Functional characterization of 102-amino acid-deleted form of human aromatase (delta102-aromatase).
A truncate form of human aromatase cDNA that corresponds to the recently identified rat cortical type aromatase mRNA variant (Yamada-Mouri et al., J. Steroid Biochem. Molec. Biol., 60: 325-329, 1997) has been generated, and the amino-terminus deleted form of the enzyme has been expressed in CHO cells. The resulting product lacking 102 residues from the N-terminus of aromatase (i.e. 102-aromatase) showed an extremely low enzyme activity using an 'In-cell' assay. A strong aromatase activity, however, was observed for the delta102-aromatase using an in vitro method on the solublized preparations. The in vitro activity was dependent on both incubation time and NADPH concentration as well as inclusion of NADPH-cytochrome P450 reductase in the assay mixture. The average turnover rate of aromatization of the reconstituted delta102-aromatase was 6.8 min(-1). The results of the immunosuppression assay suggested that delta102-aromatase still holds the epitope interactive to MAb3-2C2, a monoclonal antibody raised agaist human placental aromatase P450. Furthermore, the IC50 values of MAb3-2C2 were determined to be 24 and 23 microg/ml for the whole homogenate and the 105,000 x g precipitate fractions prepared from the truncated aromatase expressing cells, respectively, whereas an IC50 of 1.3 microg/ml was shown for the full-length human aromatase. These results indicate that the delta102-aromatase P450 can be expressed and is catalytically competent as the full-length enzyme, but the epitope structure for the monoclonal antibody MAb3-2C2 is altered from that of the native enzyme. In addition, the intracellular distribution of delta102-aromatase may be different from that of the wild-type enzyme, explaining why very low activity was measured using an 'In-cell' assay.
Kao
Y C
YC
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Higashiyama
T
T
Yarborough
C
C
Osawa
Y
Y
Chen
S
S
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001141:Aromatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006801:Humans; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D001141:Aromatase