[Purification and characterization of lathosterol 5-desaturase from rat liver microsomes by cytochrome b5-sepharose affinity column chromatography: evidence for the non-heme iron protein].
Lathosterol 5-desaturase, which catalyzes introduction of a delta 5 bond into lathosterol to form 7-dehydrocholesterol, was purified up to 2000-4000-fold with a 13-18% yield from rat liver microsomes by cytochrome b5-Sepharose affinity chromatography followed by isoelectric focusing. The final enzyme preparation was homogenous as judged by SDS-polyacrylamide gel electrophoresis and a single polypeptide of 65,000 daltons. Furthermore, the two molecular forms with isoelectric points of 6.3 and 9.5 were demonstrated by electrofocusing, though they did not show any significant difference with respect to their enzymatic properties. The desaturase was found to be a non-heme iron protein containing one atom of iron per one molecule of the enzyme. The enzyme activity was inhibited strikingly by iron chelators and cyanide. The decreased enzymatic activity, however, was recovered completely by Fe-ion to the original level, suggesting that the iron was essential for the catalytic activity.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D005260:Female; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D008667:Metalloproteins / Q000032:analysis*; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D019159:Nonheme Iron Proteins; D010088:Oxidoreductases / Q000032:analysis / Q000302:isolation & purification*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D051381:Rats; D012685:Sepharose
D008667:Metalloproteins; D019159:Nonheme Iron Proteins; C033144:lathosterol delta-5-dehydrogenase; D012685:Sepharose; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D044925:Oxidoreductases Acting on CH-CH Group Donors