM) showed that TNS probably bound to four high affinity hydrophobic sites per PA-I tetramer. Interactions between PA-I and adenine were examined by equilibrium dialysis using [3H] adenine. The results indicated the presence of at least two classes of binding sites--one high and four lower affinity sites per tetramer with dissociation constants of 3.7+/-1.5 and 42.6+/-1.2 microM, respectively. These were distinct from the TNS sites as titration of TNS-equilibrated PA-I with adenine caused TNS fluorescence enhancement. The titration curve confirmed the existence of two classes of adenine-binding sites. Conversely, when PA-I was first equilibrated with adenine and then titrated with TNS, no TNS-binding was registered. This may indicate that conformational rearrangements of the lectin molecule caused by adenine prevent allosterically TNS binding. 相似文献
4.
Proton translocation by the H+-ATPase of mitochondria. Effect of modification by monofunctional reagents of thiol residues in F0 polypeptides 总被引:1,自引:0,他引:1
A study is presented on the effect of chemical modification of thiol groups on proton conduction by the H+-ATPase complex in 'inside out' submitochondrial particles, before and after removal of the F1 moiety, and by F0 liposomes. The results obtained show that modification with monofunctional reagents [N-ethylmaleimide, 2,2'-dithiobispyridine, mersalyl and N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide] of thiol residues in membrane integral proteins of F0 results in inhibition of proton conduction. Comparison of the inhibitory effects with the binding of [14C]N-ethylmaleimide to the various F0 polypeptides indicates that the inhibition of proton conduction by thiol reagents was correlated with modification of the 25-kDa, 11-kDa and 9-kDa (N,N'-dicyclohexylcarbodiimide-binding protein) proteins. Involvement of the last component is supported by the observation that modification by thiol reagents depressed the binding of N,N'-dicyclo[14C]hexylcarbodiimide to the 9-kDa protein. 相似文献
5.
经肼解、Bio-Gel P-2柱层析、NaB^3H4和NaBH4还原,制备各种来源的、氚标记在还原末端的、还原末端为N-乙酰氨基葡萄糖醇的混合寡糖,经Bio-Gel P-4凝胶柱分离,以及用糖苷酶酶解,制备了各种不同类型的氚标记的寡糖。这些寡糖在固定化的PCL-Sepharose柱上亲和层析,根据各种类型寡糖在PCL-Sepharose柱上的层析行为,确定红花菜豆(矮生红花变种)凝集素(PCL)的 相似文献
6.
J. A. Magnuson E. R. Pandolfino G. R. Munske A. E. Namen M. S. Nissen 《Journal of biosciences》1983,5(1):83-91
Two lectins, a tetramer designated LBL4 and an octamer LBL8 designated have been purified from the lima beanPhaseolus lunatus. The tetramer appears to be nonmitogenic for human lymphocytes and is a weak mitogen for bovine cells. The octamer and a chemically cross-linked form of the tetramer are good mitogens. The lima bean lectin binds to only certain sub-populations of human lymphocytes. The primary class which does not bind appears to be a sub-population ofT-lymphocytes. Comparisons of cell binding with other lectins which bind to 2-acetamido-2-deoxy-D-galactose have been carried out. Quantitative analysis of the binding to human erythrocytes is co-operative but binding to lymphocytes is non-co-operative. These results show that there may not be a direct correlation between mitogenic stimulation and cooperative binding to membrane receptors. 相似文献
7.
N K Kella D D Roberts J A Shafer I J Goldstein 《The Journal of biological chemistry》1984,259(8):4777-4781
Measurements of the efficiency of singlet-singlet energy transfer were used to determine the distance between the hydrophobic binding site and the thiol group required for carbohydrate-binding activity of lima bean lectin. 1-Anilino-8-naphthalenesulfonate, bound to the hydrophobic binding site by noncovalent interactions, was used as the donor. Two different nonfluorescent probes were used as the acceptors: a mercurial, 2-chloromercuri-4-nitrophenol, and a maleimide, 4-dimethylaminophenylazophenyl-4'-maleimide. Acceptor was covalently attached to the thiol group at the putative carbohydrate binding site. The efficiency of energy transfer in both the 1-anilino-8-naphthalenesulfonate/2-chloromercuri-4-nitrophenol and and 1-anilino-8-naphthalenesulfonate/4-dimethylaminophenylazophenyl-4' -maleimide donor-acceptor systems indicated an apparent distance of 28 A between the two sites, assuming that the transition dipole of the donor is not correlated with respect to that of the acceptor and that each donor is quenched by a single acceptor. Using an alternate model wherein each donor is equally quenched by two acceptors on adjacent subunits, an apparent distance of 33.4 A was calculated. The fact that two donor-acceptor pairs with different F?rster's critical distance parameters yielded the same distance between the sites is consistent with our assumption of uncorrelated donor-acceptor transition dipoles. 相似文献
8.
The aspartokinase activity of the aspartokinase-homoserine dehydrogenase complex of Escherichia coli was affinity labeled with substrates ATP, aspartate, and feedback inhibitor threonine. Exchange-inert ternary adducts of Co(III)-aspartokinase and either ATP, aspartate or threonine were formed by oxidation of corresponding Co(II) ternary complexes with H2O2. The ternary enzyme-Co(III)-threonine adduct (I) had 3.8 threonine binding sites per tetramer, one-half that of the native enzyme. The binding of threonine to I was still cooperative as determined by equilibrium dialysis (nH = 2.2) or by studying inhibition of residual dehydrogenase activity (nH = 2.7). Threonine still protected the SH groups of I against 5,5'-dithiobis(2-nitrobenzoate) (DTNB) reaction but the number of SH groups reacting with thiol reagents (DTNB) was reduced by 1-2 per subunit in the absence of threonine. This suggests either that Co(III) is bound to the enzyme via sulfhydryl groups or that 1-2SH groups are buried or rendered inaccessible in I. The binding of threonine to sites not blocked by the affinity labeling produced changes in the circular dichroism of the complex comparable to changes produced by threonine binding to native enzyme and also protected against proteolytic digestion. The major conformational changes produced by threonine are thus ascribable to binding at this one class of regulatory sites. The interactions of kinase substrates with various aspartokinase-Co(III) complexes containing ATP, aspartate, or threonine and a threonine-insensitive homoserine dehydrogenase produced by mild proteolysis were studied. The inhibition of homoserine dehydrogenase by kinase substrates is not due to binding of these inhibitors at the kinase active site but was shown to be due to binding to sites within the dehydrogenase domain of the enzyme. L-alpha-Aminobutyrate, a presumed threonine analogue, also inhibits the dehydrogenase by binding at the same or similar sites in the dehydrogenase domain and not at threonine regulatory site. 相似文献
9.
藏红花凝集素分子化学修饰与其活性的关系 总被引:1,自引:0,他引:1
对甘露糖专一性结合藏红花凝集素 (Crocussativuslectin ,CSL)分子进行化学修饰 ,测定酿酒酵母 (S .cerevisiae)凝集活性和寡糖专一性结合活性的变化 .实验结果表明 ,Cys的修饰与活性无关 ,Arg、Tyr和His的修饰降低了CSL分子的酵母凝集活性和寡糖结合活性 ,但对CSL的CD光谱无显著影响 ,表明其为凝集素的活性氨基酸残基 .Glu和Asp的化学修饰可使CSL的凝集活性大幅度降低 ,与特异性寡糖的亲和力增大 ,CD光谱变化明显 ,提示CSL分子中的Glu和Asp对其空间结构影响较大 ,氨基酸羧基的修饰导致CSL构象改变 ,蛋白与寡糖的结合位点暴露 ,可有效结合的位点数增加 相似文献
10.
Brain spectrin alpha and beta chains bind 45Ca2+, as shown by the calcium overlay method. Flow dialysis measurements revealed eight high affinity binding sites/tetramer that comprise two binding components (determined by nonlinear regression analysis). The first component has one or two sites (kd = 2-30 x 10(-8) M), depending on the ionic strength of the binding buffer, with the remaining high affinity sites in the second component (kd = 1-3 x 10(-6) M). In addition, there is a variable, low affinity binding component (n = 100-400, kd = 1-2 x 10(-4) M). Magnesium inhibits calcium binding to the low affinity sites with a K1 = 1.21 mM. Proteolytic fragments from trypsin or chymotrypsin digests of brain spectrin bind 45Ca2+ if they include alpha domain IV, alpha domain III, or the amino-terminal half of the beta chain (but more than 25 kDa from the amino-terminal). These data suggest that calcium ions bind with high affinity to the putative EF-hands in alpha domain IV and to one site in the amino-terminal half of the beta chain that is associated with alpha domain IV in the native dimer. The localization is consistent with a direct calcium modulation of the spectrin-actin-protein 4.1 interaction. In addition, there appears to be one high affinity site near the hypersensitive region of alpha brain spectrin. All four proposed binding sites occur near probable calmodulin-binding or calcium-dependent protease cleavage sites. 相似文献
11.
SRIF receptors are membrane-bound glycoproteins. To structurally identify the carbohydrate components of SRIF receptors, solubilized rat brain SRIF receptors were subjected to lectin affinity chromatography. Solubilized SRIF receptors specifically bound to wheat germ agglutinin-lectin affinity columns but not to succinylated wheat germ agglutinin. This finding, as well as the ability of the solubilized receptor to interact with a Sambucus nigra L. lectin affinity column suggested that sialic acid residues are associated with SRIF receptors. The inability of the receptor to bind to concanavalin A, Dolichus biflorus agglutinin, Ulex europeaus I, and Jacalin lectin affinity columns suggests that high mannose, N-acetylgalactosamine, fucose, and O-linked carbohydrates are not associated with receptor. To investigate the functional role of the carbohydrate groups in brain SRIF receptors, specific sugars were selectively cleaved from SRIF receptors and the subsequent effect on the specific high affinity binding of the agonist [125I]MK 678 to SRIF receptors was determined. Treatment of the receptor with endoglycosidase D did not affect the specific binding of [125I] MK 678 to the solubilized SRIF receptors, consistent with the finding from lectin affinity chromatography that high mannose-type carbohydrate structures were not associated with SRIF receptors. Treatment of solubilized SRIF receptors with peptide-N-glycosidase F and endoglycosidases H and F reduced [125I]MK 678 binding to SRIF receptors indicating that either hybrid, or a combination of hybrid and complex N-linked carbohydrate structures, have a role in maintaining the receptor in a high affinity state for agonists. Treatment of solubilized SRIF receptors with neuraminidase from Vibrio cholera abolished high affinity agonist binding to the receptors, whereas treatment of the receptor with neuraminidase from Newcastle disease virus did not affect [125I]MK 678 binding to the receptor. These findings suggest that sialic acid residues in an alpha 2,6-configuration have a role in maintaining the SRIF receptor in a high affinity conformation for agonists. This is further indicated by studies on SRIF receptors in the pituitary tumor cell line, AtT-20. Treatment of AtT-20 cells in culture with neuraminidase (V. cholera) greatly reduces high affinity [125I] MK 678 binding sites, but did not alter the maximal ability of SRIF to inhibit forskolin-stimulated cAMP accumulation in intact AtT-20 cells. This finding suggests that the desialylated SRIF receptor is functionally active and remains coupled to GTP-binding proteins, but exhibits a reduced affinity for agonists. Treatment of AtT-20 cell membranes with neuraminidase from V. cholera was also able to greatly reduce the affinity of SRIF receptors for [125I]MK 678.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
12.
Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker''s yeast). 总被引:3,自引:0,他引:3
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The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin. 相似文献
13.
J W Ogilvie 《Biochemistry》1985,24(2):317-321
The smallest enzymatically active form of rabbit muscle phosphofructokinase is a tetramer of four identical or nearly identical monomers. The enzyme is inhibited by ATP, and this inhibition by ATP is relieved by the activating adenine nucleotides adenosine cyclic 3',5'-phosphate, AMP, and ADP. Each monomer contains one binding site specific for the inhibitor ATP and another site specific for the activating adenine nucleotides. The enzyme can also be activated by covalently labeling the activating adenine nucleotide binding sites with the affinity label 5'-[p-(fluorosulfonyl)benzoyl]adenosine. These activator binding sites on the enzyme have been covalently labeled to various degrees, ranging from an average value of less than one label per tetramer to four labels per tetramer, and the free-energy coupling, delta Gxy, between the covalently bound affinity label and ATP binding at the inhibitory site was determined. For enzyme preparations containing four labels per tetramer, delta Gxy is approximately 1 kcal/mol at pH 6.95 and 25 degrees C. A very significant free-energy coupling is observed in those preparations containing an average of one label per tetramer and less, and the change in delta Gxy in going from native tetramers to ones containing an average of two labels per tetramer is twice as great as the change in delta Gxy observed in going from tetramers containing an average of two labels per tetramer to ones containing four labels per tetramer, suggesting that modification of the final two monomers in the tetramer contributes much less to the antagonistic effect on ATP binding than does modification of the first two monomers in the tetramer. 相似文献
14.
Hiroyuki Takamatsu Yoshie Tani Mitoshi Akiyama Yoshihiro Nakata Tomio Segawa 《Journal of neurochemistry》1991,56(4):1452-1454
The characteristics of the carbohydrate chain on the rat cerebral cortical substance P (SP) receptor were studied. We examined the effects of pretreatment with three lectins (concanavalin A, wheat germ agglutinin, lens culinaris agglutinin) on the [3H]SP binding activities. Each lectin can bind to the specific carbohydrate chain. Among these lectins, only concanavalin A inhibited specific [3H]SP binding by reducing the affinity of the binding sites. The inhibitory action of concanavalin A was dose-dependent and diminished by the addition of alpha-methyl-D-mannoside. The present results suggest that the rat cortical SP receptor has either a biantennary complex-type or a high mannose-type of carbohydrate chain, and that the carbohydrate chain is implicated in the SP binding activity of the SP receptor system. 相似文献
15.
Unusual entropy-driven affinity of Chromobacterium violaceum lectin CV-IIL toward fucose and mannose
Pokorná M Cioci G Perret S Rebuffet E Kostlánová N Adam J Gilboa-Garber N Mitchell EP Imberty A Wimmerová M 《Biochemistry》2006,45(24):7501-7510
The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum [Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463] and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins. 相似文献
16.
Rat brain membranes were treated with different protein modifying reagents, all of which were able to reduce [3H]tryptamine binding. However, inactivation by N-ethylmaleimide and iodoacetamide only was counteracted by coincubation with tryptamine. Thus, the [3H]tryptamine binding molecule is a membrane protein with an essential sulfhydryl group at the binding site. After incubation of digitonin-solubilized membranes with seven different lectins, no precipitation of [3H]tryptamine binding sites was observed. On concanavalin A and wheat germ agglutinin affinity chromatography, no [3H]tryptamine binding activity was found to be specifically bound. Therefore, the [3H]tryptamine binding protein appears to be devoid of lectin binding carbohydrate residues. 相似文献
17.
8-Azidoadenine was employed as a photoaffinity probe of the adenine binding site of the seed lectin from lima beans and from Phaseolus vulgaris erythroagglutinin. This compound was shown to (a) bind competitively to the adenine binding site of these lectins and (b) exhibit enhanced binding in the presence of 1,8-anilinonaphthalenesulfonic acid in the same manner as adenine. The presence or absence of 1,8-anilinonaphthalenesulfonic acid during labeling caused no change in the peptide maps of either lectin when digested with trypsin. The peptide maps of each lectin showed one major peak of radioactivity. Sequencing of the corresponding tryptic peptide from lima bean lectin indicated the primary structure to be Val-Leu-Ile-Thr-Tyr-Asp-Ser-Ser-Thr-Lys. The sequence of the labeled peptide isolated from P. vulgaris erythroagglutinin was Thr-Thr-Thr-Trp-Asp-Phe-Val-Gly-Glu-Asn-Glu-Val-Leu-Ile-Thr-Tyr, which corresponded to residues 173-190 of the cDNA-derived sequence (Hoffman, L. M., and Donaldson, D. D. (1985) EMBO J. 4, 883-889). Residues 186-190 (italicized) are identical to the first five amino acids in the lima bean lectin peptide. The peptides are located at the COOH-terminal half of the lectin and show extensive homology with other legume lectins. 相似文献
18.
The techniques of electron spin resonance (ESR) and fluorescence spectroscopy have been used to study the interaction of a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine (I), with several plant lectins. While most adenine derivatives enhanced lectin-induced fluorescence of 1,8-anilinonaphthalenesulfonic acid by binding to a separate, adenine-specific site [Roberts, D.D., & Goldstein, I.J. (1982) J. Biol. Chem. 257, 11274-11277], the spin label I caused a decrease in this fluorescence with certain lectins. ESR showed the ligand to interact strongly with lectins from lima bean (Phaseolus lunatus), Dolichos biflorus, and Phaseolus vulgaris (PHA); however, no binding was observed with Griffonia simplicifolia isolectins A4 and B4, soybean agglutinin, or Amphicarpaea bracteata lectins. The spin label was highly immobilized by each of these proteins (2T magnitude of = 68 G). Apparent affinities of the spin label for the lectins decreased in the order lima bean lectin greater than PHA erythroagglutinin greater than PHA leukoagglutinin greater than D. biflorus. Spin-labeled adenine appeared to bind specifically to the adenine binding site of D. biflorus and PHA leukoagglutinin, as demonstrated by total abolition of the ESR spectrum of bound spin label by adenine. PHA erythroagglutinin and lima bean lectin bound the analogue with apparent dissociation constants of 5 X 10(-5) and 3.2 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
同步纯化人心肌肌钙蛋白T、I 总被引:4,自引:0,他引:4
同步纯化人心肌肌钙蛋白T、I李志梁付朝平钱学贤陆青王素华黎梅兰(第一军医大学珠江医院心内科,广州510282)关键词心肌肌钙蛋白T;心肌肌钙蛋白I;同步纯化收稿日期:1996-04-17;接受日期:1996-08-27。心肌肌钙蛋白包括3种不同的蛋白... 相似文献
20.
Despite years of study, a comprehensive picture of the binding of the lectin from Canavalia ensiformis, concanavalin A, to carbohydrates remains elusive. We report here studies on the interaction of concanavalin A with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, the minimum carbohydrate epitope that completely fills the oligosaccharide binding site, and the two conceptual disaccharide "halves" of the trisaccharide, methyl 3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and methyl 6-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, using titration microcalorimetry. In all cases the interaction of protein and carbohydrate is enthalpically driven, with an unfavorable entropic contribution. The choice of concentration scales has an important impact on both the magnitude and, in some cases, the sign of the entropic component of the free energy of binding. The thermodynamic data suggest binding of the two disaccharides may take place in distinct sites, as opposed to binding in a single high affinity site. In contrast to carbohydrate-antibody binding, delta Cp values were small and negative, pointing to possible differences in the motifs used by the two groups of proteins to bind carbohydrates. The thermodynamic data are interpreted in terms of solvent reorganization. Cooperativity during lectin-carbohydrate binding was also investigated. Significant cooperativity was observed only for binding of the trisaccharide, and gave a Hill plot coefficient of 1.3 for dimeric protein. 相似文献