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Two forms of 'malic' enzyme with different regulatory properties in Trypanosoma cruzi.

ABSTRACT: 1. Cell-free extracts from culture epimastigotes of Trypanosoma cruzi contained two forms of NADP+-linked 'malic' enzyme (EC 1.1.1.40), I and II, with the same molecular weight but different electrophoretic mobilities and kinetic and regulatory properties. 2. The apparent Km for L-malate was lower for 'malic' enzyme I, with hyperbolic kinetics, whereas the kinetic pattern for 'malic' enzyme II was slightly sigmoidal (h 1.4). The kinetics for NADPH were hyperbolic for 'malic' enzyme I, and very complex for 'malic' enzyme II, suggesting both positive and negative co-operativity. 3. 'Malic' enzyme II was markedly inhibited by adenine nucleotides; AMP was the the most effective, at least in the presence of an excess of MnCl2. 'Malic' enzyme I was much less affected by the nucleotides. Both enzyme forms were inhibited by oxaloacetate, competitively towards L-malate, but the apparent Ki for 'malic' enzyme I (9 microM) was 10-fold lower than the value for 'malic' enzyme II. 'Malic' enzyme II, but not 'malic' enzyme I, was activated by L-aspartate and succinate (apparent Ka of 0.12 and 0.5 mM respectively); the activators caused a decrease in the apparent Km for L-malate and, to a lesser extent, in the apparent Km for NADP+. L-Aspartate, but not succinate, increased the apparent Vmax. 4. The inhibition by AMP suggests regulation by energy charge, with the L-malate-decarboxylation reaction catalysed by 'malic' enzyme II fulfilling a biosynthetic role. The inhibition by oxaloacetate and the activation by succinate are probably involved in the regulation of the 'partial aerobic fermentation' of glucose which yields succinate as final product. The activation by L-aspartate would facilitate the catabolism of this amino acid, when present in excess in the growth medium.

SUBMITTER: Cannata JJ

PROVIDER: S-EPMC1161776 | biostudies-other | 1979 Nov

REPOSITORIES: biostudies-other

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Project description:As a central component of innate immunity, complement activation is a critical mechanism of containment and clearance of microbial pathogens in advance of the development of acquired immunity. Several pathogens restrict complement activation through the acquisition of host proteins that regulate complement activation or through the production of their own complement regulatory molecules (M. K. Liszewski, M. K. Leung, R. Hauhart, R. M. Buller, P. Bertram, X. Wang, A. M. Rosengard, G. J. Kotwal, and J. P. Atkinson, J. Immunol. 176:3725-3734, 2006; J. Lubinski, L. Wang, D. Mastellos, A. Sahu, J. D. Lambris, and H. M. Friedman, J. Exp. Med. 190:1637-1646, 1999). The infectious stage of the protozoan parasite Trypanosoma cruzi produces a surface-anchored complement regulatory protein (CRP) that functions to inhibit alternative and classical pathway complement activation (K. A. Norris, B. Bradt, N. R. Cooper, and M. So, J. Immunol. 147:2240-2247, 1991). This study addresses the genomic complexity of the T. cruzi CRP and its relationship to the T. cruzi supergene family comprising active trans-sialidase (TS) and TS-like proteins. The TS superfamily consists of several functionally distinct subfamilies that share a characteristic sialidase domain at their amino termini. These TS families include active TS, adhesions, CRPs, and proteins of unknown functions (G. A. Cross and G. B. Takle, Annu. Rev. Microbiol. 47:385-411, 1993). A sequence comparison search of GenBank using BLASTP revealed several full-length paralogs of CRP. These proteins share significant homology at their amino termini and a strong spatial conservation of cysteine residues. Alternative pathway complement regulation was confirmed for CRP paralogs with 58% (low) and 83% (high) identity to AAB49414. CRPs are functionally similar to the microbial and mammalian proteins that regulate complement activation. Sequence alignment of mammalian complement control proteins to CRP showed that these sequences are distinct, supporting a convergent evolutionary pathway. Finally, we show that a clonal line of T. cruzi expresses multiple unique copies of CRP that are differentially recognized by patient sera.

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Two forms of 'malic' enzyme with different regulatory properties in Trypanosoma cruzi.

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