Purification of oestrogen synthetase by high-performance liquid chromatography. Two membrane-bound enzymes from the human placenta.
Human placental NADPH-cytochrome P-450 reductase, obtained by 2',5'-ADP-Sepharose affinity chromatography, was separated into two reductase-active peaks on a Pharmacia Mono-Q column. By this short, two-step chromatographic procedure, the two reductases were obtained in a homogeneous state with high retention of activity and in over 900-fold purification. Aromatase-reconstituting activity was present only in the higher-molecular-weight reductase (79 000 D), not in the smaller, 70 000 D reductase, which turned out to be a proteolysis product of the former. Both proteins were eluted as a single peak in reversed-phase high-performance liquid chromatography on a Protesil-diphenyl column. Similar results were obtained with bovine hepatic NADPH-cytochrome P-450 reductases. On the other hand, starting from a reductase-free preparation, we have obtained by high-performance ion-exchange chromatography and high-performance size-exclusion chromatography, only partial purification of the aromatase cytochrome P-450, which showed the following values: aromatase activity, 3.995 nmol/min/mg protein (60-fold purification); cytochrome P-450 content, 1.376 nmol/mg protein (23-fold purification); molecular weight, 165 000 D (estimated as an aggregate by size-exclusion chromatography). Although complete purification of the aromatase component has yet to be accomplished, our results suggest that high-performance ion-exchange chromatography on a Mono-Q column is very useful for the purification of acidic, membrane-bound enzymes with good retention of activity.
D000645:Ammonium Sulfate; D001141:Aromatase / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005260:Female; D006801:Humans; D008566:Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D010088:Oxidoreductases / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology* / Q000648:ultrastructure; D011247:Pregnancy
D010088:Oxidoreductases; D001141:Aromatase; D009251:NADPH-Ferrihemoprotein Reductase; D000645:Ammonium Sulfate