Trypanosoma brucei brucei: variability in the association of some variant surface glycoproteins

In our isolation procedure, the surface antigens of the variants AnTat 1.1 and 1.10 (Trypanosoma brucei brucei) are essentially obtained as a disulfide-linked dimer while the AnTat 1.8 surface antigen is found as a mixture of monomer and disulfide-linked dimer. This observation may be related to the localization of the cysteine residues in the protein sequences. In the purification procedure using concanavalin-A Sepharose chromatography, besides the VSG elution by methyl-alpha-D-mannopyranoside, a quantitative elution of still bound VSG may be obtained by the addition of beta-mercaptoethanol to methyl-alpha-D-mannopyrannoside in the elution buffer. The surface antigen of the variant AnTat 1.1 was examined for molecular form at several different times during the release procedure. The disulfide-linked dimer could be observed within 30 min of the surface coat release, indicating its presence within the parasite.     

Molecular heterogeneity of the isolated surface glycoprotein from variant AnTat 1.1 of Trypanosoma brucei brucei

Variant surface glycoprotein (VSG) of Trypanosoma brucei brucei AnTat 1.1 was released by means of the procedure described by Baltz et al. ([1976], Ann. Immunol. [Inst. Pasteur] 127C, 761-774). The concanavalin-A chromatography yielded 3 VSG fractions according to the addition, in the elution buffer, of alpha-methyl-D-mannopyranoside, beta-mercaptoethanol, and sodium dodecyl sulfate. These VSG fractions showed heterogeneous behaviour on reverse-phase high performance liquid chromatography. The 3 VSG fractions as well as the myristylated VSG of AnTat 1.1 essentially consist of dimer VSG forms linked through a disulfide bridge, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing and nonreducing conditions.     

Study of proteolytic activities released by incubation of trypanosomes (Trypanosoma brucei brucei) in pH 5.5 and pH 7.0 phosphate/glucose buffers

Proteolytic activities released by overnight incubation of Antwerpeen Trypanozoon antigenic type (AnTat) 1.1 trypanosomes at 4 degrees C in pH 5.5 and pH 7.0 phosphate/glucose buffers were analyzed in the supernatants obtained after centrifugation of the parasite suspensions. The assays used the fluorogenic substrates N-alpha-benzyloxycarbonyl-L-phenyl-alanyl-L-arginine-7-amido-4- methylcoumarin (Z-Phe-Arg-NMec) and N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine-7- amido-4-methylcoumarin (Z-Arg-Arg-NMec) at two different pHs (6.0 and 8.3). Z-Phe-Arg-NMec hydrolysis was inhibited by 2 microM L-trans-epoxysuccinyllencylamido(4-guanidino)butane (E-64) to a greater extent in the pH 7.0 supernatant than in the pH 5.5 supernatant. Z-Arg-Arg-NMec hydrolysis by the two supernatants was not significantly inhibited by 2 microM E-64. At pH 8.3 this activity was increased more than 2-fold by the addition of dithiothreitol. The hydrolysis activities were analyzed in collected eluates after fractionation of the supernatants by gel permeation high-performance liquid chromatography. Z-Phe-Arg-NMec hydrolytic activity inhibited by 2 microM E-64 was maximal at a retention time of 33 min (approx. Mr 30,000). In addition, a hydrolytic activity against the substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec gave a peak showing a maximum at a retention time of 29 min (approx. Mr 70,000).     

Characterization of different proteolytic activities in Trypanosoma brucei brucei.

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4 degrees C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoprotein by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by an alkaline pH optimum and an estimated molecular mass of 80-100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 microM. The retained material eluted by addition of 1% methyl-alpha-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (greater than 70 kDa) and the other peak of lower molecular mass (30-70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.     

Heterogeneity in high performance liquid chromatography of a variant surface glycoprotein of Trypanosoma brucei

High performance liquid chromatography (HPLC) procedures have been used to analyze a preparation of the variant surface glycoprotein AnTat 1.1A of Trypanosoma brucei. The native preparation gives several peaks with a high reproducibility both by reverse-phase (RP-) and gel permeation (GP-) HPLC. Under RP-HPLC conditions, nine fractions are fully resolved. The RP-HPLC fractions migrate with the same molecular weight VSG band on polyacrylamide slab gel electrophoresis and no significant differences are observed in amino acid composition among these fractions. The RP-HPLC resolution is found to be related to the ability of the VSG to polymerize as shown using GP-HPLC. These results suggest the existence of a microheterogeneity of the AnTat 1.1A VSG preparation in relation to post-translational modification of the VSG molecule.     

Evidence of myristylated disulfide-linked dimer of variant surface glycoprotein of Trypanosoma brucei-brucei

1. Variant surface glycoprotein (VSGs) of Trypanosoma brucei-brucei may exist as a disulfide-linked dimer in both forms: myristylated (mfVSG) and non-myristylated (sVSG), as judge by fluorography and immunoblotting of SDS-PAGE under non-reducing conditions. 2. The dimeric VSG form is labeled with [3H]-myristic acid in our incorporation conditions. 3. AnTat 1.1 trypanosomes preincubated with tunicamycin and incubated with [3H]-myristic acid synthesized a labeled molecule that has an apparent molecular weight slightly smaller than the native form, and that also corresponds to a disulfide-linked dimer.     

Characterization of different proteolytic activities in Trypanosoma brucei brucei

The variant surface glycoprotein of African trypanosomes is released after overnight incubation of parasites at 4°C in pH 5.5 phosphate glucose buffer and may be purified by Concanavalin A Sepharose affinity chromatography [1]. The addition of proteinase inhibitors during the parasite incubation is necessary to prevent the proteolysis of the variant surface glycoproteins by the trypanosomal released proteinases. Using this procedure without the addition of proteinase inhibitors, the proteolytic activities, released from the bloodstream forms Trypanosoma brucei brucei variant AnTat 1.1, were separated by Concanavalin-A Sepharose affinity chromatography. The unretained material (F1) shows hydrolytic activity against the two synthetic substrates Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, which is stimulated by dithiothreitol, but not inhibited by E-64, and characterized by and alkaline pH optimum and an estimated molecular mass of 80–100 kDa. The Michaelis constant for the substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC was, respectively, 2.8 and 6.7 μM. The retained material eluted by addition of 1% methyl-α-D-mannopyranoside (F2) shows hydrolytic activity against the synthetic substrate Z-Phe-Arg-AMC, which is stimulated by dithiothreitol, inhibited by E-64, active between pH 6.0 and 8.0, and could be separated into two peaks of activity by HPLC, one peak of high molecular mass (> 70 kDa) and the other peak of lower molecular mass (30–70 kDa). By electrophoresis in gels containing gelatin as substrate, this fraction contains several proteins with gelatinolytic activity, whereas the unretained fraction F1 did not have any gelatinolytic activity.     

Rat albumin inhibits the formation of apoceruloplasmin during storage

Purified rat ceruloplasmin is extraordinarily unstable in storage at –70 °C. In a 20 mM phosphate buffer, pH 7.0, the ferroxidase and amine oxidase of ceruloplasmin are over 90% inactivated within two weeks. Holoceruloplasmin stored for three months in a 20 mM barbital buffer (or acetate buffer), pH 7.0 (or pH 5.5) was transformed into an apo-protein and amine (o-dianisidine) oxidase of ceruloplasmin was inactivated by 50–55%. The patterns of ferroxidase activity loss were similar to those of amine oxidase activity loss. On the contrary, when holoceruloplasmin was mixed with rat serum albumin, transformation into apoceruloplasmin was significantly prevented in a 20 mM barbital buffer, pH 7.0 (or 20 mM acetate buffer, pH 5.5). Consequently, ferroxidase and amine oxidase activities of ceruloplasmin were not inactivated and the immunochemical reactivity was not changed. These results can be applied for laboratorial and clinical purposes.     

Inactivation and reactivation of a variant-specific antigen gene in cyclically transmitted Trypanosoma brucei.

In Trypanosoma brucei, the activation of the variant-specific antigen gene AnTat 1.1 proceeds by the synthesis of an additional gene copy, the AnTat 1.1 ELC, which is transposed to a new location, the expression site, where it is transcribed. Using the AnTat 1.1 variant to infect flies, we investigated the fate of the AnTat 1.1 ELC during cyclic transmission of T. brucei. We show here that the AnTat 1.1 ELC is conserved in procyclic trypanosomes, obtained either from the midgut of infected Glossina or from cultures, and in metacyclic trypanosomes, although the AnTat 1.1 serotype is not detected among metacyclic antigen types. This same AnTat 1.1 ELC, which is thus silent as the parasite develops in the insect vector, can be reactivated without duplication during the first parasitemia wave following cyclical transmission. This re-expression of the conserved ELC accounts for the early appearance of the 'ingested' antigenic type after passage through the fly.     

Yeast glutathione reductase. Studies of the kinetics and stability of the enzyme as a function of pH and salt concentration.

1. The pH dependencies of the apparent Michaelis constant for oxidized glutathione and the apparent turnover number of yeast glutathione reductase (EC 1.6.4.2) have been determined at a fixed concentration of 0.1 mM NADPH in the range pH 4.5--8.0. Between pH 5.5 and 7.6, both of these parameters are relatively constant. The principal effect of low pH on the kinetics of the enzyme-catalyzed reaction is the observation of a pH-dependent substrate inhibition by oxidized glutathione at pH less than or equal 7, which is shown to correlate with the binding of oxidized glutathione to the oxidized form of the enzyme. 2. The catalytic activity of yeast glutathione reductase at pH 5.5 is affected by the sodium acetate buffer concentration. The stability of the oxidized and reduced forms of the enzyme at pH 5.5 and 25 degrees C in the absence of bovine serum albumin was studied as a function of sodium acetate concentration. The results show that activation of the catalytic activity of the enzyme at low sodium acetate concentration correlates with an effect of sodium acetate on a reduced form of the enzyme. In contrast, inhibition of the catalytic activity of the enzyme at high sodium acetate concentration correlates with an effect of sodium acetate on the oxidized form of the enzyme.     

Appearance of sedoheptulose 1,7-diphosphatase activity on conversion of chloroplast fructose 1,6-diphosphatase from dimer form to monomer form.

Chloroplast fructose 1,6-diphosphatase isolated at pH 5.5 as the dimer dissociated to the monomer at pH 8.5. When the pH was adjusted from 8.5 back to 5.5, the newly formed monomer partly reassociated to form the dimer. The monomer lacked the fructose diphosphatase activity characteristic of the dimer (measured in the presence of a saturating concentration of Mg⁺⁺) but retained ferredoxin-dependent activity (measured in the presence of Mg⁺⁺ plus protein factor and either reduced ferredoxin or dithiothreitol). In addition, the monomer acquired sedoheptulose 1,7-diphosphatase activity that was dependent on either reduced ferredoxin or dithiothreitol and the protein factor.     

Inactivation of aflatoxigenic Aspergilli by treatment with ozone

The D-values of conidia of aflatoxigenic Aspergillus flavus and Aspergillus parasiticus exposed to 1.74 ppm. ozone in 1 mM potassium phosphate buffer (pH 7.0 and 5.5) at 25 degrees C were determined. D-values of A. flavus conidia were 1.72 and 1.54 min at pH 5.5 and 7.0, respectively; D-values of A. parasiticus were 2.08 and 1.71 min, respectively. None of these D-values was significantly (P < or = 0.05) different from each other.     

The effect of resonance energy homotransfer on the intrinsic tryptophan fluorescence emission of the bothropstoxin-I dimer.

Bothopstoxin-I (BthTX-I) is a homodimeric Lys49-PLA2 homologue from the venom of Bothrops jararacussu in which a single Trp77 residue is located at the dimer interface. Intrinsic tryptophan fluorescence emission (ITFE) quenching by iodide and acrylamide has confirmed that a dimer to monomer transition occurs on reducing the pH from 7.0 to 5.0. Both the monomer and the dimer showed an excitation wavelength-dependent increase in the fluorescence emission maximum, however the excitation curve of the dimer was blue-shifted with respect to the monomeric form. No differences in the absorption or circular dichroism spectra between pH 5.0 and 7.0 were observed, suggesting that this curve shift is due neither to altered electronic ground states nor to exciton coupling of the Trp residues. We suggest that fluorescence resonance energy homotransfer between Trp77 residues at the BthTX-I dimer interface results in excitation of an acceptor Trp population which demonstrates a red-shifted fluorescence emission.     

A protease-like permeability factor in the guinea pig skin. 2. In vitro activation of the latent form permeability factor by weakly acidic phosphate buffer.

Conditions for the in vitro activation of the latent form of a protease-like permeability factor in the pseudoglobulin fraction from guinea pig skin were examined. (1) The factor was activated by dialysis against 67 mM phosphate buffer at pH 5.8--6.4, not at pH 7.0--8.0. (2) High salt concentration (200 mM or greater phosphate buffer or 67 mM phosphate buffer containing 200 mM or greater KCl or NaCl) prevented the activation at pH 6.2. (3) High osmotic pressure (sucrose at 1 M) did not affect activation at pH 6.2. (4) Reconversion of the activated permeability factor into an inactive form was not observed under high salt conditions, under which the latent permeability factor was stable in its own form. (5) The molecular size of the latent permeability factor was estimated as approx. 80 000 by Sephadex G-100 gel filtration at high salt concentration.     

Apparent monomer activity of saturated fatty acids im micellar bile salt solutions measured by a polyethylene partitioning system

Partitioning of saturated fatty acids between discs of polyethylene film and aqueous buffer has been characterized and subsequently used to measure monomer activities of fatty acids in micellar solutions of bile salt. Partitioning of fatty acids between polyethylene and buffer achieved equilibrium in about 24-48 hr. Partition coefficients for fatty acids 10:0 and 16:0 were essentially independent of concentration, as expected for true partitioning. Experiments with various pH buffers showed that only the protonated form of fatty acids 12:0 and 16:0 participated in partitioning, and the midpoints of the partition coefficients vs. pH curves were 4.5-5.0 and 6.5-7.0, respectively. Experimentally determined partition coefficients at pH 7.4 ranged from 2.03 +/- 0.09 for 9:0 to 56,100 +/- 13,850 for 17:0. The addition of each methylene group increased the partition coefficient by a factor of about 3.75, corresponding to an incremental free energy change for each methylene group of -3433 J.mole(-1) (-820 cal.mole(-1)). Monomer activities of solutions of 14:0 and 16:0 dissolved in 20 mM taurodeoxycholate were linearly dependent on the total fatty acid concentration. 1 mM 14:0 and 16:0 in 20 mM taurodeoxycholate had monomer activities of 1.3 x 10(-5) M and 5.6 x 10(-7) M, respectively. Solutions prepared with a constant concentration ratio of fatty acid to taurodeoxycholate had essentially constant monomer activities between 8 and 20 mM taurodeoxycholate. These studies support the hypothesis that fatty acid interaction with bile acid micelles is similar to a phase distribution system, so that some measurable monomer activity of fatty acid exists in equilibrium with the mass of fatty acid contained in the micelle.     

2.9 A resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma brucei

The variant surface glycoprotein (VSG) of Trypanosoma brucei forms a coat on the surface of the parasite; by the expression of a series of antigenically distinct VSGs in the surface coat the parasite escapes the host immune response. The 2.9 A resolution crystal structure of the N-terminal domain of one variant, MITat 1.2, has been determined. The structure was solved using data collected from two crystal forms. Initially a partial model was built into an electron density map based on multiple isomorphous replacement phases and improved by phase combination methods. Subsequently this model was used to obtain the molecular replacement solution for a second crystal form, providing starting phases which were refined using 2-fold non-crystallographic symmetry averaging. The current model includes 362 residues and has been refined using X-PLOR to an R value of 0.22 for data between 7 and 2.9 A. The molecule is a dimer, approximately 100 A long, having an asymmetrical cross section with maximum dimensions of approximately 40 A x 60 A. Two long, approximately 70 A, alpha-helices from each monomer pack together to form, with several other helices, a core helix bundle that extends nearly the full length of the molecule. The "top" of the protein, which in the surface coat may be exposed to the external environment, is formed from the ends of the two long helices, a short three-stranded beta-sheet, and a strand having irregular conformation that packs above these secondary structure elements. Two conserved disulfide bridges are in this part of the molecule. Several elements of the MITat 1.2 sequence, which contribute to the formation of the helix bundle structure, have been identified. These elements can be found in the sequences of several different VSGs, suggesting that to some extent the VSG structure is conserved in those variants.     

Structural competition involving G-quadruplex DNA and its complement

Structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex has been implicated for repetitive DNA sequences, but the competitive mechanism of these multistranded structures still needs to be elucidated. We investigated the effects of sequence context, cation species, and pH on duplex formation by the G-quadruplex of dG(3)(T(2)AG(3))(3) and its complement the I-motif of d(C(3)TA(2))(3)C(3), using ITC, DSC, PAGE, CD, UV, and CD stopped-flow kinetic techniques. ITC and PAGE experiments confirmed Watson-Crick duplex formation by the complementary strands. The binding constant of the two DNA strands in the presence of 10 mM Mg(2+) at pH 7.0 was shown to be 5.28 x 10(7) M(-1) at 20 degrees C, about 400 times larger than that in the presence of 100 mM Na(+) at pH 5.5. The dynamic transition traces of the duplex formation from the equimolar mixture of G-/C-rich complementary sequences were obtained at both pH 7.0 and pH 5.5. Fitting to a single-exponential function gave an observed rate of 8.06 x 10(-3) s(-1) at 20 degrees C in 10 mM Mg(2+) buffer at pH 7.0, which was about 10 times the observed rate at pH 5.5 under the same conditions. Both of the observed rates increased as temperature rose, implying that the dissociation of the single-stranded structured DNAs is the rate-limiting step for the WC duplex formation. The difference between the apparent activation energy at pH 7.0 and that at pH 5.5 reflects the fact that pH significantly influences the structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex, which also implies a possible biological role for I-motifs in biological regulation.