204 research outputs found

    Glycolytic enzymes of parasites as drug targets.

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    Topogenesis of glycolytic enzymes in Trypanosoma brucei.

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    The protozoan haemoflagellate Trypanosoma brucei, differs from other eukaryotic cells in that it contains nine enzymes involved in glucose and glycerol metabolism which are associated with microbody-like organelles called glycosomes. The information available to date indicates that glycosomal enzymes are synthesized as polypeptides of mature size. For three of them, glyceraldehyde-phosphate dehydrogenase, aldolase and glycerol-3-phosphate dehydrogenase, it has been shown that they are made on free polysomes in the cytosol and are subsequently transferred to the glycosome without any secondary modification. The topogenic signal responsible for import into the glycosome must, therefore, be present in the mature protein. Remarkable differences exist between the latter proteins and other glycolytic enzymes: (i) most glycosomal proteins have an apparent Mr which is 1-5 kDa larger than their homologous counterparts from the cytosol, or from other organisms; (ii) they have a high net positive charge. Based on the modelling of three glycosomal sequences in the respective homologous structures, it is thought that the topogenic signal may consist of a unique insertion, containing one or more basic amino acids which, together with additional positive charges elsewhere, constitute two positive hot spots approximately 4 nm apart on the surface of the protein. Such common elements, unique for the glycolytic enzymes from the Trypanosomatidae, lend themselves as excellent targets for the development of new drugs

    [The Rational Design of New Drugs for Sleeping Sickness]

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    In the Trypanosomatidae, contrary to all other organisms, the glycolytic enzymes are located inside a microbody-like organelle called glycosome. these glycosomal enzymes play an essential role in the energy metabolism of the parasites, and because of their unique location in the cell they are attractive targets for new chemotherapeutic approaches. Most of the glycosomal enzymes and several of their cytosolic isoenzymes from Trypanosoma brucei and Leishmania mexicana have been purified to homogeneity or have been over-expressed. Two of the glycosomal enzymes have been crystallized and their three-dimensional structure solved. some characteristics common to the majority of the glycosomal enzymes and absent from all other glycolytic enzymes have been identified and studied. One typical aspect is the existence of unique peptide insertions or of N- and C-terminal extensions in the glycosomal proteins. These peptides are often characterized by and abundance of the positively charged amino acids: arginine and lysine. Three-dimensional modelling has revealed that these positively charged amino acids cluster on the surface of the proteins in two so-called hot spots that are about 40 Angstrom apart. Another important structural difference has been found in the NAD-binding region of the glycosomal glyceraldehyde-phosphate dehydrogenase. We have exploited some of these differences and this has resulted in the synthesis of compounds that specifically inhibit glycosomal enzymes and leave the glycolytic enzymes from other organisms unharmed

    Compartmentation of carbohydrate metabolism in trypanosomes.

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    The trypanosomatidae: amazing organisms

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    Phylogenetic analysis using protein sequences

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    A rapid method for the isolation of intact glycosomes from Trypanosoma brucei by Percoll -gradient centrifugation in a vertical rotor.

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    A rapid method for the isolation of intact glycosomes from trypanosoma brucei is described. A post large-granule extract is subjected to density-gradient centrifugation on isotonic Percoll in a vertical rotor. This results in a good separation of the glycosomes, which equilibrated at 1.09 g/cm3, from other cellular components. Glycosomes isolated from such a gradient are 9-fold purified relative to the starting homogenate with a yield of 25%. Their glycolytic enzymes exhibit a high latency indicative of an intact glycosomal membrane
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