Characterisation and properties of cholesterol desaturases from the ciliate Tetrahymena thermophila

Abstract

Live Tetrahymena thermophila transforms exogenous cholesterol into 7,22-bis, dehydrocholesterol (DHC) by desaturation at positions C7(8) and C22(23) of the cholesterol moiety. In this first report on expression, isolation, characterization, and reconstitution of at positions C7(8) and C22(23) of the cholesterol moiety. In this first report on expression, isolation, characterization, and reconstitution of Tetrahymena thermophila transforms exogenous cholesterol into 7,22-bis, dehydrocholesterol (DHC) by desaturation at positions C7(8) and C22(23) of the cholesterol moiety. In this first report on expression, isolation, characterization, and reconstitution of Tetrahymena’s cholesterol desaturases in cell-free extracts, we describe conditions for increasing the expression of both desaturases based on the addition of specific sterols to the culture medium. Reactions performed in vitro, with isolated microsomes, yield only the monounsaturated derivatives, 7-DHC and/or 22-DHC. However, selectivity towards one product can be improved with the addition of specific compounds: b-mercaptoethanol inhibited C22(23) desaturase activity completely, while ethanol selectively increased this activity. Detergent- solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroylphosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b5. NADH Q1 or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroylphosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b5. NADH Q1 or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. on the addition of specific sterols to the culture medium. Reactions performed in vitro, with isolated microsomes, yield only the monounsaturated derivatives, 7-DHC and/or 22-DHC. However, selectivity towards one product can be improved with the addition of specific compounds: b-mercaptoethanol inhibited C22(23) desaturase activity completely, while ethanol selectively increased this activity. Detergent- solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroylphosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b5. NADH Q1 or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroylphosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b5. NADH Q1 or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. s cholesterol desaturases in cell-free extracts, we describe conditions for increasing the expression of both desaturases based on the addition of specific sterols to the culture medium. Reactions performed in vitro, with isolated microsomes, yield only the monounsaturated derivatives, 7-DHC and/or 22-DHC. However, selectivity towards one product can be improved with the addition of specific compounds: b-mercaptoethanol inhibited C22(23) desaturase activity completely, while ethanol selectively increased this activity. Detergent- solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroylphosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b5. NADH Q1 or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroylphosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b5. NADH Q1 or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. b-mercaptoethanol inhibited C22(23) desaturase activity completely, while ethanol selectively increased this activity. Detergent- solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroylphosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b5. NADH Q1 or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions. b5. NADH Q1 or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b5 in these reactions.b5 in these reactions.