We examined the interfaces of homodimeric triosephosphate isomerase (TIM) from eight different species. The crystal structures of the enzymes showed that a portion of the interface is markedly similar in TIMs from Trypanosoma cruzi (TcTIM), Trypanosoma brucei, and Leishmania mexicana and significantly different from that of TIMs from human, yeast, chicken, Plasmodium falciparum, and Entamoeba histolytica. Since this interfacial region is central in the stability of TcTIM, we hypothesized that it would be possible to find agents that selectively affect the stability of TIMs from the three trypanosomatids. We found that 6,6'-bisbenzothiazole-2,2' diamine in the low micromolar range causes a desirable irreversible inactivation of the enzymes from the three trypanosomatids and has no effect on the other five TIMs. Thus, the data indicate that it is possible to find compounds that induce selective inactivation of the enzymes from three different trypanosomatids.

After tissue homogenization, 43% of the total hexokinase activity found in maize radicles was recovered in the mitochondrial fraction and 35% was soluble, in the cytosol. The maize submitochondrial particles obtained after mitochondrial sonication retained a high hexokinase activity. The mitochondrial respiration (state 4 rate) was activated by glucose. This activation was blocked by carboxyatractyloside (0.5 mM) and by oligomycin (2 micrograms/ml). The affinities for ATP and glucose of both soluble and membrane-bound maize hexokinases are similar to those of yeast hexokinase. The Km for ATP of these different forms of hexokinase varied between 0.15 and 0.37 mM, and the Km for glucose between 0.05 and 0.13 mM. A major difference between the two maize hexokinase forms is that only the mitochondrial enzyme was strongly inhibited by ADP (Ki 0.04 mM). The soluble forms of hexokinase found both in the cytosol of maize radicles and in yeast are not inhibited by ADP. In a previous report [de Meis, Grieco and Galina (1992) FEBS Lett. 308, 197-201] it was shown that the mitochondrial F1-F0-ATPase can use glucose 6-phosphate and yeast hexokinase as an ATP regenerating system. We now show that the membrane-bound hexokinase and glucose 6-phosphate can also serve as an ATP regenerating system for the mitochondria of maize radicles provided that the ADP concentration is kept below 0.05 mM. Higher ADP concentrations inhibit the reverse reaction of the mitochondrial hexokinase.

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The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report that Trichomonas vaginalis contains 2 fully functional tpi genes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located in α-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed that tpi2 transcript is approximately 3·3-fold more abundant than tpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement an Escherichia coli strain carrying a deletion of its endogenous tpi gene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM from Saccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. As T. vaginalis depends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicated tpi copy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein.

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