[Partial purification and characterization of delta 7-sterol 5-desaturase from rat liver microsomes]

The terminal oxygenase of the NADH-depending lathosterol (cholest-7-en-3 beta-ol) 5-desaturase system was partially purified from rat liver microsomes, by Triton X-100 solubilization, DEAE-cellulose column chromatography, and hydrophobic affinity chromatography with Aminohexyl-Sepharose. The terminal oxygenase activity was approximately 18 fold greater than the starting microsome, and the yield was 18.4%, nevertheless, the terminal enzyme activity was almost free from other electron transfer components in microsomes. It was demonstrated that NADH, molecular oxygen, phospholipid, and three enzymes: NADH-cytochrome b5 reductase, cytochrome b5, and the terminal oxygenase, were absolutely essential for lathosterol 5-desaturation in the reconstituted system. Furthermore, the rate of the NADH-depending lathosterol 5-desaturation in the reconstitution system, was proportional to the concentration either of the terminal desaturase, cytochrome b5, or NADH-cytochrome b5 reductase, under conditions in which other enzymes were present in excess.