Characterization of guinea pig estrogen sulfotransferase expressed by Chinese hamster ovary cell-K1 stable transfectants.
Estrogen sulfotransferase (EST) activity expressed by Chinese hamster ovary (CHO)-K1 cells stably transfected with a plasmid containing a guinea pig EST complementary DNA insert was subjected to biochemical characterization, and the EST protein was further examined by nondenaturing isoelectric focusing and immunoblot analysis. CHO-K1 cells transfected with the same plasmid without the EST complementary DNA insert as well as untransfected CHO-K1 cells did not demonstrate either EST activity or the presence of an immunologically related protein. The EST expressed by the stably transfected CHO-K1 cells was found to manifest Michaelis-Menten kinetics and would use only estrogenic steroids as substrates, whereas other forms of steroids, such as pregnenolone, dehydroepiandrosterone, cortisol, and testosterone, were not acted on. When 17 beta-estradiol was used as a substrate, sulfonation occurred exclusively at the 3 position; 17-sulfonate was not formed. Thus, the expressed EST acted selectively on the 3-hydroxyl group of phenolic steroids. The apparent Km values for estrone, 17 beta-estradiol, and estriol were 60, 70, and 40 nM, respectively. The maximum velocity (Vmax) determinations for estrone and 17 beta-estradiol were equivalent, whereas the Vmax for estriol was reduced by 33%. Of the three estrogens, only 17 beta-estradiol caused substrate inhibition at a high concentration. Steroid sulfonation requires 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the active sulfonate donor, and the Km value for PAPS was 1.2 microM. In steroid sulfotransferase reactions, two products are formed: the sulfonated steroid product and the desulfonated cofactor, 3'-phosphoadenosine-5'-phosphate (PAP). The sulfonation of 17 beta-estradiol was inhibited by PAP in a dose-dependent manner. In addition, the Km for PAPS was increased by PAP, whereas the Vmax was unaffected, indicating competitive inhibition (Ki, approximately 0.52 microM). The EST protein expressed by the CHO-K1 cell stable transfectants demonstrated a mol wt of 34 kilodaltons, as determined by sodium dodecyl sulfate-gel electrophoresis. Additionally, when the expressed EST protein was subjected to isoelectric focusing, it was found to consist of multiple charge isoforms. These findings are comparable to what has been previously reported for native guinea pig adrenocortical EST. Furthermore, the charge isoform pattern that was demonstrated for the expressed EST was similar to the pattern observed for the native protein.
Tomizuka
T
T
Second Department of Internal Medicine, Chiba University School of Medicine, Japan.
Oeda
T
T
Tamura
Y
Y
Yoshida
S
S
Strott
C A
CA
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000031:analogs & derivatives / Q000096:biosynthesis; D006168:Guinea Pigs; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000378:metabolism; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D007527:Isoenzymes; D004958:Estradiol; D000244:Adenosine Diphosphate; C010840:adenosine 3'-phosphate-5'-phosphate; D015238:Sulfotransferases; C020122:estrone sulfotransferase