2Centro de Biologia Molecular "Severo Ochoa," Universidad Autonoma de Madrid, Madrid, Spain, and 3Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
Received on February 12, 2001; revised on April 24, 2001; accepted on May 15, 2001.
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Abstract |
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The results clearly show a differential specificity toward high-mannose glycans between the CBPs from the two developmental stages. Thus, the isolated CBP from epimastigotes exhibited stronger affinity for higher mannose oligomers containing the Man1-2Man
1-6Man
1-6 structure. Its affinity decreased, as did the number of mannose residues on the oligomer or removal of the terminal Man
1-2-linked mannose. By contrast the CBP isolated from the trypomastigote stage showed about 400-fold lower avidity than the epimastigote form, and contrary to it, it was slightly more specific toward Man5GlcNAc than Man9GlcNAc. Analysis of the interaction of epimastigote-Man-CBP with its ligands by UV difference spectroscopy indicates the existence of an extended binding site in that protein with a large enthalpic contribution to the binding. The thermodynamic parameters of binding were obtained by isothermal titration calorimetry and been found that the
H values to be in good agreement with the vant Hoff values. The binding reactions are mainly enthalpically driven and exhibit enthalpy-enthropy compensation.
In addition, analysis of the high-mannose glycans from different parts of the digestive tract of the reduviid insect vector of T. cruzi suggest a role of the CBP in the retention of the epimastigote stage in the anterior portion of the gut.
Key words: Trypanosoma cruzi/carbohydrate-binding protein/mannose-specific
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Introduction |
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The parasite life cycle can be divided into four stages (Brener, 1973; de-Souza, 1984
): the parasite is taken in the blood meal of the insect as trypomastigote, which differentiates into the epimastigote that multiply extracellularly in the midgut of reduviid insects. Then, epimastigotes transform into infective nondividing metacyclic trypomastigotes, which are released in the feces (Kollien and Schaub, 1998
; Schaub and Losch, 1988
; Schaub, 1988
, 1991). Metacyclic trypomastigotes are able to invade a wide variety of host mammalian cells, phagocytic and nonphagocytic (de-Souza, 1984
; Pereira, 1990
). Once inside the cells, the metacyclic forms escape from endocytic vacuoles to the cytoplasm where they transform into amastigotes, which multiply intracellularly. On rupture of host cells, they differentiate into trypomastigotes that circulate in the blood until they encounter appropriate target cells and then go through another intracellular cycle or are taken up by the insect again. This complex developmental cycle requires the mutual recognition between the parasite and the host cell previous to the adhesion as an essential requisite for parasite penetration (Piras et al., 1983
; Pereira, 1990
; Ortega-Barria and Pereira, 1991
). There is relevant evidence pointing to the surface glycans in both the parasite and the host cell as the prospective "ligands" for target cell receptors (Villalta and Kierszenbaum, 1983
, 1985a,b, 1987; Bonay and Fresno, 1995
). To evaluate this potential role a detailed knowledge of the specificities of any carbohydrate-binding proteins (CBPs) identified on the surface of the cells is required.
Here we report the results of the fine specificity of binding of the recently described CBPs from both the epimastigote and metacyclic forms of T. cruzi (Bonay and Fresno, 1995). On the other hand, recent results indicate that UV difference spectroscopy is a useful and powerful method to study the sugar binding to lectins. By measuring directly the changes in the ultraviolet spectrum of the CBPs on binding to a glycan, it eliminates the need for a reporter group (fluorescent or spectroscopic) attached to the glycan together with their possible perturbations (Neurohr et al., 1980
, 1982). This method allowed us the study of the thermodynamics of disaccharides and oligosaccharides binding to the epimastigote form of the CBP. The results show a considerable higher enthalpy change for oligomers with a higher number of mannose compared to that linear di- or trisaccharides, on binding to the CBP, strongly suggesting the existence of an extended binding region on this protein. In addition we present data on the composition of high-mannose glycans present on the surface lining the digestive tract of the reduviid insect vector of T. cruzi, suggesting a putative role for those CBPs in the active retention of the epimastigote form.
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Results |
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Inhibition of thyroglobulin-bCBP binding by mono- and oligosaccharides
Aliquots of the biotinylated man-CBP at a concentration enough to give a 50-70% maximal response (0.17 nM) in the binding assay (Figure 1) were incubated with increasing concentrations of various haptens and the binding to the immobilized thyroglobulin was measured. Figure 2 (panel A, epimastigote man-CBP; panel B, trypomastigote man-CBP) shows representative inhibition curves; the hapten concentrations required for 50% inhibition derived from those curves are summarized in Table I.
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Concerning the trypomastigote-isolated CBP, there were some differences to the epimastigote one as shown in Table I; in particular it is noticed a general lower binding avidity to glycans.
Interaction of CBPs with mannooligosaccharides, high-mannose, and hybrid glycans
Branched trisaccharide mannotriose (Man1-3(Man
1-6)Man) and mannopentaose were almost four times as good as inhibitors as Man
1-3Man and Man
1-6Man for the epimastigote CBP. However, when tested against the metacyclic form, a threefold increase in potency passing from mannotriose to mannopentaose was observed, suggesting that the binding site of this protein may better accommodate a branched manno-trisaccharide when the branch is found farther from the mannose ß-linked at the reducing end.
Likewise the linear mannosaccharides, the attachment of a chitobiose residue at the reducing end of a branched saccharide did not increase the binding affinity. A mannose substitution in 1-2 linkage at the nonreducing end of Man
1-3Manß1-4GlcNAc as in Man
1-2Man
1-3Manß1-4GlcNAc increased threefold the binding affinity to the epimastigote CBP as compared with Man
1-2Man, the best mannobiose inhibitor. This suggests the presence of an extended binding site in this protein. Indicative of the differences exhibited by the binding sites of the two CBP forms was the fact that the same substitution, instead of increasing, reduced the binding to the metacyclic form twofold. Substitution of the
1-3 and
1-6-linked mannose residues of mannotriose by GlcNAc, which reduced the inhibitory potency by almost 50-fold, provided evidence that the epimastigote man-CBP binding site was sensitive to changes at the nonreducing end.
The effect of extending the non-reducing end of the oligosaccharides was better shown when testing the inhibitory effect of high-mannose glycans (Figure 2 and summarized in Table I). The epimastigote man-CBP (Figure 2A) was highly specific for higher number mannose oligomers, obtaining 50% inhibition with 6, 15, and 30 µM for the man9, man8, and man7 oligomers (isomer mixture), respectively. Isomer D1D2 and D1D3 of man8 and D1 of man7 were the best inhibitors along man9 (ID50 6 µM). The reduction in binding affinity on removal of the terminal Man1-2-linked mannose residue of the Man
1-6 branch was significant, as seen with the man8 isomer D2D3 and man7 isomers D2 and D3, thus providing support to the notion of preference of the epimastigote CBP for a Man
1-2Man
1-6Man
1-6 structure. Further evidence derives from the fact that a significant decrease in specificity was observed when the mannose oligomers decrease to 6 and 5 (50% inhibition at 120 and 150 µM, respectively). Conversely, the extension on the nonreducing end by a Man
1-2 reduced the binding for the metacyclic CBP form as shown by comparing the affinity for the isomers D1 and D3 of Man7. Thus the trypomastigote-isolated CBP shows a different binding specificity and lower avidity than the epimastigote form (Figure 2B and Table I). This form exhibits a better specificity for lower number of mannose oligomers but with a lower affinity; the 50% inhibition was reached with 0.75 and 2 mM for man5 and man9, respectively.
Analysis of data shown in Table I, in particular the inhibition of high-mannose isomers, evidence the subtle differences in the binding specificity of the two CBPs. Taken together, these data suggest a preference of epimastigote man-CBP for a free extended man1-2man
1-6man
1-6man structure found in Man9 and the isomers D1D2 and D1D3 of man8 and man7, while the metacyclic man-CBP preferentially binds man
1-3man as found in the trimannosyl core, and hence exhibits almost the same affinity for all the high-mannose glycans.
The number of carbohydrate binding sites on the purified man-CBPs was determined by equilibrium dialysis, using [3H]-man6. The data are shown in Figure 3, plotted according Scatchard. The number of binding sites, obtained from that curve was found to be 1 ± 0.2 and 0.9 ± 0.3 for epimastigote and trypomastigote man-CBPs, respectively. Because both lectins behave as monomers (Bonay and Fresno, 1995) under those conditions of ionic strength and pH, our data indicate one binding site per molecule of 6570 kDa.
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In that sense, we decided to carry out a limited set of isothermal calorimetry determinations. Taking into account the limited availability of the CBPs from T. cruzi (the highest concentration that could be achieved was about of 25 µM), and the low binding constant for some of the glycans tested, the determinations were restricted to the glycans with the highest binding constant to be sure to work in an adequate window of C values. The results of a typical measurement consisting of 5 µl addition of man9 (200 µM) to epimastigote man-CBP (10 µM) in buffer MES 10 mM, NaCl 100 mM, pH 7.2, at 292 K are shown in Figure 8A, exhibiting a monotonic decrease in the exothermic heat of binding until saturation is achieved. The nonlinear least squares fit of the total heat released as a function of the man9 concentration to the one site model described by Equation 1 is shown in Figure 8B. The thermodynamic parameters for the binding of man9 to the epimastigote man-CBP calorimetrically derived (Table III) show that the binding reaction is essentially enthalpically driven with little dependence of the enthalpy value on temperatures from 280 to 292 K. The remarkable point is that the value of Hb for man9 and the other high-mannose glycans tested were in agreement with the value obtained by the vant Hoff plots.
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Discussion |
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The relative affinity of each glycan was determined by its ability to inhibit the binding of biotinylated-CBP (b-CBP) to immobilized thyroglobulin and quantitating the amount of b-CBP bound by secondary binding of streptavidin-HRP. The results presented in this report confirm the binding specificity of the CBP tested for mannose containing glycans because this was the agent used to elute the CBP from the affinity resin (Bonay and Fresno, 1995). Further confirmation was provided by the lack of inhibition of glucose derivatives (methyl-ß-glucoside, maltose, kojibiose), galactose, galactosamine, or N-acetyl-galactosamine, which were totally ineffective as inhibitors. Exclusively based on the activity of monosaccharides it is clear that the equatorial hydroxyl groups at C3 and C4 are necessary for the monosaccharide to be active as epimers of mannose like altrose (C3), talose (C4), and galactose (C2 and C4) were inactive. Furthermore, N-acetylmannosamine was also not inhibitory (not shown), suggesting that substitution of the axially oriented hydroxyl at C2 by a bulky acetamido group was not tolerated.
Introduction of nonpolar aglycon as methyl- or methylumbelliferyl increased twofold the inhibitory ability of the resulting glycosides, as occurs in other mannose-specific lectins like concanavalin A (Kaku et al., 1991) but contrary to Artocarpus integrifolia lectin (Misquith et al., 1994
). Concerning the mannooligosaccharides, mannotriose and mannopentaose were the strongest inhibitors, followed by linear Man
1-2Man
1-3Manß1-4GlcNAc and Man
1-2Man. Additions of a GlcNAc residue at the nonreducing end of the linear mannooligosaccharides did not increase the inhibitory activity, leading us to presume that the epimastigote man-CBP does not bind to it through the reducing end of the saccharide. Furthermore, the sharp differences of binding distinguishing the disaccharide Man
1-2Man much better than Man
1-4Man also confirm this view. Interestingly enough, extension of the mannopentaose core through the nonreducing end with further mannose residues, thus generating high-mannose oligosaccharides potentiates the inhibitory activity in particular for the epimastigote man-CBP. Those data preliminarily define the binding site and suggest the existence of an extended binding site for saccharides on this protein.
The best inhibitor for the epimastigote man-CBP was Man9 followed by the Man8 (D1D2 and D1D3) and Man7 (D1) isomers that maintain the terminal nonreducing 1-2-linked mannose to the
1-6mannose of the
1-6mannose branch, showing all of them almost equal binding affinity. Removal of the terminal
1-2-linked mannose on that branch, as in isomers D2 and D3 of Man7, reduced the inhibitory potency by 10-fold but it was still twofold higher than mannopentaose or mannotriose, thus indicating that it is not the only determinant of binding specificity. Further confirmation of this was provided when comparing the relative potency of Man
1-2Man
1-3Man
1-6(Man
1-2Man
1-3)Man
1-4GlcNAc with mannopentaose and the Man7 isomers where can be evidenced that the
1-3linked mannose residue of the
1-6 branch attached to the core mannose did not determine the binding specificity by itself, but it has an essential contribution to it in combination with the
1-6 branch terminating with an
1-2-linked mannose residue. Altogether those results define the common structural features recognized by the epimastigote man-CBP as depicted in Figure 9.
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On the other hand, as can be seen from Table III, the binding of Man9 or Man1-2Man
1-3Manß1-4GlcNAc was associated with a much larger enthalpy change than those found for the binding of mannotriose or methyl
-D-mannopyranoside. For the Man9 and the linear mannooligosaccharide the entropy contribution was considerably more unfavorable than those of the monosaccharide or mannotriose. Regarding the Man
1-2Man
1-3Manß1-4GlcNAc, the large negative value of T
S counterbalances the increase in
Hbv, resulting in values of free energy and K only slightly larger than those for mannotriose. However, in the case of Man9 the larger value of
Hbv (and
Hb) as compared to the other glycans tested, leads to a larger free energy change and a considerably greater association constant. For the glycans studied in this report, the increases in
Hbv over those seen for monosaccharide (three- to fourfold), are clearly too large as to be assigned to nonspecific hydrophobic interactions of another mannopyranosyl residue as shown by the fact that addition of a 4-methylumbelliferyl group did not increases the affinity for the monosaccharide. This is in contrast to what is seen in another mannose specific lectin as concanavalin A (Van Landschoot et al., 1980
; Williams et al., 1981
), but resembles to what has been reported for the binding of chitooligosaccharides to lysozyme (Banerjee et al., 1973
; Banerjee and Rupley, 1973
) or wheat germ agglutinin (Allen et al., 1973
; Wright, 1980
, 1984), where there is a steady increase in
H values for binding in the series N-acetyl-D-glucosamine, N,N'-diacetyl-chitobiose, N,N,N''-triacetyl-chitotriose, that outweighs the increasingly unfavorable T
S values, so there is an approximately 1000-fold increase in association constants for N,N'-diacetyl-chitobiose over that N-acetyl-D-glucosamine. Considered together, the results of hapten inhibition and difference spectra clearly suggests the existence in the epimastigote mannose-specific CBP of an extended binding site for the glycans able to accommodate the high-mannose glycans containing the minimal determinants shown in the model (Figure 9).
Based on those results, it is possible to suggest one possible functional role of the man-CBP in parasitehost interaction, as an additional factor mediating it. Analysis of the high-mannose glycan composition on sections of the intestinal tract of the insect vector for T. cruzi shows that almost 70% of the high-mannose glycans present at the anterior portion of the intestinal tract are glycans toward which the epimastigote man-CBP exhibits high affinity (Man9 and Man8) in contrast to less than 10% found in the hindgut. The anterior portion of the intestinal tract is where the epimastigote form of the parasite binds to the cells lining it before transforming into the metacyclic trypomastigote form that accumulates in the rectum and eventually is discharged with the faeces to infect a new mammalian host (Schaub and Losch, 1988; Schaub, 1988
, 1991; Kollien and Schaub, 1998
). Interestingly, the man-CBP from metacyclic trypomastigote, although having similar specificity, have lower overall affinities for similar oligosaccharides. The mode of attachment of epimastigotes has gained interest, because adhesion of T. cruzi as well as other trypanosomes is considered to be a necessary step previous to differentiation to the infective stage (Bonaldo et al., 1988
). There are several reports in the literature proposing different mechanisms to explain the interaction of the different developmental stages of the parasite with the insect vector (Kleffmann et al., 1998
; Schaub and Boker, 1986
, 1987; Schaub and Losch, 1989
; Schmidt et al., 1998
). Based on the observation that foregut and hindgut epithelia are lined with an extracellular cuticle, it was proposed that chitin could serve as the natural substrate for binding of T. congolense, T. vivax, and T. cruzi (Wallbanks et al., 1989
; Bonaldo et al., 1995
; Kleffmann et al., 1998
; Schmidt et al., 1998
). It should be stressed that one report (Schmidt et al., 1998
), provided evidence of a hydrophobic attachment mechanism to the superficial layer of the Triatomine rectum, not involving chitin or heparin as the attachment substrates, but not ruling out the involving of mannose residues on glycoproteins or glycolipids. Furthermore, lectins of diverse specificity identified in the intestinal tissues of reduviids, tsetse, and sandflies have been proposed to interact with glycans exposed at the parasite cell surface (Pereira et al., 1981
) and also heparin receptors have been identified at the flagellum of T. cruzi and Leishmania species (Butcher et al., 1990
; Ortega-Barria and Pereira, 1991
, 1992; Herrera et al., 1994
). Not until the identification and characterization of CBPs present on the parasite cell surface (Bonay and Fresno, 1995
), an active recognition from the parasite to the host or vector cells was considered. The data presented in this report regarding the fine binding specificity of the mannose-CBP isolated from the epimastigote stage together with the analysis of the high-mannose glycan composition exposed to the environment where the parasites reside provides a plausible framework that allows to consider that the T. cruzi CBPs participate in mediating the active adhesion and retention of epimastigotes to the anterior parts of the intestinal tract probably as a requisite to their differentiation.
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Materials and methods |
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Insects
Specimens of Rhodnius prolixus were maintained at 21°C and 5060% relative humidity under a light:dark cycle of 17:7 h in an environmental cabinet.
The insects (third and fourth stage) were dissected after feeding on a guinea pig and the digestive tract removed under a stereomicroscope washing continuously with phosphate buffered saline (PBS). The isolated organ was divided in three portions: anterior and posterior midgut and hindgut.
Parasites
The strain of T. cruzi used was originally obtained from a patient with Chagas disease at the Instituto Nacional de la Salud, Madrid, Spain. It was cloned and named strain G (Alcina and Fresno, 1988). Epimastigotes were continuously cultured in liver infusion-tryptose (LIT) medium supplemented with 10% fetal calf serum (FCS) as described previously (Alcina and Fresno, 1988
). Metacyclic trypomastigotes were obtained by metacyclogenesis induced by incubating late-log epimastigotes in TAU3AAG medium plus 0.035% sodium carbonate for 96 h (Goldenberg et al., 1987
). Transformation of the parasite was assessed by resistance to complement lysis using horse serum. After 96 h in metacyclogenesis medium, the parasites were washed with PBS and resuspended to 1 x 108 per ml in PBS. Then, 500-µl samples of parasites were mixed with equal volumes of 70% (v/v) fresh serum and incubated for 1 h at 37°C. The mixture was washed in PBS and the surviving parasites (metacyclics) counted in a hemocytometer.
CBPs
The mannose-specific CBPs from epimastigotes and trypomastigotes were isolated as described before (Bonay and Fresno, 1995). The purified CBPs were biotin-labeled using NHS-LC-biotin as described by the manufacturer (Pierce Europe, Netherlands) in the presence of mannose to avoid labeling of the binding site.
Binding specificity of the CBPs
This was done by quantitative hapten inhibition using immobilized thyroglobulin as a model glycoprotein. Briefly, thyroglobulin dissolved in 50 mM sodium carbonate buffer, pH 9.6, containing 0.02% sodium azide (Buffer A) at 50 pmol/ml was applied (50 µl) to each well of a 96-well microtiter plate and incubated at least 2 h at 4 °C. The plate was then rinsed three times with 50 mM sodium phosphate buffer, pH 7.4, containing 0.05% Tween-20 (Buffer B). The remaining sites on the plate were coated by incubating with 350 µl of Buffer A containing 1% bovine serum albumin (BSA) at 25°C for 1 h. A 50-µl aliquot of b-CBP (at about 50 pmol/ml in 50 mM Tris buffer, pH 7.4, containing 150 mM NaCl) was mixed with aliquots of the different glycans at 4°C for 2 h and then applied to each well plate and incubated at room temperature for 4 h. The plate was washed three times with Buffer A and streptavidinhorseradish peroxidase (HRP) conjugate (2 µg/ml) was added to each well (100 µl) and incubated for 1 h. Finally the plate was washed four times with buffer B and 100 µl of the substrate solution (ABTS 0.3 mg/ml dissolved in 50 mM citrate buffer, pH 5.0, containing 0.012% H2O2) was added and incubated at 25°C for 1020 min. The reaction was terminated by addition of 100 µl of 5% sodium dodecyl sulfate and the absorbance read at 405 nm.
UV difference spectroscopy
This analysis was performed by using Yankeelov cells on an Aminco double-beam spectrophotometer using an extinction coefficient for the epimastigote man-CBP E1% = 18.2 at 279.8 nm. For the titrations, aliquots of saccharide solutions were added to the man-CBP solution (12 µM), while the same amount of sugar was added to the reference semi-cell. The solution in the sample cell was gently mixed and kept in a thermostat for 5 min and the difference spectra were measured in 250330 nm region.
Glycan isolation
The high-mannose glycans were isolated by endoglycosidase H digestion of the insect intestinal tract. Briefly, the three portions of the intestinal tract were washed extensively with PBS after cutting longitudinally. The washed portions were incubated with 50 mU of endoglycosidase H (Streptomyces plycatus, recombinant, Calbiochem) and endoglycosidase D (Diplococcus pneumoniae, Calbiochem) in a final volume of 75 µl as described by the manufacturers, over 24 h at 37°C with continuous mixing in a thermomixer. The reaction mixture was centrifuged and buffer exchanged by the use of a Centricon-10 (Amicon,USA). The released glycans were fluorescently labeled with 8-amino-1,3,6-trisulfonic acid as described by Klock and Starr (1998).
Titration calorimetry
Isothermal titration calorimetry measurements were carried out using an OMEGA titration calorimeter from Microcal Inc. as described before in the literature (Wiseman et al., 1989; Schwarz et al., 1991
; García-Hernández et al., 1997
) and according to the manufacturer recommendations. To stabilize the temperature, a circulating water bath was used. After an overnight period to equilibrate the instrument, aliquots (510 ml) of the ligand solution (840 x protein-binding sites) were added via the computer-controlled 100-ml rotating syringe stirring at 395 rpm to the solution cell containing 0.8 ml of the epimastigote man-CBP solution at 515 mM. The total concentration of ligand was from 50 to 400 mM. The heat of dilution was determined to be negligible in separated titrations of the ligand solution into buffer solution. The total heat, Qt, was then fitted via a nonlinear least squares minimization method to the total ligand concentration (Xt) using Equation 1 (Wiseman et al., 1989
):
(Eq. 1) Qt = nMt HbV{1 + Xt /nMt + 1/nKb Mt [(1 + Xt /nMt + 1/nKb Mt )2 4Xt /nMt]1/2}/2,
where n is the number of binding sites per monomer, and V is the cell volume. The expression for the heat released per ith injection, dQ(i), is then given by Equation 2 (Yang, 1990):
(Eq. 2) Q(i) = Q(i) + dVi/2V[Q(i) + Q(i 1)] Q(i 1)
where dVi is the volume of titrant added to the solution. The parameters G°b, and
Sb are calculated from the basic equations of thermodynamics according to the Equations 3 and 4:
(Eq. 3) G0b =
Hb T
Sb
(Eq. 4) G0b = nRTLn{Kb},
where n is number of moles, T is the absolute temperature, and R = 1.99 cal mol1 K1.
The binding enthalpies were also calculated from the vant Hoff plots. Values for Hbv were calculated from the best fit of Ln{Kb} to 1/T.
Equilibrium dialysis
Solutions of purified epimastigote man-CBP (0.8 mg/ml) and of man6 containing [3H]-man6 were made in TrisHCl 100 mM, pH 6.8. Each chamber of the dialysis cells received 100 µl of the protein or glycan solution. After equilibration during 50 h at 4°C, aliquots of 80 µl were removed from each chamber and counted on a Beckman scintillation counter after mixing with 10 ml aqueous scintillation fluid.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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