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M P Barrett

Publications and source records attributed to M P Barrett.

43 records · Page 3Linked to original sources
PubMed1995

Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2.

The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.

Animals↗
PubMed1995

A diamidine-resistant Trypanosoma equiperdum clone contains a P2 purine transporter with reduced substrate affinity.

Following the demonstration that the transport of melaminophenyl arsenical drugs in Trypanosoma brucei is dependent upon an unusual adenosine nucleoside transporter (Carter and Fairlamb, Nature 361 (1993) 173-175) we have investigated adenosine transport in the related parasite Trypanosoma equiperdum (Botat1.1) and a cloned derivative resistant to the diamidine drug berenil (diminazene aceturate) with limited cross-resistance to the melaminophenyl arsenical cymelarsen. The parental strain possesses a bipartite adenosine transport system consisting of one component which is inhibited in a dose-dependent and saturable manner with increasing concentrations of inosine and a second component which is similarly inhibited by adenine. Uptake of adenosine on this second transporter is also inhibited in a dose-dependent fashion by berenil and cymelarsen. Both transporters have high affinity for adenosine (apparent Km values of 0.60 and 0.70 mM and Vmax values of 8.4 and 6.9 pmol (s (10(8) trypanosomes))-1 at 25 degrees C, respectively). Thus T. equiperdum shares with T. brucei a system comprising two adenosine transporters named P1 and P2, respectively. The P1 transporter is similar in the sensitive and resistant T. equiperdum clones, whereas the P2 transporter has reduced transport capacity at physiological adenosine concentration and decreased affinity for adenosine in the drug-resistant clone.

Adenosine↗
PubMed1994

Characterization of glucose transport and cloning of a hexose transporter gene in Trypanosoma cruzi.

A gene from Trypanosoma cruzi, TcrHT1, which encodes a member of the glucose transporter superfamily has been cloned. The gene is similar in sequence to the T. brucei hexose transporter THT1 and the Leishmania transporter Pro-1 and is present in the T. cruzi genome as a cluster of at least eight tandemly reiterated copies. Northern blot analysis revealed two mRNA transcripts which differ in size with respect to their 3' untranslated regions. When injected with in vitro transcribed TcrHT1 mRNA, Xenopus oocytes express a hexose transporter with properties similar to those of T. cruzi. Glucose transport in T. cruzi is mediated via a carrier with unique properties when compared with the other glucose transporters already characterized among the Kinetoplastida. It is a facilitated transporter with a high affinity for D-glucose (Km = 84.1 +/- 7.9 microM and Vmax = 46 +/- 9.4 nmol/min per mg of protein) that shares with other kinetoplastid hexose transporters the ability to recognize D-fructose, which distinguishes these carriers from the human erythrocyte glucose transporter GLUT1.

Amino Acid Sequence↗
PubMed1994

Overexpression in Escherichia coli and purification of the 6-phosphogluconate dehydrogenase of Trypanosoma brucei.

The gene encoding 6-phosphogluconate dehydrogenase (EC 1.1.1.44) from Trypanosoma brucei was cloned into the overexpression vector pET3a which utilizes the T7 polymerase gene expression system. Up to 40% of total cell protein consisted of the trypanosome enzyme when expression was induced in Escherichia coli host strains at 28 degrees C. The enzyme was rapidly degraded at temperatures higher than 30 degrees C. The T. brucei enzyme was purified to near homogeneity (as judged by SDS-polyacrylamide gel electrophoresis) using a two-step purification method, involving a DE-52 cellulose batch preparation followed by 2' AMP-agarose affinity chromatography. The purified protein crystallized readily. A molecular model of the trypanosome enzyme based on its mammalian counterpart revealed differences between the two enzymes in residues involved in cofactor binding.

Animals↗
PubMed1993

Preliminary crystallographic study of 6-phosphogluconate dehydrogenase from Trypanosoma brucei.

We have obtained well-ordered single crystals of 6-phosphogluconate dehydrogenase, an enzyme of the oxidative branch of the pentose phosphate pathway, from Trypanosoma brucei. The crystals are trigonal rhombs with unit cell dimensions a = b = 135.1 A, c = 116.7 A and belong to one of the enantiomorphic pair of space groups P3(1)21/P3(2)21. X-ray diffraction to better than 2.8 A has been recorded using a rotating anode CuK alpha source. Elucidation of the three-dimensional structure of the T. brucei enzyme will, by indicating structural differences from the known sheep enzyme structure, aid the design of mutants to probe the active site. Knowledge of the structure will also assist in assessing the potential use of rationally designed compounds to inhibit this enzyme specifically.

Animals↗
PubMed1993

A 6-phosphogluconate dehydrogenase gene from Trypanosoma brucei.

A Trypanosoma brucei gene encoding 6-phosphogluconate dehydrogenase (6-PGDH) (EC 1.1.1.44) was identified and cloned by functional complementation of Escherichia coli gnd mutants with genomic trypanosome DNA. The T. brucei gnd gene is present as a single copy. In Northern blot experiments a probe derived from the gene hybridises to 2 transcripts (2.9 kb and 3.1 kb) which are found in both bloodstream and procyclic form organisms; the larger transcript is more abundant in bloodstream form organisms. The derived amino acid sequence of the protein is 479 amino acids in length, with a molecular weight of 52,000. It is homologous to 6-PGDHs from bacterial and mammalian sources, but diverges significantly from these other enzymes.

Amino Acid Sequence↗