Kinetic parameters for the binding of p-nitrophenyl α-d-mannopyranoside to concanavalin A

Lewis, Sidney D.; Shafer, Jules A.; Goldstein, Irwin J.

doi:10.1016/0003-9861(76)90125-9

Cited by 48 publications

(23 citation statements)

References 11 publications

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“…The dissociation of C and M from CMPL has been previously shown to be small (Sherry et al, 1975;Brown et al, 1977). The on-off rate of PNPM with CMPL is also rapid (Lewis et al, 1976) in comparison with the time course changes we observe in Figure 1. Thus, the decrease in diffusion current traced in Figure 1 (curve B) is a direct measure of the first-order rate constant, k2, for the conformational transition from C M P to CMPL.…”

Section: Resultssupporting

confidence: 84%

“…It should be noted that the association constants determined by the polarographic technique are somewhat smaller than the literature values and the reasons for these differences are not presently clear. However, a search of the literature finds a considerable variation in the reported PNPM-Con A binding constants ( K , = 0.9 X lo4 MI' at 25 "C, pH 5, Lewis et al, 1976;3.6 X lo4 M-' at 25 "C, Hassing & Goldstein, 1970; 1.5 X lo4 M-l at 27 "C, pH 7, Bessler et al, 1974) and this variation may simply reflect minor differences in experimental conditions. The temperature dependence of our equilibrium constants yields an enthalpy change of -7.9 f 0.4 kcal/mol for the PNPM-CMPL interaction.…”

Section: Discussionmentioning

confidence: 99%

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Sugar binding properties of various metal ion induced conformations in concanavalin A

Sherry¹,

Buck²,

Peterson³

1978

Biochemistry

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Concanavalin A is known to undergo a first-order conformational transition when metals are added to the demetallized protein at pH 5.6 (Brown, R.D., III, et al. (1977) Biochemistry 16, 3883--3896). The rate constants for this process, which wer have measured using a polarographic technique, are identical when zinc, cobalt, or manganese occupies S1 and calcium occupies S2. The reducible sugar, p-nitrophenyl alpha-D-mannopyranoside, binds only to the locked conformational structure which is formed upon the addition of metals. The affinity of the protein for sugars is dependent upon occupancy of S1 and S2 and quite sensitive to the identity of the metal in S2. The metals may be removed from the locked protein structure and the protein temporarily retains its ability to bind with sugars but with a considerably lower affinity. The locked form of concanavalin A is unstable at a pH near 2 and unfolds to the unlocked structure with a half-life of 25 min resulting in simultaneous loss of metal and sugar binding.

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Section: Resultssupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Sugar binding properties of various metal ion induced conformations in concanavalin A

Sherry¹,

Buck²,

Peterson³

1978

Biochemistry

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“…As the plot (Fig.2) is linear up to 200 pM, the upper limit of KI (allowing an error in the linearity within 10 % for the high concentration points) should be about 500 M-'. This is in agreement with the value deduced for MeUmbManp and dimeric con A [2] and for other glycosides [15,19]. Under these limiting conditions given above a bimolecular two-step binding process of the scheme in Eqn (2) could be operative, and be consistent with our results.…”

Section: Possible Alternative Mechanismssupporting

confidence: 93%

“…The results obtained for the tetrameric con A are practically identical to those for the native (and derivatized) dimeric forms [2] and are entirely consistent with the experimental evidence obtained with stopped-flow, using p-nitrophenyl a-D-mannopyranoside [14,15]. The analyses using relaxation amplitudes Fig.3 and 4) are in accordance with the scheme (Eqn 1).…”

Section: Single-step Mechanismsupporting

confidence: 82%

Binding of 4-Methylumbelliferyl alpha-d-Mannopyranoside to Tetrameric Concanavalin A. Fluorescence Temperature-Jump Relaxation Study

et al. 1977

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The kinetics of saccharide binding to the tetramer form of concanavalin A have been studied at pH 7.2 with the temperature-jump method. 4-Methylumbelliferyl a-D-mannopyranoside was used as a ligand; its fluorescence is totally quenched upon binding. A single relaxation of ligand fluorescence (z = 20-400 ms) was observed and was investigated at three different temperatures, using kinetic titration and dilution types of experiments. The concentration dependence of the relaxation time and amplitude were consistent with a single-step bimolecular association and independent binding sites. In the temperature range 13 -24 "C the association and dissociation rate parameters are in the range (6-10) x lo4 M-l s-' and (1.4-3.2) s-' respectively, corresponding to activation energies for the forward and reverse reactions equal to approx. 13 and 8 kcal/mol (54 and 33 kJ/mol) respectively. Two additional relaxations of protein fluorescence (3 ms and larger than 1 s at 25 "C) were unaffected by carbohydrate binding. Tetrameric concanavalin A shows carbohydrate binding parameters that are almost identical to those of native or derivatized dimeric concanavalin A.We have recently investigated in detail [1,2] the binding kinetics of saccharides to native and chemically modified dimeric concanavalin A (con A). We now report on a temperature-jump relaxation study using the fluorescent [3] 4-methylumbelliferyl CI-Dmannopyranoside (MeUmb-Manp) and tetrameric con A. This tetravalent form of the lectin is prevalent at neutral pH [4,5] and is therefore of greater biological interest [6]. Analysis of the kinetic data in terms of relaxation times and amplitudes shows that as in the case of dimeric con A, the binding of MeUmbManp is consistent with a bimolecular single-step association, which is not diffusion controlled. No kinetic evidence for interaction between the saccharide binding sites or for a resulting conformational change in the protein could be obtained. EXPERIMENTAL PROCEDUREConcanavalin A, composed of intact polypeptide chains [7], was used under conditions of apparent full . Protein concentrations were determined using A;% = 11.4 and were expressed as binding sites on the basis of a molecular weight of 25 500 for the protomer. MeUmb-Manp solutions were free of 7-hydroxy-4-methylcoumarin and concentrations were determined at 318 nm using E = 1 . 3 6~ 104 M-' cm-l. The temperature-jump apparatus has been described [9, 101. Solutions containing MeUmb-Manp were excited at 313 nm and fluorescence was measured above 360 nm. A temperature jump of 3.2 "C was applied in all cases. Further experimental conditions, data acquisition and data analysis were as described for the dimeric protein [1,2]. RESULTSUpon perturbing mixtures of tetrameric con A and MeUmb-Manp by temperature an intense relaxation of ligand fluorescence was observed due to dissociation of the fluorescent glycoside from the totally

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“…5, the K value was determined to be 1.31 × 10 3 M −1 . This constant obtained is almost as high as that (1.25 × 10 3 M −1 ) between free Con A and glucose (Pai et al, 1992) but slightly lower than that (1.44 × 10 4 M −1 ) between free Con A and p-nitrophenyl ␣-mannopyranoside under the conditions at 25°C and pH 7.0 (Lewis et al, 1976). The fact indicates that sugar moiety of pAPM plays a role as a ligand for Con A.…”

Section: Apparent Adsorption Equilibriummentioning

confidence: 72%

Preparation of a new thermo‐responsive adsorbent with maltose as a ligand and its application to affinity precipitation

Hoshino

Taniguchi

Kitao

et al. 1998

Biotechnol. Bioeng.

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A thermo-responsive polymer on which maltose was covalently immobilized as an affinity ligand was newly synthesized for purification of thermolabile proteins from the crude solution by affinity precipitation. Among the thermo-responsive polymers synthesized as carriers for adsorbent, poly(N-acryloylpiperidine)-cysteamine (pAP) has a lower critical solution temperature (LCST) of around 4 degrees C, at which its solubility exhibits a sharp change. Adsorbent for affinity precipitation was prepared by combining pAP with maltose using trimethylamine-borane as a reducing reagent. This adsorbent (pAPM) obtained showed a good solubility response: pAPM in the basal buffer (pH 7.0) became soluble below 4 degrees C and was completely insoluble above 8 degrees C. The affinity precipitation method using pAPM consisted of the following four steps: adsorption at 4 degrees C, precipitation of the complex at 10 degrees C, desorption by adding the desorption reagent at 4 degrees C, and recovery of a target protein at 10 degrees C. In the affinity precipitation of Con A from the crude extract of jack bean meal, 82% of Con A added was recovered with 80% purity by addition of 0.2 M methyl-alpha-D-mannopyranoside as a desorption reagent. In the repeated purification of Con A from the crude extract, pAPM could be satisfactorily reused without decrease in the affinity performance. Moreover, when pAPM was used for the purification of thermolabile alpha-glucosidase from the cell-free extract of Saccharomyces cerevisiae, 68% of total activity added was recovered and the specific activity per amount of protein of the purified solution was enhanced 206-fold higher than that of the cell-free extract without thermal deactivation of the enzyme.

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Kinetic parameters for the binding of p-nitrophenyl α-d-mannopyranoside to concanavalin A

Cited by 48 publications

References 11 publications

Sugar binding properties of various metal ion induced conformations in concanavalin A

Sugar binding properties of various metal ion induced conformations in concanavalin A

Binding of 4-Methylumbelliferyl alpha-d-Mannopyranoside to Tetrameric Concanavalin A. Fluorescence Temperature-Jump Relaxation Study

Preparation of a new thermo‐responsive adsorbent with maltose as a ligand and its application to affinity precipitation

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