Expression of human placental aromatase in Saccharomyces cerevisiae.
A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone. Nucleotide sequence microheterogeneity was found in the 3'-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones. Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase. Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with the two mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing. Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed. The enzyme was expressed in Saccharomyces cerevisiae. The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione. A level of 2 micrograms aromatase/mg partially purified yeast microsomes was estimated by analyses of carbon monoxide difference spectra on microsomal fractions from yeast carrying plasmid pHARK/VGAL. Using [1 beta, 2 beta-3H]androst-4-ene-3,17-dione as the substrate, an apparent Michaels-Menken constant (Km) of 34 nM and a maximum velocity (Vmax) of 23 pmol [3H]water formed per min/mg protein were obtained for the yeast synthesized aromatase by transformation with plasmid pHARK/VGAL. The kinetic results are similar to those determined for human placental aromatase, and suggest that the yeast synthesized aromatase will be useful for further structure-function studies.
Pompon
D
D
Beckman Research Institute of the City of Hope, Division of Immunology, Duarte, California 91010.
Liu
R Y
RY
Besman
M J
MJ
Wang
P L
PL
Shively
J E
JE
Chen
S
S
D000595:Amino Acid Sequence; D001141:Aromatase / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D003579:Cytochrome Reductases / Q000502:physiology; D015342:DNA Probes; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010920:Placenta / Q000201:enzymology*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D015342:DNA Probes; D015345:Oligonucleotide Probes; D003577:Cytochrome P-450 Enzyme System; D001141:Aromatase; D003579:Cytochrome Reductases