Comparison of estrogen-derived ortho-quinone and para-quinol concerning induction of oxidative stress

Ortho-quinones formed from catechol estrogens are considered prooxidants due to the production of superoxide radical anions through redox cycling via semiquinones. Para-quinols have been identified as novel metabolites of and as the major products of hydroxyl-radical scavenging by estrogens. Cycling of these compounds has also been discovered, because they are converted back to the parent estrogen via reductive aromatization in vitro and in vivo. We hypothesized that, unlike ortho-quinones, para-quinols do not induce oxidative stress due to this cycling. Like the estrogen itself, the 17β-estradiol-derived para-quinol (10β,17β-dihydroxyestra-1,4-diene-3-one) did not induce oxidative stress, as the rate of hydrogen peroxide production during the incubations of the compounds in various tissue homogenates was not significantly different from that of the control experiments performed without the addition of a test compound. We also confirmed that the estrogen metabolite estra-1,5(10)-dien-3,4,17-trione (estrone 3,4-quinone) was a profound prooxidant due to redox cycling, especially in uterine tissue. Therefore, we concluded that para-quinols do not induce oxidative stress.     

Involvement of dynorphin and the kappa opioid receptor in feeding

Dynorphin-(1–17) produces a highly specific increase in food ingestion. Similar enhancement of food ingestion is found with dynorphin fragments (1–10), (1–11), (1–13) and (3–13) but not with (1–8) and (1–9). Dynorphin B (rimorphin) also enhances food intake. The highly specific kappa agonist U-50,488 also enhances food intake as do a number of other kappa-opiate receptor agonists. These studies provided further support for the role of a highly specific dynorphin-kappa opioid receptor in the modulation of feeding.     

Formation of triton WR 1339-filled rat liver lysosomes : II. Involvement of autophagy and of pre-existing lysosomes

The participation of endocytosis in the formation of Triton-filled lysosomes was followed after injecting Triton WR 1339 simultaneously with colloidal gold or horseradish peroxidase. According to cytomorphometric measurements Triton WR 1339 also significantly enhances autophagy. The analysis of the influence of Triton WR 1339 subfractions shows that autophagy is only augmented by the polymer component (‘macrotriton’) but not by the low molecular component (‘microtriton’) alone. When the latter was injected combined with either macrotriton or colloidal gold particles, autophagy increased to a level observed with Triton WR 1339 and beyond the levels determined for either component alone. Thus, autophagy appears to be stimulated by endocytosis. Autolysosomes are transformed within a few hours to peribiliary ‘dense bodies’. These do not display any significant turnover rate within a period of several days; therefore, dense bodies could be prelabelled with colloidal gold in order to show that they are the target organelles ingesting (macro-)Triton. According to difference spectra autophagy leads to a considerable concentration of microsomal and mitochondria! cytochromes in (macro-)Triton-filled lysosomes far beyond the level detected in ‘normal’ lysosomes. Because of the participation of both hetero- and autophagy in the formation of Triton WR 1339-filled lysosomes they have to be classified as telolysosomes.     

Caspase-independent apoptosis,in human MCF-7c3 breast cancer cells,following photodynamic therapy,with a novel water-soluble phthalocyanine

A new water-soluble phthalocyanine derivative, 2,3,9,10,16,17,23,24-octakis(3-aminopropyloxy) phthalocyaninato zinc II (PoII) was studied as a photosensitizer for photodynamic therapy (PDT) in MCF-7c3 cells. We report here that PoII and red light induces apoptosis. However, the precise mechanism appears to differ from that induced by PDT with other known phthalocyanines. The present study provides evidence that in the case of PoII, caspases do not participate in the apoptotic response. PoII-PDT-treated cells exhibited chromatin condensation and phosphatidylserine (PS) externalization. In the absence of light activation, PoII had no detectable cytotoxic effect. An early event upon PoII-PDT was photodamage to lysosomes, suggesting that they are the primary sites of action. Moreover, the treatment induces Bid activation, mitochondrial swelling and translocation of apoptosis-inducing factor (AIF) to the nucleus. An atypical proteolysis of poly(ADP-ribose) polymerase (PARP) indicative of calpain-like activation was observed. These data support the notion that an alternative mechanism of caspase-independent apoptosis was found in PoII-photosensitized cells.     

Lack of effect of vasoactive intestinal peptide antagonists on blood flow in the rat thyroid

We used three putative vasoactive intestinal peptide (VIP) antagonists: 1) [4Cl-D-Phe⁶,Leu¹⁷]VIP, 2) [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂, and 3) VIP(10–28) to assess the involvement of endogenous VIP in the regulation of thyroid hormone secretion and thyroid blood flow (BF). We measured thyroid BF in ketamine-pentobarbital-anesthetized rats using the microsphere technique. Increases in thyroid BF induced by VIP administration (30 pmol-1.5 nmol/100 g b.wt.) were not affected by any of the three compounds tested at doses 10–100 times higher than that of VIP. These compounds (3–15 nmol/100 g b.wt.) also failed to affect basal thyroid BF or hormone secretion. Increases in pancreatic and salivary gland BFs induced by VIP (30 pmol/100 g b.wt.) were also not affected by [4Cl-D-Phe⁶,Leu¹⁷]VIP or [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂ (3 nmol/100 g b.wt.). These results indicate that the three compounds tested are not effective inhibitors of VIP receptors in the thyroid vasculature and, therefore, they cannot be used in the investigation of the functional significance of endogenous VIP in the regulation of thyroid BF.     

Antigenic complexes of steroid hormones formed by coupling to protein through position 7: Preparation from Δ -3-oxosteroids and characterization of antibodies to testosterone and androstenedione

A general method for rendering Δ³-3-oxosteroids antigenic by coupling to a macromolecule through position 7 is described. It involves nucleophilic attack on the 6, 7-dehydroderivatives of the steroids by ambidentate reagents to form 7-thioether alkanoic acids. These were covalently attached to bovine serum albumin (BSA) by use of the carbodiimide reagent. Addition products with mercapoacetic acid and β-mercaptopropionic acid and their BSA-conjugates, were thus obtained from testosterone androst-4-ene-3, 17-dione, progesterone and 17-hydroxyprogesterone through the respective 4, 6-dienes.

Immunization of rabbits with testosterone-7-carboxymethyl-thioether-BSA and the homologous testosterone-7-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for testosterone (Ka=9. 4×10⁹ 1/mol) that showed little cross-reaction with androstenedione (< 1%) and with a variety of 17-oxoandrostane compounds ( 0.5%). Conversely, immunization with androstenedione-7 -carboxyethyl-thioether-BSA yielded an antiserum with high affinity for androstenedione (Ka = 1. 04 × 10¹⁰ I/mol) but minimal cross-reaction with testosterone (< 0.5%) and 17β-hydroxy-androstane compounds ( 1%). The reaction of anti-testosterone and anti-androstenedione sera with their homologous haptens was not significantly inhibited by the closely related steroids 17- estosterone and dehydroepiandrosterone, or by 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, estrone and estradiol-17β. However, anti-testosterone sera cross-reacted with 5-dihydrotestosterone (40–50%) and to a lesser extent with 5β-dihydrotestosterone (5%). Analogously, the anti-androstenedione sera cross-reacted with 5-dihydroandrostenedione (71%) and to a minor extent with 5β-dihydroandrostenedione (8%).

A radioimmunoassay procedure for the determination of testosterone in plasma is described, which makes use of the new anti-testosterone serum. Preliminary results suggest that it can be applied to ether extracts from human sera without Chromatographic purification.     

Purification and characterization of human lysosomes from EB-virus transformed lymphoblasts

A method was developed for the isolation of unmodified lysosomes of human origin using cultured EB-virus transformed lymphoblasts. The cells were lysed carefully by repeated resuspension in buffered isotonic sucrose. A crude granular fraction derived from this lysate was further purified by isopyknic centrifugation in an isotonic colloidal silica gel gradient and by free-flow electrophoresis. The following relative specific activities (mean ± S.D.) of lysosomal marker enzymes were measured in a pooled lysosomal fraction obtained from the final electrophoresis step (representing less than 0.1% of the initial protein): β-N-acetylglucosaminidase 85.6 ± 15.5; β-galactosidase 87.6 ± 13.4; acid β-glycerophosphatase 41.7 ± 3.5; β-glucuronidase 36.6 ± 6.1. With respect to the final two enzymes the recovery within this pooled fraction was 5–6% of the initial lysate. The great differences in relative specific activities achievable may be due mainly to different extralysosomal portions of the lysosomal marker enzymes, as was found for acid β-glycerophosphatase which was largely distributed within non-lysosomal structures in lymphoblasts when studied by histochemical staining. The final fraction consisted almost exclusively of lysosomes when examined by electron microscopy. Most lysosomes appeared club-shaped immediately after cell lysis and throughout the preparation procedure. Examination by electron microscopy and measurement of the latency of lysosomal enzyme activity revealed an exceptional integrity of the lysosomal membrane. This method provides the opportunity to study highly purified lysosomes from patients with lysosomal disorders.     

Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves Bid cleavage

Photodynamic therapy (PDT) protocols employing lysosomal sensitizers induce apoptosis via a mechanism that causes cytochrome c release prior to loss of mitochondrial membrane potential (DeltaPsi(m)). The current study was designed to determine how lysosomal photodamage initiates mitochondrial-mediated apoptosis in murine hepatoma 1c1c7 cells. Fluorescence microscopy demonstrated that the photosensitizer N-aspartyl chlorin e6 (NPe6) localized to the lysosomes. Irradiation of cultures preloaded with NPe6 induced the rapid destruction of lysosomes, and subsequent cleavage/activation of Bid, pro-caspases-9 and -3. Pro-caspase-8 was not activated. Release of cytochrome c occurred at about the time of Bid cleavage and preceded the loss of DeltaPsi(m). Extracts of purified lysosomes catalyzed the in vitro cleavage of cytosolic Bid, but not pro-caspase-3 activation. Pharmacological inhibition of cathepsin B, L and D activities did not suppress Bid cleavage or pro-caspases-9 and -3 activation. These studies demonstrate that photodamaged lysosomes trigger the mitochondrial apoptotic pathway by releasing proteases that activate Bid.     

A new method for the preparation of rat liver lysosomes. Separation of cell organelles of rat liver by carrier-free continuous electrophoresis

A combination of differential centrifugation and carrier-free continuous electrophoresis is introduced as a new method for the isolation of animal cell organelles. Various buffers were systematically checked in order to find the system which preserves the organelles and gives as well a good separation in the free-flow electrophoresis apparatus. Triethanolamine-acetate buffer (10 mM), pH 7.4 was used. The isolated lysosomes were pure according to marker enzymes and electron micrographs. A heterogeneity of the lysosomes in electrophoretic mobility was demonstrated with respect to the marker enzymes arylsulfatase and β-glucuronidase. The lysosomes with higher mobility showed a maximum enrichment of 240-fold with respect to arylsulfatase. The lysosomes with lower electrophoretic mobility showed a 65-fold enrichment with respect to β-glucuronidase. The ratio of β-glucuronidase to arylsulfatase varied from 2:1 to 1:2 in lysosomes of different mobility. The yield amounted to approximately 1 mg of lysosomal protein per gram of liver protein. 5–8 mg of lysosomes can be obtained in one experiment. The electrophoretic separation proves to be an effective tool in obtaining pure and well preserved lysosomes.     

Biotransformation of 14-deacetoxyl sinenxan A by Ginkgo cell suspension cultures and the cytotoxic activity evaluation

14-Deacetoxyl sinenxan A [2,5,10β-triacetoxy-4(20),11-taxadiene, 1] was converted to two new products, 10β-hydroxy-2,5-diacetoxy-4(20),11-taxadiene (2) and 10β-butyloxy-2,5-diacetoxy-4(20),11-taxadiene (3) both about in 20% yields by Ginkgo cell suspension cultures. Their structures were identified on the basis of their chemical and spectroscopic data. The three compounds (1–3) were preliminarily evaluated for their in vitro cytotoxic activities against two solid tumor cell lines and their drug-resistant counterparts (KB and KB/V, MCF-7 and MCF-7/ADR), and the decreased activities were observed in the case of the two products. The results suggested that biotransformation might be a valuable approach to diversifying natural products and provide some useful information on the study on the structure–activity relationships of the type of compounds.     

A new member of YER057c family in Trypanosoma cruzi is adjacent to an ABC-transporter

Tcp17 is a Trypanosoma cruzi gene located contiguous to the ABC-transporter tcpgp2. The protein contains 160 amino acid residues with a predicted molecular mass of 16.5 kDa. Western blot analysis using a polyclonal antiserum against recombinant TCP17 revealed that the protein is only expressed in the epimastigote form of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. A sequence comparison of TCP17 showed a remarkable homology with a conserved family of prokaryotic and eukaryotic proteins called YER057c whose function has not yet been characterized. Here, we propose a new signature of this family considering the N-terminal: [IV]–X(4)–[AV]–[AP]–X–[AP]–X(3)–Y–X(9)–[LIVF]–X(2)–[SA]–G–[QS], and the C-terminal: [AT]–R–X(2)–[IVFY]–X–[VC]–X(2)–L–P–X(4)–[LIVM]–E–[IVM]–[DE] motifs. Immunofluorescence and immunoelectron microscopy studies suggest that the protein has a wide distribution in the cell, with a higher concentration in the external side of the plasma membrane, on the Golgi complex and on cytoplasmic vacuoles. Although the physiological function of TCP17 is unknown, its conservation in evolution suggests biological relevance in the parasite.     

Immuno-electronmicroscopical localization of a microvillus membrane disaccharidase in the human small-intestinal epithelium with monoclonal antibodies

The cellular localization of the human intestinal disaccharidase, sucrase-isomaltase, was visualized in ultrathin cryosections by the use of specific monoclonal antibodies [25] followed by protein A-gold. The principle site of immunoreaction concerned the microvillus membrane, which supports current concepts of the localization of these hydrolases. One antibody against sucrase-isomaltase also showed labeling of the Golgi apparatus, apical vesicles, and lysosomes, but not of the basolateral membrane. The labeling of the Golgi complex was uniform, suggesting the absence of accumulation of sucrase-isomaltase in cisternae during its passage through this organelle. Absence of labeling of the basolateral membrane appears to support the view that newly synthesized sucrase-isomaltase is transferred directly from the Golgi complex to the microvillus membrane, bypassing the basolateral membrane. However, the results do not exclude the possibility of a very rapid passage through the basolateral membrane. A substantial fraction of the sucrase-isomaltase occurred in lysosomes, which indicates that this organelle plays a major role in the catabolism of microvillar hydrolases. Transport of sucrase-isomaltase to lysosomes might occur by endocytosis or via the crinophagic pathway. The latter was previously postulated to reflect a regulatory mechanism at the post-Golgi level for the surface expression of microvillar membrane proteins.     

Large and prolonged in vivo response of pancreatic secretion to phenylethylamide and phenylethylester derivatives of Boc-[Nle-Nle]CCK(26–33) in the rat

Supramaximal doses of cholecystokinin (CCK) induce in vitro submaximal biological responses (i.e., smaller by 50% than the response to a maximal dose of CCK), desensitization and residual stimulation, and in vivo secretory inhibition and edematous pancreatitis. It has been reported previously that supramaximal doses of Boc-[Nle²⁸-Nle³¹]CCK(27–32)/-phenylethylester (JMV180) do not produce these effects. The aim of this study was to analyze the in vivo response of pancreatic secretion of the rat to a wide dose range of Boc-[Nle²⁸-Nle³¹]CCK(26–33) (JMV118), an analog of CCK8 with the same activity spectrum as CCK8, to JMV180 and to Boc-[Nle²⁸-Nle³¹]CCK(27–32)-phenylethylamide (JMV170). The three peptides were administered as intravenous infusions and as bolus intravenous injections. In the case of infusions, the same maximal effect was observed with all three peptides. It was obtained with 22.5 pmol/kg · min of JMV118; JMV180 and JMV170 were about 700 times less potent. In the case of bolus injections, the maximal response to JMV118 was observed with 450 pmol/kg, and the response peaked 10–15 min after the injection. Higher doses of JMV118 induced a secretory peak that was smaller and delayed relative to the moment of injection. JMV180 and JMV170 were about 500 times less potent: the maximal response was observed with 218700 pmol/kg and peaked 10–15 min after the injection. Larger doses of JMV180 and JMV170 produced neither supramaximal inhibition nor a delayed peak response, but induced a sustained stimulation of pancreatic secretion that could last more than 3 h after the injection. These data indicate that single large doses of JMV180 and JMV170 can produce a large and long-lasting stimulation of pancreatic secretion in vivo, a goal that cannot be reached with JMV118 or CCK8.     

Domain-dependent photodamage to Bcl-2. A membrane anchorage region is needed to form the target of phthalocyanine photosensitization

Photodynamic therapy using the photosensitizer Pc 4 and red light photochemically destroys the antiapoptotic protein Bcl-2 and induces apoptosis. To characterize the requirements for photodamage, we transiently transfected epitope-tagged Bcl-2 deletion mutants into DU-145 cells. Using confocal microscopy and Western blots, wild-type Bcl-2 and mutants with deletions near the N terminus were found in mitochondria, endoplasmic reticulum, and nuclear membranes and were photodamaged. A mutant missing the C terminus, including the transmembrane domain, spread diffusely in cells and was not photodamaged. Bcl-2 missing alpha-helices 5/6 was also not photodamaged. Bcl-2 missing only one of those alpha-helices, with or without substitutions of the singlet oxygen-targeted amino acids, behaved like wild-type Bcl-2 with respect to localization and photodamage. Using green fluorescent protein (GFP)-tagged Bcl-2 or mutants in live cells, no change in either the localization or the intensity of GFP fluorescence was observed in response to Pc 4 photodynamic therapy. Western blot analysis of either GFP- or Xpress-tagged Bcl-2 revealed that the photodynamic therapy-induced disappearance of the Bcl-2 band was accompanied by the appearance of bands indicative of heavily cross-linked Bcl-2 protein. Therefore, the alpha(5)/alpha(6) region of Bcl-2 is required for photodamage and cross-linking, and domain-dependent photodamage to Bcl-2 offers a unique mechanism for activation of apoptosis.     

Subcellular localization of a fluorescent derivative of CuII(atsm) offers insight into the neuroprotective action of CuII(atsm)

Copper complexes of bis(thiosemicarbazone) (Cu(II)(btsc)s) have been studied as potential anti-cancer agents and hypoxia imaging agents. More recently, Cu(II)(btsc)s have been identified as possessing potent neuroprotective properties in cell and animal models of neurodegenerative disease. Despite their broad range of pharmacological activity little is known about how cells traffic Cu(II)(btsc)s and how this relates to potential anti-cancer or neuroprotective outcomes. One method of investigating sub-cellular localization of metal complexes is through confocal fluorescence imaging of the compounds in cells. Previously we harnessed the fluorescence of a pyrene group attached to diacetyl-bis(N4-methylthiosemicarbazonato)copper(ii)) (Cu(II)(atsm)), (Cu(II)L(1)). We demonstrated that Cu(II)L(1) was partially localized to lysosomes in HeLa cancer epithelial cells. Here we extend these studies to map the sub-cellular localization of Cu(II)L(1) in M17 neuroblastoma cells. Treatment of M17 or HeLa cells led to rapid association of the Cu-complex into distinct punctate structures that partially co-localized with lysosomes as assessed by co-localization with Lysotracker and acridine orange. No localization to early or late endosomes, the nucleus or mitochondria was observed. We also found evidence for a limited association of Cu(II)L(1) with autophagic structures, however, this did not account for the majority of the punctate localization of Cu(II)L(1). In addition, Cu(II)L(1) revealed partial localization with ER Tracker and was found to inhibit ER stress induced by tunicamycin. This is the first report to comprehensively characterize the sub-cellular localization of a Cu(II)(atsm) derivative in cells of a neuronal origin and the partial association with lysosome/autophagic structures and the ER may have a potential role in neuroprotection.     

Chemical variation within and between individuals of the lichenized ascomycete Tephromela atra

HPLC-analysis was used to determine the concentrations of the lichen compounds alectoronic acid (depsidon), -collatolic acid (depsidon) and atranorin (depsid) in the lichenized ascomycete Tephromela atra (syn. Lecanon atra) (Hudson) Hafeliner from limestone walls on the Baltic island of Öland, Sweden. In 24 individuals of T. atra sampled on a stone wall, the pre-reproductive and reproductive tissue did not differ in the concentrations of alectoronic acid, collatolic acid and atranorin. The concentrations of the three lichen compounds were inter-correlated in the reproductive tissue, but not in the pre-reproductive tissue. Single individuals of T. atra ranged in area covered from 10.1 to 147.4 cm² (mean: 38.5 cm²; N=24); 38.6% of this area was pre-reproductive tissue. However, the concentrations of the three lichen compounds were correlated neither with the total area covered by the lichen nor with the percentage of pre-reproductive tissue. This suggests that the concentrations of the lichen compounds do not change with increasing size (age) of the lichen. Analysis of specimens of T. atra from eight localities revealed a significant variation in lichen compounds (range between localities: alectoronic acid 0.60–3.26 μg/mg lichen dry weight (DW); collatolic acid 2.14–11.59 μg/mg lichen DW; atranorin 0.58–4.16 μg/mg lichen DW). The level of grazing observed in the lichens differed significantly among localities. However, no correlations between the concentrations of the three lichen compounds and the grazing damage to the lichens were found.     

Prosomatostatin is proteolytically processed at the amino terminal segment by subtilase SKI-1

Processing of prohormones to generate active products typically occurs at basic residues via cleavage by proprotein convertases. A less common type of cleavage is mediated at hydrophobic (L, V, F, N) or small amino acid (A, T, S) residues. Efforts to identify the proteinases responsible for processing precursors at their hydrophobic amino acids has led to the recent cloning of a new type-1 membrane-bound subtilase called SKI-1. The NH₂-terminal region of prosomatostatin, previously shown to contain a sorting signal for the regulated secretory pathways, is processed to generate PSST_[1–10]. The exact cleavage mechanism is unknown, but has been assumed to involve monobasic processing at Lys¹³ followed by carboxypeptidase trimming. We found that K13A mutation did not block PSST_[1–10] production. Since the prosomatostatin sequence R⁸–Q⁹–F¹⁰–L¹¹↓ qualifies as a potential SKI-1 substrate, using a vaccinia virus expression system along with HPLC and radioimmunoassays, we observed that overexpression of recombinant SKI-1 in COS-1 and HEK-293 cells significantly increased the production of PSST_[1–10]. Additionally, in CHO cells lacking SKI-1, there was a significant reduction in PSST_[1–10] production which could be increased upon SKI-1 stimulation. Mutagenesis studies showed that efficient processing of PSST to PSST_[1–10] required the RXRXXL motif. However, this NH₂-terminal cleavage was not a prerequisite for the formation of SST-14 and SST-28.     

Superovulation using recombinant human FSH and ultrasound-guided transabdominal follicular aspiration in baboon (Papio anubis)

The response of baboon females to a modified human ovarian stimulation protocol incorporating start of pituitary suppression in the luteal phase of the cycle with a GnRH agonist (GnRHa) and recombinant human FSH (rhFSH) was studied. A long-acting GnRHa implant supplying goserelin acetate was administered s.c. to six adult female baboons experiencing regular menstrual cycles (33–34 days) on days 22–24 of the cycle. Follicular development was monitored by transabdominal ultrasonography and serum levels of E2 and progesterone (P4) and rhFSH were determined by ELISA. Menses occurred 9–10 days after GnRHa administration. Daily i.m. administration of 75 IU rhFSH commenced 9–10 days after menses and continued for 9–10 days. When most follicles were ≥5 mm diameter and serum E2 had reached its maximum level, 2000 IU hCG was administered i.m. to induce follicle maturation. Transabdominal ultrasound-guided follicular aspiration of follicles ≥2 mm diameter was performed 30–34 h after hCG administration.

One baboon did not show an adequate response to rhFSH stimulation. This animal did not receive further treatment and no data for it are presented. The number of follicles aspirated was 21±4 and 17.2±3.8 oocytes were recovered per animal with an average recovery rate of 82% (86/105). The number of oocytes collected from five animals were 14, 21, 16, 15, and 20 (n=86). Most of the oocytes recovered were in metaphase II and 3 h after recovery 91% (78/86) were considered suitable for in vitro fertilization. It was concluded that recombinant human FSH can successfully induce follicular recruitment and oocyte maturation in baboon females during pituitary suppression with a GnRHa     

The membrane-bound 17β-estradiol dehydrogenase of porcine endometrial cells: purification, characterization and subcellular localization

The membrane-bound 17β-estradiol dehydrogenase of porcine endometrial cells was purified to homogeneity as judged by SDS-PAGE and silver staining of a single 32 kD band. A second, more hydrophobic product of the purification protocol contained additional bands at 45 and 80 kD. The 17β-estradiol dehydrogenase activities of both products exceeded those for 17-one reduction by more than 260-fold. Activities of 3-, 3β- and 20-dehydrogenases were absent in either fraction. Polyclonal and monoclonal antibodies raised against the 32 kD protein and the more hydrophobic product precipitated the enzymatic activity and reacted with the 32 and 80 kD bands, but not with the 45 kD band in Western blots. The subcellular localization of the enzyme was studied in sections of intact cells and of isolated organelles using gold sol coated with F(ab′)₂ fragments of monoclonal antibody F1. Gold particles were found exclusively over cytoplasmic vesicles of 120–150 nm diameter with electron-dense contents.