Assignment of estradiol-17β dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells

Abstract

Homogenates of porcine endometrium contain substantial activity for the dehydrogenation of estradiol-17\g=b\ but little for estrone reduction. Both activities are associated with cytoplasmic structures. The dehydrogenase is characterized by a pH 7.7 optimum, Km 2.2 \m=x\ 10\m=-\7 mol/l for estradiol and Km 4.4 \m=x\10\m=-\5mol/l for the cosubstrate NAD+. The corresponding figures for the reductase are pH 6.6, Km 1.1 \m=x\10\m=-\6mol/1 for estrone and Km 2.1 \m=x\10\m=-\5mol/l for the cosubstrate NADPH. The (mitochondrial/lysosomal) 17 000 \m=x\ g particulate fraction contains a 52-fold higher dehydrogenase than reductase activity. The (microsomal) 200 000 \m=x\ g particulate fraction is only 16-fold richer in dehydrogenase. Isopycnic centrifugations of the two fractions in Percoll gradients reveal that estrone reductase and the coequilibrating marker enzyme cytochrome c reductase occur in constant proportions, whereas the dehydrogenase/cytochrome c reductase ratios are different. Both, the kinetic data and the structural assignments speak in favour of individual enzymes catalyzing the dehydrogenation of estradiol and the reduction of estrone. All gradient fractions exhibiting dehydrogenase activity feature small, electrondense vesicles of 0.15\p=n-\0.20\g=m\min diameter as a common structural element which might harbour the dehydrogenase.Estradiol is metabolized after nuclear passage in porcine endometrium cells by dehydrogenation to estrone (1,2), and subsequent hydroxylation at either 6a-or 7a-(3). Our previous results sug¬ gested a localization of the 17ß-ol dehydrogenase in organdíes resembling lysosomes in size and density, although the pH optimum of the enzyme was in the neutral range. We re-investigated this discrepancy by employing novel techniques for homogenization and organeile separation (4). The ex¬ periments were also thought to clarify whether the prevailing oxidation of estradiol to estrone and the less prominent reversal are catalyzed by one and the same enzyme or by individual entities at differ¬ ent localizations. This might help in exploring the mechanism by which the cell terminates the hor¬ monal stimulus. Materials and MethodsThiamine pyrophosphate was bought from Sigma (Mün¬ chen, FRG); NAD+ and NADPH from Boehringer Mann¬ heim (Mannheim, FRG); Percoll from Pharmacia (Frei¬ burg, FRG); Triton X-100 from Serva (Heidelberg, FRG); xylene-based fluor, estrone, estradiol, estriol and all other grade A chemicals were from E.Merck (Darmstadt, FRG). Tritium-labelled steroids [2,4,6,7-3H]estrone,specific activity 95 Ci/mmol and [6,7-3H] estradiol, specific activity 59 Ci/mmol were from New England Nuclear (Dreieich/FRG) and purified before use. Bond Elut RP-18 (100 mg) solid-phase extraction columns were from Analytichem International (Harbor City, CA); reversed-phase columns LiChrosorb RP-18, RT 250-4, 5 um, and guard columns, RT 4-4, 10 um, were from E.Merck; acetonitrile HPLC-grade was bought from Zinsser (Frankfurt, FRG). Milli-Q H20, Millipore (Eschborn, FRG) was used for HPLC.Endometrium was ...

Molecular cloning and amino acid sequence of the porcine 17β‐estradiol dehydrogenase

“…However, the cDNA-derived amino acid sequence and Edman sequencing of additional peptides of the 80-kDa VHF protein suggest that the latter is a mixture of a 32-kDa EDH/actin complex and of the primary-translation product of EDH-mRNA. Interestingly, the monoclonal antibody W1, which binds to the 32-kDa enzyme but not to its 80-kDa actin complex, does also not attach to the 80-kDa translation product (Adamski et al, 1992).…”

“…The purification of EDH activity from porcine uteri resulted in a homogeneous 32-kDa protein (EDH) and a mixture of 32, 45 and 80kDa proteins (VHF; Adamski et al, 1992). Partial amino acid sequencing and immunotyping with specific antibodies identified the 80-kDa protein of VHF as a covalently linked complex of the 32-kDa enzyme and 45-kDa actin (Adamski et al, 1993b).…”

“…The 32-kDa enzyme and the SO-kDa band of the veryhydrophobic-fraction-(VHF)-containing enzymic activity were purified by conventional chromatography (Adamski et al, 1992) or by immunoadsorption (Adamski et al, 1993b). Protease digestions were performed with either native or electroeluted material or in situ on poly(viny1idene difluoride) membranes (Adamski et al, 1993b).…”

“…The 17P-estradiol dehydrogenases of human and porcine endometrium are associated with cytoplasmic structures (Tseng and Gurpide, 1974;Entenmann et al, 1980). The porcine enzyme performs oxidation 360-fold more efficiently than reduction under optimal conditions (Adamski et al, 1989, Adamski et al, 1992. It was isolated as a 32-kDa protein from endometrial cells (Adamski et al, 1992), which feature specialized enzyme-filled organelles (Adamski et al, 1993a).…”

“…The porcine enzyme performs oxidation 360-fold more efficiently than reduction under optimal conditions (Adamski et al, 1989, Adamski et al, 1992. It was isolated as a 32-kDa protein from endometrial cells (Adamski et al, 1992), which feature specialized enzyme-filled organelles (Adamski et al, 1993a). Some of the porcine 17p-estradiol dehydrogenase (EDH) was found in combination with 45-kDa p-actin forming an 80-kDa covalent EDH-actin complex linked through a y-glutamyl-elysine bond (Adamski et al, 1993b).…”