Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization.
A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B coupled with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-alpha-phosphatidylcholine with [1 beta-3H,4-14C]-androstenedione as substrate. The specific activity of purified aromatase was 65.0 (50.6-74.3) nmol.min-1.(mg of protein)-1 or a turnover rate of 5.0 (4.3-5.9) min-1. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The Km of immunoaffinity-purified aromatase was 12, 210, 41, and 2830 nM for androstenedione, 16 alpha-hydroxyandrostenedione, testosterone, and 16 alpha-hydroxytestosterone, respectively. The very high Km value for 16 alpha-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at -80 degrees C.
Yoshida
N
N
Endocrine Biochemistry Department, Medical Foundation of Buffalo Research Institute, New York 14203.
Osawa
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal; D001141:Aromatase / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002417:Cattle; D002846:Chromatography, Affinity; D003579:Cytochrome Reductases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D001141:Aromatase; D003579:Cytochrome Reductases