CHARACTERIZATION OF THE CHOLESTEROL DESATURASE COMPLEX FROM TETRAHYMENA THERMOPHILA

NUSBLAT ALEJANDRO; NUDEL CLARA

Bariloche Argentina

Congreso; SAIB XXXIX Annual Meeting; 2003

SAIB - SAB

CHARACTERIZATION OF THE CHOLESTEROL DESATURASE COMPLEX FROM TETRAHYMENA THERMOPHILA Nusblat Alejandro and Nudel Clara Cátedra de Microbiología  Industrial y Biotecnología, FFyB, UBA. Buenos Aires, Argentina. E- mail: anusblat@ffyb.uba.ar   Two cholesterol desaturase activities that convert cholesterol into D7 and D22 dehydrocholesterol were isolated in Tetrahymena microsomal fractions. Both desaturases react on cholesterol forming the double unsaturated derivative 7,22 bisdehidrocholesterol as the major product in live cultures. We have characterized both enzyme activities present in the microsomal fractions with respect to substrates, cofactors and lipids requirements. From the saturation curve for cholesterol, we have calculated a Km = 30 uM and Vmax = 0.087 nmol/min for delta 7. The stability of the desaturase was assayed with respect to temperature and pH. Optimal conditions were 30º C and pH 7.2-7.4. Both enzymes have an absolute requirement for oxygen and  reduced pyridine nucleotides and are inhibited by cyanide. The microsomal fraction also displays NADH-Cytochrome b5 reductase activity and contains cytochrome b5, both required for the enzymatic activities. The desaturases were solubilized from microsomal membranes by means of a detergent and their activities reconstituted in artificial liposomes. Similar properties with respect to cofactors, oxygen and cytochrome dependence has been observed in 4-methyl sterol oxidase and D5 sterol desaturase in plants, mammals and yeast. We assume therefore that the desaturase complex from Tetrahymena is microsome-bound and consists sequentially of NADH-Cytochrome b5 reductase, cytochrome b5 and the final oxidase.