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M Callens

Publications and source records attributed to M Callens.

At least 37 records · Page 2Linked to original sources
PubMed1993

Overexpression of trypanosomal triosephosphate isomerase in Escherichia coli and characterisation of a dimer-interface mutant.

In this paper, the successful expression of trypanosomal triosephosphate isomerase (TIM) from Trypanosoma brucei brucei to high yield in Escherichia coli, using a T7-polymerase-based expression system, is described. Overexpressed trypanosomal TIM is fully active. The measured physicochemical properties of this recombinant TIM and TIM purified from trypanosomes are indistinguishable. Crystals of recombinant TIM have been grown in the presence of 2.4 M ammonium sulphate under the same conditions as for trypanosomally expressed TIM. The recombinant TIM crystal structure has been refined at 0.23 nm resolution; no differences were detected between this structure and the original crystal structure. A TIM mutant was made in which a unique dimer-interface histidine residue (His47) was changed into an asparagine. This variant ([H47N]TIM) could be expressed and purified to homogeneity by a procedure which was somewhat different from the purification of recombinant wild-type TIM. It is shown that the [H47N]TIM dimer is considerably less stable than wild-type trypanosomal TIM. The catalytic activity of [H47N]TIM is concentration dependent. The dilution-dependent inactivation is reversible. His47 is involved in a water-mediated hydrogen bond with Asp385 of the other subunit. The lower stability of the [H47N]TIM dimer implies that this water-mediated hydrogen bond is important for the stability of the TIM dimer.

Amino Acid Sequence↗
PubMed1993

Inhibition of the glycolytic enzymes in the trypanosome: an approach in the development of new leads in the therapy of parasitic diseases.

Glycolysis in the trypanosome represents an important target for the development of new therapeutic agents due to the fact that this metabolism is essential for the parasite, glucose being its sole source of energy. In addition, different features of this metabolism and those associated with glycolytic enzymes offer opportunities for the development of efficient and selective compounds. Examples are given in this work of inhibitors directed to the enzymes aldolase and glyceraldehyde-phosphate-dehydrogenase and also of molecules acting specifically on the clusters of basic amino-acids present at the surfaces of the glycolytic enzymes in the parasite.

Amino Acid Sequence↗
PubMed1993

Synthesis and activity of inhibitors highly specific for the glycolytic enzymes from Trypanosoma brucei.

Most glycosomal enzymes of Trypanosoma brucei carry a relatively high number of positive charges. In at least 3 of the enzymes some of the charges unique to these enzymes are concentrated in 2 distinct areas on the enzymes' surface, about 4 nm apart [4] and these positively charged structural elements have been suggested to be the site of interaction with the trypanocidal drug Suramin. We have synthesized a series of symmetrical long chain molecules with negative charges or strong dipoles at each end. Several of these compounds inhibited the glycosomal enzymes more strongly than Suramin. They also exhibited a specificity for the trypanosome enzymes, when compared with homologous enzymes from other organisms. By varying the chain length of the active compounds, a 4-nm distance between the molecules' extremes proved optimal for inhibition. Tetra-substituted compounds were better than di-substituted. Modifications introduced at the two ends indicated that a planar orientation, with an amide bond linking a phenyl ring to the chain, is preferred. Inhibition kinetics for some of the enzymes indicated the existence of multi-site interactions with the inhibitors.

Animals↗
PubMed1992

Binding characteristics of Mn2+, Co2+ and Mg2+ ions with several D-xylose isomerases.

D-Xylose isomerases are metal-ion (Mn2+, Co2+, Mg2+)-requiring tetrameric enzymes. Both the stoichiometry and the binding constants have been determined by titrating the metal-ion-free enzymes from five organisms (Actinomycetaceae and more divergent bacteria) with the respective metal ions using the enzyme activity as indicator of active complex-formation. The following characteristics have been observed for each specific isomerase: (i) two essential metal ion sites (one structural and one catalytic) exist per subunit; (ii) the metal ion binding at one site does not affect the binding at the other site; (iii) of the four possible configurations E, aE, Eb and aEb, only the double-occupied enzyme is active; (iv) the metal ion activation is a time-dependent process; (v) the dissociation constants for both the structural and catalytic sites may be identical or may differ by one or higher orders of magnitude; (vi) metal ion binding is stronger in the order Mn2+ greater than Co2+ much greater than Mg2+; (vii) pronounced increases in Km values concomitant with decreasing equivalents of metal ion added are only observed in the presence of Mg2+ ions.

Aldose-Ketose Isomerases↗
PubMed1992

Structure of the complex between trypanosomal triosephosphate isomerase and N-hydroxy-4-phosphono-butanamide: binding at the active site despite an "open" flexible loop conformation.

The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N-hydroxy-4-phosphono-butanamide was determined by X-ray crystallography to a resolution of 2.84 A. Full occupancy binding of the inhibitor is observed only at one of the active sites of the homodimeric enzyme where the flexible loop is locked in a completely open conformation by crystal contacts. There is evidence that the inhibitor also binds to the second active site of the enzyme, but with low occupancy. The hydroxamyl group of the inhibitor forms hydrogen bonds to the side chains of Asn 11, Lys 13, and His 95, whereas each of its three methylene units is involved in nonpolar interactions with the side chain of the flexible loop residue Ile 172. Interactions between the hydroxamyl and the catalytic base Glu 167 are absent. The binding of this phosphonate inhibitor exhibits three unusual features: (1) the flexible loop is open, in contrast with the binding mode observed in eight other complexes between triosephosphate isomerase and various phosphate and phosphonate compounds; (2) compared with these complexes the present structure reveals a 1.5-A shift of the anion-binding site; (3) this is the first phosphonate inhibitor that is not forced by the enzyme into an eclipsed conformation about the P-CH2 bond. The results are discussed with respect to an ongoing drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.

Amino Acid Sequence↗
PubMed1992

Some kinetic properties of pyruvate kinase from Trypanosoma brucei.

We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg2+ had the highest catalytic activity, but also the highest S0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn2+. The reaction rates of Mg(2+)-pyruvate kinase and Mn(2+)-pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co(2+)-pyruvate kinase were hyperbolic. However, Co(2+)-pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg2+ and Mn2+ as the activating metal ions. Moreover, with Co2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K0.5 values for the former 3 compounds increased when Mg2+ was replaced by Co2+, while the K0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1992

Levamisole plus 5-fluorouracil inhibits the growth of human colorectal xenografts in nude mice.

Fragments of human colorectal adenocarcinomas were inserted under the renal capsule of nude mice. The growth of these tumour grafts was significantly inhibited by the combination of 5-fluorouracil (5-FU) and levamisole. An alternating regimen of levamisole 2.5 mg/kg and 5-FU 20 mg/kg decreased the size of tumour implants by 33-59% and/or increased the number of macroscopically disappeared fragments in the combined group compared with ineffective monotherapy with saline, levamisole or 5-FU. This model could be valuable for investigating the mechanism of action of levamisole and to evaluate the effects of this adjuvant therapy in other oncological settings.

Adenocarcinoma↗
PubMed1992

Aromatase inhibition by R 83 842, the dextro isomer of R 76 713, in JEG-3 choriocarcinoma grown in ovariectomized nude mice.

The effects of repeated (5 days) dosing with the non-steroidal aromatase inhibitor R 83 842 (the dextro isomer of R 76 713) on tumor aromatase and uterus weight in ovariectomized nude mice bearing JEG-3 tumors were examined. In animals bearing an androstenedione implant the presence of a JEG-3 tumor significantly increased uterus weight, proving that tumor aromatase indeed converted androgens to estrogens. Oral administration of R 76 713 (10 mg/kg) for 5 days reduced the increase in uterus weight by 84% in tumor bearing mice revealing true in vivo aromatase inhibition by R 76 713. Experiments performed in the absence of exogenously added androgens gave similar results. Uterus weights in tumor bearing mice were significantly higher than in control mice. Oral administration of R 83 842 (5 mg/kg) for 5 days reduced uterus weight in the tumor bearing animals. Ex vivo aromatase measurements performed in JEG-3 tumors from these animals showed an aromatase inhibition of 93.9% in treated mice as compared to untreated mice. Five days oral treatment with R 83 842 dose-dependently lowered both aromatase activity and uterus weight. Doses of 5 and 0.5 mg/kg inhibited tumor aromatase by 94.1 and 74.7%, respectively, and reduced uterus weight. After a dose of 0.05 mg/kg aromatase activity and uterus weight were similar to those in the control group.

Animals↗
PubMed1991

Kinetic properties of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolase from rabbit muscle and Staphylococcus aureus.

The kinetic properties of aldolase from Trypanosoma brucei were studied in comparison with aldolase from rabbit muscle and Staphylococcus aureus. The 3 enzymes displayed a similar broad pH optimum for the cleavage of fructose 1,6-bisphosphate (Fru(1,6)P2) and a similar narrow pH optimum for the cleavage of fructose 1-phosphate (Fru-1-P). However, small alterations in the maximal cleavage rate at more extreme pH values yielded disparities between the pH curves. The reaction catalyzed by the aldolases from T. brucei and S. aureus proceeded via an ordered sequence, as described for the rabbit-muscle enzyme. We determined for the 3 enzymes the kinetic parameters for both the cleavage and the formation of Fru(1,6)P2 and for the cleavage of Fru-1-P. The trypanosomal enzyme differed in its higher ratio of the maximal rate of Fru(1,6)P2-cleavage vs. the maximal rate of Fru(1,6)P2-formation, its higher affinity towards dihydroxyacetone phosphate, and its higher turnover number for the cleavage of Fru-1-P. At ionic strengths above 0.1 M the kinetic parameters of the trypanosomal enzyme followed the limited form of the Debye-Hückel equation. At ionic strengths below 0.1 M the enzyme revealed a characteristic deviation: the apparent Km for Fru(1,6)P2 increased with decreasing salt concentration. The trypanosomal aldolase was competitively inhibited by adenine nucleotides and phosphates. This inhibition occurred in the same concentration range as observed for the rabbit-muscle enzyme, while the bacterial enzyme was less affected.

Adenosine Diphosphate↗
PubMed1991

Chemical modification of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolase from rabbit muscle and Staphylococcus aureus.

Chemical modifications of Class I aldolases from Trypanosoma brucei, rabbit muscle and Staphylococcus aureus with carboxypeptidase A, glyceraldehyde 3-phosphate and cysteine-specific reagents revealed the following differences between the three homologous enzymes. Aldolase from S. aureus was not affected by any of these reagents. Carboxypeptidase-A treatment of rabbit-muscle and T. brucei aldolase inhibited the activity of both enzymes towards fructose-1,6-bisphosphate (Fru(1,6)P2), while the activity towards fructose-1-phosphate (Fru-1-P) was affected only in the case of the trypanosomal enzyme. Moreover carboxypeptidase-A treatment reduced the turnover numbers of these two aldolases for both Fru(1,6)P2 and Fru-1-P to a similar level. Glyceraldehyde 3-phosphate, in the absence of dihydroxyacetone phosphate, also inactivated aldolases from rabbit muscle and T. brucei with second order rate constants of 1054 and 254 min-1 M-1, respectively. Using 5,5'-dithiobis-(2-nitrobenzoic acid) with rabbit-muscle aldolase, a total of 4 thiol groups could be titrated per subunit, resulting in a total inactivation. The presence of substrate completely protected the enzyme from inactivation. Methyl methanethiosulfonate also reacted with four cysteine residues, but this led to very little inactivation. This indicates that the inactivation by modification with DTNB is due to conformational changes in the enzyme. In T. brucei aldolase only one thiol group could be titrated with methyl methanesulfonate and there was no loss of activity. With 5,5'-dithiobis-(2-nitrobenzoic acid) five cysteines were titrated with an immediate and complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Characterization of pyruvate kinase of Trypanosoma brucei and its role in the regulation of carbohydrate metabolism.

Pyruvate kinase from Trypanosoma brucei is a labile enzyme, losing its activity within several hours. In mixtures containing 50 mM triethanolamine buffer, pH 7.2, 25% glycerol and 0.5 mM inorganic phosphate the enzyme remained active and could be purified to homogeneity with a specific activity of 417 units mg-1 and a yield of 65%. The enzyme has an activation energy of 31.9 kJ mol-1. Magnesium and potassium ions are essential for activity. Cobalt or manganese ions replace Mg2+ but this leads to a decrease in maximal velocity. Potassium ions can be substituted by ammonium ions, while sodium ions behave as a competitive inhibitor with respect to both K+ and NH4+. All metal ions studied displayed sigmoidal kinetics. The enzyme is activated, with decreasing efficiency by fructose 2-phosphorothioate 6-phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. They all display hyperbolic kinetics. Glycerate 2,3-bisphosphate, glyceraldehyde 3-phosphate, CoASAc, oxalate, AMP, ADP, and ATP inhibit the enzyme. At substrate saturation PK was activated by Pi up to a concentration of 0.8 mM. At higher Pi concentrations the enzyme is inhibited. The enzyme is unaffected by most amino acids, only phenylalanine stimulates and tyrosine inhibits.

Amino Acids↗
PubMed1989

Reaction of Woodward's reagent K with D-xylose isomerases. Modification of an active site carboxylate residue.

D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.

Aldose-Ketose Isomerases↗
PubMed1988

Evidence for an essential histidine residue in D-xylose isomerases.

Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.

Aldose-Ketose Isomerases↗
PubMed1988

Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber.

The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 x 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 x 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 x 10(5) M-1 and 5 x 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.

Aldose-Ketose Isomerases↗
PubMed1986

Nutritive and non-nutritive sucking in preterm infants.

Nutritive and non-nutritive sucking was studied in 9 preterm infants with postmenstrual ages ranging from 28 to 33 weeks and postnatal ages ranging from 0 to 8 weeks. During nutritive sucking, sucking bursts were longer than sucking pauses. During non-nutritive sucking the opposite was seen. The sucking rate was lower during nutritive sucking. During nutritive sucking the respiratory rate was higher during the pauses than during the bursts. During non-nutritive sucking the respiratory rate was higher during sucking. It is concluded that non-nutritive sucking cannot serve as a model for studying feeding mechanisms in the preterm infant.

Electromyography↗