Studies on the biochemical and genetic characteristics of a key enzyme of carbohydrate metabolism, triosephosphate isomerase (TIM), in trophozoites of Giardia lamblia, were continued this year. In collaboration with Dr. Levenbook, purification of the enzyme was undertaken. Ammonium sulfate fractionations followed by chromatographic separation of the enzyme from other soluble proteins resulted in a 100-fold enrichment of TIM activity. Chromatography revealed that the parasite enzyme has properties which differ markedly from mammalian and trypanosome counterparts. In collaboration with Dr. Mowatt, transfection experiments with the TIM gene from G. lamblia and a TIM chromosomal deletion mutant of Escherichia coli showed that TIM activity was now expressed by the mutant bacterium. Complementation with TIM genes suggests the general applicability of the method to cloning other genes for which E. coli mutants are available. Collaborative studies with Dr. Diamond on the respiratory activities of Entamoeba spp. were completed this year. Only the low temperature reptilian species of amoeba exhibit specific and marked stimulation of NAD(P)H oxidase by Mn2+. Collaboration with Dr. Clark, who has cloned and partially sequenced the gene for transhydrogenase in E. histolytica, showed that this enzyme is associated with the particulate fraction of the parasite. Since this enzyme previously has been found only in cells containing mitochondria, these observations may indicate a pre- mitochondrial component of a parasite lacking this organelle.
