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Biotechnology and Applied Biochemistry (2002) 36, (195–204) (Printed in Great Britain)

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Thiol compounds from a free-living pathogenic opportunistic amoeba, Acanthamoeba polyphaga

Raúl N. Ondarza*†‡¹, Angélica Iturbe*, Eva Hernández* and Gerardo Hurtado*

*Center of Research on Infectious Diseases, National Institute of Public Health, Cuernavaca, Mor., Mexico, 62508, †Faculty of Medicine, Department of Biochemistry, National Autonomous University of Mexico, University City, Mexico, 04510, and ‡Center of Research for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, UCSD, La Jolla, CA 92093-0204, U.S.A.

Key words: cysteine, disulphide-reducing enzyme, glutathione, novel thiol, potential drug target.

Abbreviations used: Gspd, glutathione-spermidine; DTT, dithiothreitol; NEM, N-ethylmaleimide; mBBr, monobromobimane; PCA, perchloric acid.

¹To whom correspondence should be addressed, Olivar de los Padres 941, Mexico, D.F. 01780 (e-mail ondarza@servidor.unam.mx).

New bimane-reacting compounds from perchloric acid extracts have been detected by HPLC from Acanthamoeba polyphaga. The main compounds detected are cysteine, glutathione and other novel thiol compounds. All of these compounds must be thiols, since they disappear or decrease substantially when treated by N-ethylmaleimide prior to acetonitrile/bimane derivatization. Cysteine and glutathione increase in quantity when dithiothreitol reduction is applied to the fresh extract. This means that they are likely to be present in their oxidized and reduced form and indicates the possible presence of a corresponding thiol/disulphide enzymic system. There are other compounds that have a different behaviour, since although they can react with bimane, they do not disappear if treated previously by N-ethylmaleimide. This shows that they are not thiols but can react with bimane. The main thiol compounds found to be present, in both the parasite and the host lymphocyte cells, were cysteine and glutathione. We were unable to detect ovothiol A in Acanthamoeba but instead we found another thiol compound that could be structurally related to trypanothione. The new thiol compounds unique to this parasite and not present in lymphocytes will permit the study of disulphide-reducing enzymes as potential drug targets.

Received 3 December 2001/21 August 2002; accepted 22 August 2002

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