Partial Purification and Characterization of Lathosterol 5-Desaturase from Rat Liver Microsomes

• Published in: Journal of biochemistry (Tokyo) Vol. 97; no. 3; pp. 955 - 959
• Main Authors: HONJO, Kyosuke, ISHIBASHI, Teruo, IMAI, Yoh
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.01.1985
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Electron Transport; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; lathosterol 5-desaturase; liver; Male; microsomes; Microsomes, Liver - enzymology; Oxidoreductases; Oxidoreductases - isolation & purification; Oxidoreductases Acting on CH-CH Group Donors; Potassium Cyanide - pharmacology; Rats; Rats, Inbred Strains; Solubility; Steroid; Purification; Rat; Enzyme; Oxidoreductase; Liver; Rodentia; Biosynthesis; Microsome; Metabolism; Cholesterol; Characterization; Vertebrata; Mammalia; Hepatocyte; Lathosterol oxidase; Animal; Catalyst activity
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a135137

The terminal oxidase of the NADH-dependent lathosterol 5-desaturation system was solubilized from rat liver microsomes with 2% Triton X-100, and partially purified approximately 18-fold with 19% yield after DEAE-cellulose and 6-amino-hexyl-Sepharose column chromatography. The final enzyme preparation was free from other electron transfer components and phospholipids in microsomes, and the desaturation reaction was reconstituted with the following components: NADH, molecular oxygen, phospholipids and, three proteins, i.e., NADH-cytochrome b5 reductase, cytochrome b5 and the terminal oxidase. Omission of one of these components led to an almost complete loss of the desaturase activity. Under the reconstitution conditions, the desaturase activity was significantly inhibited by potassium cyanide but was not affected by -SH reagents such as N-ethylmaleimide and dithiothreitol.