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PubMed1999

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase.

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.

Animals↗
PubMed1984

Oxidation of lactaldehyde by cytosolic aldehyde dehydrogenase and inhibition of cytosolic and mitochondrial aldehyde dehydrogenase by metabolites.

An enzyme fraction which oxidizes lactaldehyde to lactic acid has been purified from goat liver. This enzyme was found to be identical with the cytosolic aldehyde dehydrogenase. Lactaldehyde was found to be primarily oxidized by this enzyme. Almost 90% of the total lactaldehyde-oxidizing activity is located in the cytosol. Methylglyoxal and glyceraldehyde 3-phosphate were found to be strong competitive inhibitors of this enzyme. Aldehyde dehydrogenase from goat liver mitochondria has also been partially purified and found to be strongly inhibited by these metabolites. The inhibitory effects of these metabolites on both these enzymes are highly pH dependent. The inhibitory effects of both the metabolites have been found to be stronger for the cytosolic enzyme at pH values higher than the physiological pH. For the mitochondrial enzyme, the inhibition with methylglyoxal was more pronounced at higher pH values, whereas stronger inhibition was observed with glyceraldehyde 3-phosphate at physiological pH.

Aldehyde Dehydrogenase↗
PubMed2003

Replacement of a cytosolic copper/zinc superoxide dismutase by a novel cytosolic manganese superoxide dismutase in crustaceans that use copper (haemocyanin) for oxygen transport.

The blue crab, Callinectes sapidus, which uses the copper-dependent protein haemocyanin for oxygen transport, lacks the ubiquitous cytosolic copper-dependent enzyme copper/zinc superoxide dismutase (Cu,ZnSOD) as evidenced by undetectable levels of Cu,ZnSOD activity, protein and mRNA in the hepatopancreas (the site of haemocyanin synthesis) and gills. Instead, the crab has an unusual cytosolic manganese SOD (cytMnSOD), which is retained in the cytosol, because it lacks a mitochondrial transit peptide. A second familiar MnSOD is present in the mitochondria (mtMnSOD). This unique phenomenon occurs in all Crustacea that use haemocyanin for oxygen transport. Molecular phylogeny analysis suggests the MnSOD gene duplication is as old as the origin of the arthropod phylum. cytMnSOD activity in the hepatopancreas changes during the moulting cycle of the crab. Activity is high in intermoult crabs and non-detectable in postmoult papershell crabs. mtMnSOD is present in all stages of the moulting cycle. Despite the lack of cytCu,ZnSOD, crabs have an extracellular Cu,ZnSOD (ecCu,ZnSOD) that is produced by haemocytes, and is part of a large, approx. 160 kDa, covalently-linked protein complex. ecCu,ZnSOD is absent from the hepatopancreas of intermoult crabs, but appears in this tissue at premoult. However, no ecCu,ZnSOD mRNA can be detected, suggesting that the protein is recruited from the haemolymph. Screening of different taxa of the arthropod phylum for Cu,ZnSOD activity shows that those crustaceans that use haemoglobin for oxygen transport have retained cytCu,ZnSOD. It appears, therefore, that the replacement of cytCu,ZnSOD with cytMnSOD is part of an adaptive response to the dynamic, haemocyanin-linked, fluctuations in copper metabolism that occur during the moulting cycle of the crab.

Amino Acid Sequence↗
PubMed1988

Effects of lowering extracellular and cytosolic pH on calcium fluxes, cytosolic calcium levels, and transmitter release in presynaptic nerve terminals isolated from rat brain.

We examined the effects of extracellular and intracellular pH changes on the influx of radioactive 45Ca, the concentration of ionized Ca (pCai) as monitored with the Ca-sensitive fluorescent indicator fura-2, and the efflux of dopamine in presynaptic nerve endings (synaptosomes) isolated from rat brain corpora striata and preloaded with [3H]dopamine. Cytosolic pH (pHi) was monitored by loading the synaptosomes with the H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (see Nachshen, D. A., and P. Drapeau, 1988, Journal of General Physiology, 91:289-303). An abrupt decrease of the pH of the external medium, from 7.4 to 5.5, produced a slow decrease of pHi (over a 5-min period) from an initial value of 7.2 to a steady state level of approximately 5.8. When 20 mM acetate was present in acidic media, pHi dropped as fast as could be measured (within 2 s) to a level similar to that reached (more slowly) in the absence of acetate. It was therefore possible to lower pHi over short time periods to different levels depending on whether or not acetate was present upon extracellular acidification. Extracellular acidification to pH 5.5 (in the absence of acetate) had no significant effect on pCai and dopamine release over a 30-s period (pHi = 6.4). Acidification in the presence of acetate lowered pHi to 5.8 without affecting pCai, but dopamine efflux increased approximately 20-fold. This increase in basal dopamine release was also observed in the absence of extracellular Ca. Thus, intraterminal, but not extracellular, acidification could stimulate the efflux of dopamine in a Ca-independent manner. The high Q10 (3.6) of acid-stimulated dopamine efflux in the presence of nomifensine (which blocks the dopamine carrier) was consistent with an activation of vesicular dopamine release by H+. When synaptosomes were both depolarized for 2 s in high-K (77.5 mM) solutions and acidified (in the absence of acetate), there was a parallel block of 45Ca entry and evoked dopamine release (50% block at pH 6.0 with 0.2 mM external Ca). When acetate was included in the acidic media to further reduce pHi, Ca entry remained blocked, but evoked dopamine release was increased. Therefore, extracellular, but not cytosolic, acidification inhibited the release of dopamine by blocking voltage-gated Ca channels. The stimulation by cytosolic acidification of both basal and evoked dopamine release suggests that vesicular release in resting and depolarized synaptosomes was directly activated by cytoplasmic H+.

Animals↗
PubMed1993

The cytosolic acidification in rat parotid cells is associated with an increase in cytosolic Ca2+ concentration.

A transient decrease in cytosolic pH ([pH]i) in rat parotid cells was evoked by the addition of carbachol (CCh), phenylephrine, or substance P, whereas isoproterenol and dibutyryl cyclic AMP had little or no effect on [pH]i. The decrease in [pH]i induced by the Ca(2+)-mobilizing agonists was also observed in Ca(2+)-free medium, but not when the intracellular Ca2+ stores were previously depleted. Ionomycin and thapsigargin elicited a decrease in [pH]i with an increase in cytosolic Ca2+ concentration ([Ca2+]i). The protein kinase C activator and inhibitor had no effect on the agonist-induced decrease in [pH]i. These results suggest that the cytosolic acidification is associated with an increase in [Ca2+]i.

Animals↗
PubMed1989

Changes in cytosolic 3,5,3'-tri-iodo-L-thyronine (T3) binding activity during administration of L-thyroxine to thyroidectomized rats: cytosolic T3-binding protein and its activator act as intracellular regulators for nuclear T3 binding.

Changes in the amount of cytosolic 3,5,3'-tri-iodo-L-thyronine (T3)-binding protein (CTBP) and its activator during administration of L-thyroxine (T4) to thyroidectomized rats were investigated. Thyroidectomy decreased the amount of CTBP in the kidney, whereas the activator was not significantly modified by thyroidectomy. The activator was increased by administration of T4 to thyroidectomized rats. The amount of CTBP was also increased by administration of T4. The activator increased the maximal binding capacity (MBC) without changes in the affinity constant for T3 binding in CTBP. A T4-induced increase in MBC in cytosol inhibited nuclear T3 binding in vitro by competition of T3 binding between CTBP and the nuclear receptor. These results suggest that thyroid hormone increases the capacity for cytosolic T3 binding through increasing the amount of CTBP and its activator, and that these increases play a role in regulating the amount of T3 that binds to its nuclear receptor.

Animals↗
PubMed1995

Callose synthesis in spirostanol treated carrot cells is not triggered by cytosolic calcium, cytosolic pH or membrane potential changes.

Carrot (Daucus carota L.) cell suspensions were treated with a spirostanol saponin from Yucca. This saponin is an elicitor of callose synthesis. Irrespectively of the mode of action of spirostanol on the callose synthase activity itself, the spirostanol-induced callose synthesis in carrot is not preceded by changes in membrane potential, cytosolic free calcium or cytosolic pH. The inability of modulators of cytosolic free calcium content (verapamil, nifedipine and Br-A23187), EGTA and a proton pump inhibitor (vandate) to inhibit or induce callose formation is consistent with a calcium- and pH-independent mechanism for callose deposition.

Calcium↗
PubMed1994

Copurification of cytosolic fructose-1,6-bisphosphatase and cytosolic aldolase from endosperm of germinating castor oil seeds.

The cytosolic isozymes of fructose-1,6-bisphosphatase (FBPasec) and aldolase (ALDc) from germinating castor oil seed endosperm (COS) (Ricinus communis L.; cv Hale) were purified to homogeneity and final specific activities 49 and 2.8 (mumol product produced/min)/mg protein, respectively. Nondenaturing polyacrylamide gel electrophoresis of the final FBPasec preparation resolved a single protein-staining band which comigrated with FBPase activity. Two protein-staining bands of 41 and 39 kDa that occurred in an approximate 1:1 ratio were observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final FBPasec preparation. Rabbit anti-(FBPasec) immune serum immunoprecipitated the activities of FBPasec, but not that of the plastidic isozyme of FBPase from germinated COS. Immunoblot analysis utilizing affinity purified anti-(COS FBPasec) immunoglobulin G established that the 39-kDa subunit of FBP-asec did not arise via proteolytic cleavage of the 41-kDa subunit during tissue extraction and enzyme purification. However, FBPasec was susceptible to degradation by endogenous protease(s) during incubation of an acidic (pH 5.9) clarified COS extract at 25 degrees C. This proteolysis caused the production of a 32-kDa antigenic polypeptide and resulted in FBPase inactivation. Gel filtration indicated that purified FBPasec exists in at least 8 different oligomeric forms ranging in size from > 2 million to < 34 kDa. The majority of FBPasec, however, eluted as a 143-kDa heterotetramer. Sodium dodecyl sulfate gel electrophoresis of the final ALDc preparation yielded a single 40-kDa protein-staining polypeptide that cross-reacted with anti-(carrot ALDc) IgG. FBPasec copurified with ALDc through polyethylene glycol fractionation, Q-Sepharose, and phosphocellulose chromatographies, and the intensity of the fluorescence emission spectrum of ALDc was greatly reduced in the presence of COS FBPasec, but not rabbit muscle FBPase. These findings suggest that these two metabolically sequential enzymes might specifically interact in the cytosol of the highly gluconeogenic germinating COS. Our results also demonstrate that endogenous nonspecific acid phosphatase activity can interfere with the spectrophotometric assay for FBPase and can thus result in overestimations of FBPase activity in impure plant extracts.

Ricinus communis↗
PubMed1988

Identification and characterization of a cytosolic 30 kDa histamine stimulated phosphoprotein in parietal cell cytosol.

Histamine stimulated acid secretion is mediated by an increase in intracellular cAMP. Cytosolic protein phosphorylation stimulated by histamine was investigated in isolated rabbit parietal cells. Histamine stimulated the phosphorylation of a 30 kDa phosphoprotein with an isoelectric point of 5.6. Cimetidine completely inhibited histamine-stimulated pp30 phosphorylation. However, omeprazole had no effect on the phosphorylation of pp30. Forskolin and 8-bromo-cAMP also stimulated the phosphorylation of pp30. The results suggest that pp30 is a histamine-stimulated, cAMP-dependently phosphorylated protein substrate in parietal cell cytosol.

Animals↗
PubMed1993

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. The cytosolic enzyme has a novel 3 alpha/17 beta-hydroxysteroid dehydrogenase activity.

1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively. 2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low Km values. 3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase. 4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

17-Hydroxysteroid Dehydrogenases↗
PubMed1990

Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+.

A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.

Aniline Compounds↗
PubMed1997

Dynamic compartmentation of vacuolar amino acids in Penicillium cyclopium. Cytosolic adenylates act as a control signal for efflux into the cytosol.

The regulation of amino acid transport from the vacuolar reservoir into the cytoplasm has been studied in hyphal cells of Penicillium cyclopium. To avoid artifacts caused by the isolation of vacuoles, efflux was examined "in situ," i.e. in cells whose plasma membranes were permeabilized for micromolecules by a treatment with nystatin. The ATP-dependent proton gradient and amino acid transport activities at the vacuolar membrane remained intact under these conditions. Accumulation of amino acids in the vacuole proved to be the result of a dynamic equilibrium of active, ATP-dependent uptake and energy-independent efflux. The latter was strongly accelerated after the vacuolar amino acid content had surpassed a threshold level. Efflux of vacuolar amino acids was specifically controlled by extravacuolar adenylates: ATP, 5'-adenylyl imidodiphosphate (an ATPase-resistant ATP-analogue), ADP, or AMP caused a strong inhibition in the concentration range around 200 micromol/liter, whereas both lower and higher concentrations allowed significant efflux rates. Estimates of the cytosolic adenylates (which consisted mainly of ATP) were close to 2 mmol/liter in glucose-metabolizing cells, which concentration allowed maximum rates of both vacuolar uptake and efflux. During 24 h of carbon and nitrogen starvation, the adenylate level decreased toward the efflux-inhibiting region around 200 micromol/liter, whereas 3-4 d of carbon and nitrogen starvation caused a further decline of the adenylate content, leading again to efflux-permitting concentrations. Thus, the cytosolic adenylate pool appears to effectively control the availability of vacuolar amino acids for the cellular metabolism.

Adenine Nucleotides↗
PubMed2003

The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex.

In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.

Benzoquinones↗
PubMed1992

Changes of cytosolic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2.

In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are much less influenced by changes in extracellular Mg2+ (Mg2+o). To evaluate interference with measurement of cytosolic Mg2+ (Mg2+i) by changes in Ca2+i, we determined the dissociation constant (Kd) and Hill coefficient (NH) of the Ca(2+)-mag-fura-2 and Mg(2+)-mag-fura-2 complexes in standard solutions, in intact acini after loading with the acetoxymethyl ester of mag-fura-2 (mag-fura-2/AM), and in lysates derived from acini loaded with mag-fura-2/AM. The Kd of the Ca(2+)-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20 microM, and NH was 1.44 +/- 0.16 (n = 24). Kd of the Mg(2+)-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and NH was 1.20 +/- 0.20 (n = 25). Mean Kd values were slightly lower in acinar lysates and in solutions of standard mag-fura-2. In acini from either gland, 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (BAPTA) suppressed carbachol-induced Ca2+i transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg2+o was increased from less than 10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg2+i during exposure to carbachol. Under these conditions, pancreatic acinar Mg2+i is 0.53 +/- 0.14 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Silica induces changes in cytosolic free calcium, cytosolic pH, and plasma membrane potential in bovine alveolar macrophages.

The mineral-dust induced activation of pulmonary phagocytes is thought to be involved in the induction of severe lung diseases. The activation of bovine alveolar macrophages (BAM) by silica was investigated by flow cytometry. Short-term incubation (< 10 min) of BAM with silica gel and quartz dust particles induced increases in the cytosolic free calcium concentration ([Ca2+]i), decreases in intracellular pH (pHi), and increases in plasma membrane potential (PMP). The extent of these changes was concentration dependent, related to the type of dust and was due to Ca2+ influx from the extracellular medium. An increase in [Ca2+]i was inhibited, when extracellular Ca2+ was removed. Furthermore the calcium signal was quenched by Mn2+ and diminished by the calcium channel blocker verapamil. The protein kinase C specific inhibitor bisindolylmaleimide II (GF 109,203 X) did not inhibit the silica-induced [Ca2+]i rise. In contrast, silica-induced cytosolic acidification and depolarization were inhibited by GF 109,203 X but not by removal of extracellular calcium. Addition of TiO2 particles or heavy metal-containing dusts had no effect on any of the three parameters. Our data suggest the existence of silica-activated transmembrane ion exchange mechanisms in BAM, which might be involved in the specific cytotoxicity of silica by Ca(2+)-dependent and independent pathways.

Animals↗
PubMed2006

The C-terminal flavin domain of gp91phox bound to plasma membranes of granulocyte-like X-CGD PLB-985 cells is sufficient to anchor cytosolic oxidase components and support NADPH oxidase-associated diaphorase activity independent of cytosolic phospholipase A2 regulation.

We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient PLB-985 cells and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and NADPH-dependent diaphorase activity. The present study focuses on the C-terminal cytoplasmic domain of gp91(phox) (residues 283-570), which contains the NADPH binding and flavin adenine dinucleotide-reducing center, to determine if this portion is regulated by AA. The gp91(phox) C-terminal reductase domain was expressed in X-CGD PLB-985 cells lacking normal gp91(phox) (X-CGD PLB 91CT cells) and was detected in the plasma membrane. It appears to be bound electrostatically to the plasma membrane, as it is eluted by high salt. Permeabilized, granulocyte-like X-CGD PLB 91CT cells lacking cPLA(2) protein and activity, as well as AA release after stimulation, supported NADPH-dependent diaphorase activity after stimulation, similar to granulocyte-like X-CGD PLB 91CT cells. Normal translocation of p47(phox) and p67(phox) to the membrane fractions of both stimulated cell types indicated that the gp91(phox) C-terminal region is sufficient to anchor the cytosolic oxidase components to the membranes. cPLA(2) translocated to membranes and bound the assembled oxidase in granulocyte-like X-CGD PLB 91CT cells after stimulation. Therefore, the assembled membrane-bound oxidase complex encompassing the flavin domain of gp91(phox) provides a docking site for cPLA(2) but is not the site of AA-based regulation of oxidase activity.

Binding Sites↗
PubMed1983

Analysis of binding of [3H]Estradiol to the cytosol fraction of rat pancreas: comparison with sites in the cytosol of uterus.

The cytosol fraction of rat pancreas [100,000 X g (for 1 h) supernatant] demonstrated specific but nonsaturable binding of [3H]estradiol in the concentration range of 2-50 X 10(-9) M. Scatchard analysis of specifically bound [3H]estradiol, determined by the isotope dilution technique (competition with excess unlabeled estradiol), indicated a single class of binding sites (approximately 4.4 pmol/mg protein) with an apparent Kd of 5 X 10(-8) M. Such cytosol fractions, prepared from homogenates that contained protease inhibitors, when prelabeled with [3H]estradiol, demonstrated a single sharp eluate peak of radioactivity after Sephadex G-200 chromatography that corresponded to a molecular weight of 120,000. When protease inhibitors were omitted, [3H]estradiol was associated with material of considerably lower molecular weight. Routinely, the protease inhibitors leupeptin (1 mM), phenylsulfonyl fluoride (0.5 mM), and tosylphenylalanylchloromethyl ketone (0.05 mM) were included in the buffer used for homogenizing both the pancreas and uterus. The protein that binds [3H]estradiol in uterus differed from that in pancreas in a number of ways: 1) in the range of 10-20 X 10(-9) M [3H]estradiol, specific binding of the hormone to uterine sites was saturable, and Scatchard analysis indicated a single class of binding sites, (approximately 0.5 pmol/mg protein) having an apparent Kd of 3 X 10(-10) M; 2) the rate constants of dissociation of the [3H] estradiol-bound complexes in pancreas and uterus were 3.1 X 10(-4) sec-1 (t1/2, 37 min) and 8.7 X 10(-5) sec-1 (t1/2, 134 min), respectively; 3) the molecular weight of the estrogen-binding protein in freshly prepared uterine supernatant fractions appeared to be at least 240,000; this was unaltered regardless of whether protease inhibitors were present during initial homogenization of the tissue; and 4) when uterine supernatants prepared in the absence of protease inhibitors were kept at 8 C for 24 h and then analyzed by Sephadex G-200 chromatography, a second peak of [3H]estradiol-binding activity appeared at the same eluate volume as the low molecular weight binding fractions of pancreas. These data suggest that although the binding proteins in pancreas and uterus are different, there may be some common features at the hormone-binding locus.

Animals↗
PubMed2003

Glucocorticoids induce cytosolic phospholipase A2 and prostaglandin H synthase type 2 but not microsomal prostaglandin E synthase (PGES) and cytosolic PGES expression in cultured primary human amnion cells.

This study examines the regulation of major enzymes in prostaglandin E(2) (PGE(2)) synthesis by glucocorticoids in separate cultures of human amnion epithelial and fibroblast cells at term. Cytosolic phospholipase A(2) (cPLA(2)), cytosolic PGES (cPGES), and microsomal PGES (mPGES) mRNA were expressed at similar levels in both cell types, whereas a greater prostaglandin H synthase type 2 (PGHS-2) mRNA expression was observed in amnion fibroblasts than in epithelial cells. Amnion fibroblasts produced 50-fold more PGE(2) per cell than epithelial cells. Dexamethasone (0.01-1 microM) increased PGE(2) production in amnion fibroblasts in a concentration-dependent manner but did not affect PGE(2) production in amnion epithelial cells. Both mRNA and protein expression of cPLA(2) and PGHS-2 but not cPGES and mPGES were increased in a dose-dependent manner by dexamethasone (0.01-1 microM) in amnion fibroblasts. Induction of cPLA(2) and PGHS-2 mRNA by dexamethasone was blocked by RU486. Dexamethasone did not affect PGHS-2, cPGES, and mPGES mRNA expression in amnion epithelial cells. In conclusion, amnion fibroblasts express a higher level of PGHS-2 mRNA and produced more PGE(2) per cell than amnion epithelial cells at term of human pregnancy. Glucocorticoids increase PGE(2) production only in the amnion fibroblasts mainly through induction of cPLA(2) and PGHS-2 expression.

Amnion↗