PARTIAL PURIFICATION AND CHARACTERIZATION OF CHOLESTEROL DESATURASES FROM TETRAHYMENA THERMOPHILA

NUSBLAT ALEJANDRO; VALCARCE GERMAN; OTERMIN FRANCISCO; NUDEL CLARA

XXXVIII Annual Meeting of the SAIB, Villa Carlos Paz, Cordoba

Congreso; SAIB 2002, 38 Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2002

SAIB

A peculiar trait of the protozoan Tetrahymena thermophila is its ability to desaturate sterols. Cholesterol, for example, is transformed into the unsaturated sterols D7-dehydrocholesterol, D22- dehydrocholesterol and D 7,22-bis dehydrocholesterol. Based on this property, we set up to study the enzymes that are involved in the process. We found two putative enzymes that converted cholesterol into the final product D7,22- bis dehydrocholesterol. One had activity for D7 desaturase and the other for D22 desaturase. In order to separate these two activities, we developed a method based on differential growing conditions, thus allowing measurement of each enzyme separately. We found that precursors added to the growth media could selectivity increase the activity of each enzyme recovered from microsomal preparations. For instance, growth of Tetrahymena in the presence of D22-dehydrocholestrol increased  D7 desaturase activity, while growth with cholesterol induced D22 desaturase activity. Other differential traits between both enzymes were their activities in the presence of 2-mercaptoethanol and ethanol. In addition, both enzymes required reduced cofactors. We also aimed to purify these two enzymes by means of various chromatographic techniques. We assayed ion exchange chromatography (IEC), pseudo-affinity chromatography, hydrophobic interaction chromatography (HIC) and size exclusion chromatography, and found that the bests suitable were IEC and HIC. By using these two techniques, we achieved a partial purification of each enzyme separately, with a final yield of 10 to 30 % in enzyme activity and a purification factor of 8 to 20.