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1.
Some compounds derived from plants have been known to possess estrogenic properties and can thus alter the physiology of higher organisms. Genistein and daidzin are examples of these phytoestrogens, which have recently been the subject of extensive research. In this study, genistein and daidzin were found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 μM by binding to the estrogen receptor (ER). Results have shown that this enhancement was effectively blocked by the known estrogen receptor antagonist tamoxifen, indicating the involvement of the ER in AChE induction. That genistein and daidzin are estrogenic were confirmed in a cell proliferation assay using the human breast cancer cell line MCF7. This proliferation was also blocked by tamoxifen, again indicating the involvement of the ER. On the other hand, incubating the PC12 cells in increasing concentrations of 17 β-estradiol (E2) did not lead to enhanced AChE activity, even in the presence of genistein or daidzin. This suggests that mere binding of an estrogenic compound to the ER does not necessarily lead to enhanced AChE activity. Moreover, the effect of the phytoestrogens on AChE activity cannot be expressed in the presence of E2 since they either could not compete with the natural ligand in binding to the ER or that E2 down-regulates its own receptor. This study clearly suggests that genistein and daidzin enhance AChE activityin PC12 cells by binding to the ER; however, the actual mechanism of enhancement is not known. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

2.
The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.  相似文献   

3.
Acetylcholinesterase (AChE) in the clonal NG108-15 cell line has been previously characterized. This cell line represents an in vitro system to study AChE regulation and effects of chemical compounds that may alter AChE activity. Recently, glycyl-L-glutamine (GLG) was demonstrated to function as a neurotrophic factor for maintenance of AChE content in cat denervated superior cervical ganglion cells. In the present study, regeneration of AChE activity in cultures of undifferentiated NG108-15 cells after soman inhibition was investigated in the presence and absence of GLG. Cells were treated with soman (5.5 × 10–6 M) for 15 min and then washed to remove excess soman. Culture medium containing either GLG (10–6, 10–5, or 10–4M) or glycyl-L-glutamic acid (10–6 M) was added to cultures after soman treatment and remained in the medium until cell harvest. Cells were physically detached at various times after soman treatment and specific AChE activity was determined. After soman, AChE activity dramatically decreased to less than 1% of untreated cellular activity at 1 hr. AChE activity gradully increased after 5 hr, while untreated cell AChE activity was regained 20 hr after soman. The t1/2 for AChE regeneration was approximately 10 hr. GLG did not increase the rate of AChE regeneration after soman inhibition. These results indicate that GLG is not a directly acting neurotrophic factor for AChE synthesis in NG108-15 cells after chemical AChE inactivation.Abbreviations AChE acetylcholinesterase - NG108-15 cell neuroblastoma-glioma 108-15 cell - DMEM Dulbecco's modified Eagles minimal essential medium - FBS fetal bovine serum - GLGA glycyl-L-glutamic acid - L-GA L-glutamic acid - GLG glycyl-L-glutamine - GD soman The opinions or assertions contained herein are the private views of the authors and are not to be construed as reflecting the view of the Department of the Army or the Department of the Army or the Department of Defense.  相似文献   

4.
To obtain information about the evolution of acetylcholinesterase (AChE), we undertook a study of the enzyme from the skeletal muscle of the lamprey Petromyzon marinus, a primitive vertebrate. We found that the cholinesterase activity of lamprey muscle is due to AChE, not pseudocholinesterase; the enzyme was inhibited by 1,5-bis(4-allyldimethylammonium phenyl) pentane-3-one (BW284C51), but not by tetramonoisopropyl pyrophosphortetramide (iso-OMPA) or ethopropazine. Also, the enzyme had a high affinity for acetylthiocholine and was inhibited by high concentrations of substrate. A large fraction of the AChE was found to be glycoprotein, since it was precipitated by concanavalin A-agarose. Optimal extraction of AChE was obtained in a high-salt detergent-containing buffer; fractional amounts of enzyme were extracted in buffers lacking salt and/or detergent. These data suggest that globular and asymmetric forms of AChE are present. On sucrose gradients, enzyme that was extracted in high-salt detergent-containing buffer sedimented as a broad peak of activity corresponding to G4; additionally, there was usually a peak corresponding to A12. Sequential extraction of AChE in conjunction with velocity sedimentation resolved minor forms of AChE and revealed that the G1, G2, G4, A4, A8, and A12 forms of AChE could be obtained from the muscle. The identity of the forms was confirmed through high-salt precipitation and collagenase digestion. The asymmetric forms of AChE were precipitated in low ionic strength buffer, and their sedimentation coefficients were shifted to higher values by collagenase digestion.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
The activity of acetylcholinesterase (AChE) isolated from coleoptiles of etiolated oat seedlings is strongly inhibited by neostigmine and less so by eserine. The optimum of the enzyme activity occurs at pH 7.2 and a temperature of + 36 °C. The enzyme Michaelis constant is 280 μM. Choline within the range of concentration from 0.001 to 10 mM does not affect the enzyme activity. Calcium ions at 5 mM concentration cause inhibition, while magnesium and manganese ions do not affect the enzyme activity. AChE isolated from oat seedlings differs in a number of properties from AChE occurring in the tissues of other plants. This research was supported in part by grant CPBP 05.02.4.07.  相似文献   

6.
Summary Variations in the (2′–5′) oligoadenylate synthetase (2–5 A synthetase) level were examined prior to and during the differentiation in culture of the human monocyte cell line U937 and the promyelocytic cell line HL60 in an attempt to reveal whether the enzyme is actively involved in hematopoietic cell maturation. The basal level of this enzyme was much higher in U937 than in HL60 cells. The activity of 2–5 A synthetase was enhanced in both cell lines in response to α, β interferons. During cell differentiation, ten markers were measured. The level of the enzyme rose during the process of cellular maturation in both cell lines. The 2–5 A synthetase activity observed in differentiated HL60 and U937 cells was comparable to that observed in mature normal granulocytes and monocytes respectively. Induction of U937 differentiation by chemicals was associated with detectable production of IFN. The increase in enzyme activity observed was mostly dependent on endogenous production of interferon, since it was inhibited by interferon antibodies. Kinetic studies showed that in U937 cells 2–5 A synthetase was expressed prior to several of the differentiation markers. The rise in the enzyme's level observed during the differentiation of HL60 cells was independent of endogenous production of interferon, since it was not inhibited by the addition of anti-interferon antibodies. These results suggest that different biochemical and molecular mechanisms are responsible for the induction of 2–5 A synthetase observed during the differentiation of hematopoietic cells. In any case, 2–5 A synthetase can be considered as a biochemical marker of cell status and differentiation in hematopoietic cells.  相似文献   

7.
Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about 1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn2+, Co2+ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating the potential role of this enzyme for detergent application.  相似文献   

8.
1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.  相似文献   

9.
It was found that in medium with low ionic strength nucleoside diphosphate kinase (NDPK) solubilization from the outer membrane of liver mitochondria could be partially reversed by the addition of 3.3 mM MgCl2. Complete rebinding of the enzyme after the addition of MgCl2 was observed when the mitochondrial washing and storage medium contained leupeptin, an inhibitor of cathepsins. It was demonstrated that leupeptin and another inhibitor of cysteine proteinases, E-64, do not influence the rate of NDPK solubilization as well as its solubilized and membrane-associated activity. We conclude that NDPK becomes sensitive to proteolysis only after its solubilization; proteolysis does not affect the part of the enzyme molecule that is responsible for catalysis. After solubilization of NDPK in the absence of leupeptin, cathepsins damage sites of its binding on the membranes. The rate of the enzyme solubilization is dependent on the pH of the storage medium (pH 6.0–8.0); it decreases with increase in pH. It was shown that in the medium with high ionic strength, MgCl2 does not reverse pH-dependent NDPK solubilization, but solubilization could be reversed by increase in medium pH in the presence of E-64 and BSA. The physiological importance of these results is discussed. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 5, pp. 710–720.  相似文献   

10.
1. Xenopus laevis oocytes express endogenously two components of the cholinergic system: the muscarinic receptors and the acetylcholinesterase (AChE). 2. A biochemical characterization of this enzyme was carried out. 3. The results established that the activity found in the oocytes correspond to 'true' AChE with a molecular weight of 65,000 Da and a sedimentation coefficient of 3-4 S. 4. The enzyme aggregates in the absence of detergent suggesting that it possess an hydrophobic character; despite that, it is not sensitive to PIPLC. 5. A comparison with the Xenopus brain and muscle AChE shows different post-translational modifications and catalytic properties with the oocyte AChE.  相似文献   

11.
Terminal sialylation of therapeutic glycoprotein is important for biological activity and in vivo stability. The enzyme α2,3-sialyltransferase is the key enzyme that links sialic acids to the termini of glycans in the Chinese hamster ovary (CHO) cell line. Terminal sialylation is affected by numerous factors, but the elements that regulate α2,3-sialyltransferase are not known. We investigated the relationship between α2,3-sialyltransferase activity, ammonium concentration, and cell attachment area-based cell concentration in a recombinant human erythropoietin (rhEPO)-producing CHO cell line. We found that ammonium in the culture medium had almost no effect on α2,3-sialyltransferase activity, but that the activity was affected by cell attachment area-based cell concentration; α2,3-sialyltransferase activity and terminal sialylation of rhEPO decreased with increasing the cell concentration. These results demonstrate that the cell attachment area-based cell concentration is an important factor that affects 2,3-sialyltransferase activity and terminal sialylation of CHO cells.  相似文献   

12.
Amphiphile dependency, solubility in aqueous solutions, and sensitivity to proteolysis of acetylcholinesterase (AChE) and nonspecific cholinesterase (nsChE) in the rat superior cervical ganglion were studied and compared to properties of soluble plasma cholinesterases. Ganglion AChE shows strong amphiphile dependency: an amphyphilic substance must be present in the homogenizing medium in order to obtain maximal apparent enzyme activity. Apparent activity of AChE solubilized in Ringer's solution was also increased after subsequent addition of a detergent. The 4 S molecular form, predominant in this extract (corresponding to the fastest electrophoretic band), is very sensitive to papain proteolysis but can be protected by a detergent. This molecular form therefore carries an important hydrophobic domain and is probably membrane bound in situ. The 10 S form of ganglionic AChE, extracted in Ringer's solution, is probably a soluble enzyme since, like soluble plasma enzymes, it is not amphiphile dependent and is rather resistant to proteolysis. Ganglion nsChE is more water soluble, less amphiphile dependent and more protease resistant than AChE.  相似文献   

13.
Abstract— A study was made of the effect of various solubilization procedures on the release of AChE from electric organ tissue of the electric eel and on the molecular state of the enzyme. The procedures employed included homogenization in different ionic media or in the presence of detergents, etuymic treatment and chemical modification. Studies were performed on intact electroplax, tissue homogenates and membrane fractions. The apparent AChE activity of intact cells, homogenates and membrane fractions was shown to be governed by diffusion-controlled substrate and hydrogen ion gradients, generated by AChE-catalyscd hydrolysis, leading to a lower substrate concentration and a lower pH in the vicinity of the particulate enzyme.
Treatment of homogenates with NaCl solutions or with NaCl solutions containing the nonionic detergent Triton X-100 causes release of the native'molecular forms of the enzyme (primarily the 18 S species) which aggregate at low ionic strength. For optimal extraction both high ionic strength (e.g. 1 M-NaCl) and the detergent are needed AChE is also solubilized by treatment of tissue homogenates with trypsin, bacterial protease or collagenase. The first two enzymes caused its release as an 11 S non-aggregating form, while collagenase also produces a minor non-aggregating - 16 S component. Treatment of tissue homogenates with maleic anhydride causes release of AChE as a non-aggregating 18 S species. On the basis of the solubilization experiments it is concluded that the interaction of AChE with the excitable membrane is primarily electrostatic. The possible orientation of the enzyme within the synaptic gap is discussed.  相似文献   

14.
Fraser PD  Schuch W  Bramley PM 《Planta》2000,211(3):361-369
 Phytoene synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, phytoene synthase has been purified 600-fold from tomato (Lycopersicon esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa, with an isoelectric point of 4.6. Although phytoene synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) synthase. Both Mn2+ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with K m values for Mn2+, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 μM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including β-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of phytoene synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences. Received: 20 January 2000 / Accepted: 16 February 2000  相似文献   

15.
Circulating acetylcholine, substrate of membrane acetylcholinesterase (AChE), is known to enhance the band 3 protein degree of phosphorylation. The purpose of this study was to verify whether the band 3 phosphorylation status is associated with a G protein and whether it is an influent factor on AChE enzyme activity. From blood samples of healthy donors, erythrocyte suspensions were prepared and incubated with AChE substrate (acetylcholine) and inhibitor (velnacrine), along with protein tyrosine kinase (PTK) and tyrosine phosphatase (PTP) inhibitors. AChE activity was determined by spectrophotometry and extract samples were analyzed by western blotting using primary antibodies to different G protein subunits. Our results with phosphorylated band 3 (PTP inhibitor) show an increase in erythrocyte AChE (p < 0.0001). A dephosphorylated band 3 state (PTK inhibitor) shows a significant decrease. We identified a potential linkage of protein subunits Gαi1/2 and Gβ with band 3 protein. Gαi1/2 and Gβ may be linked to the band 3 C-terminal site. Gαi1/2 is associated with the band 3 N-terminal domain, except for the control and ACh aliquots. Gβ is associated with both phosphorylated and dephosphorylated band 3 in the presence of velnacrine. We conclude that an erythrocyte G protein with subunits Gαi1/2 and Gβ is associated with band 3. AChE depends on the degree of band 3 phosphorylation and its association with Gαi1/2 and Gβ.  相似文献   

16.
The membrane-bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was solubilized by Triton X-100 or by treatment with proteinase K and purified to apparent homogeneity by affinity chromatography. Although the two forms differed only slightly in their subunit molecular weight (66,000 and 65,000 daltons, respectively), considerable differences existed between native and digested detergent-soluble AChE. The native enzyme sedimented at 6.5 S in the presence of Triton X-100 and formed aggregates in the absence of detergent. The digested enzyme sedimented at 7.5 S in the absence and in the presence of detergent. In contrast to the detergent-solubilized AChE, the proteolytically derived form neither bound detergent nor required amphiphilic molecules for the expression of catalytic activity. This led to the conclusion that limited digestion of detergent-soluble AChE results in the removal of a small hydrophobic peptide which in vivo is responsible for anchoring the protein to the lipid bilayer.  相似文献   

17.
18.
Summary The ADAMs (A Disintegrin And Metalloprotease-like) family is a large and rapidly expanding group of metallo-proteinases with structural similarity. The are normally characterized by the presence of a proteolytic domain and disintegrin and signalling domains. Although 21 ADAMs proteins have been already cloned to date, in most cases their natural substrates are unknown. The best characterized representative of the mammalian ADAMs family is the TNF-α converting enzyme (TACE). TACE is an integral membrane metalloproteinase that causes the secretion of the active form of TNF-α from its plasma membrane precursor and thus can be regarded as a membrane protein secretase. Secretion of membrane proteins is a very well documented biological phenomenon and was demonstrated for a diverse range of membrane proteins, two examples being angiotensin converting enzyme (ACE) and Alzheimer's amyloid precursor protein (APP). ACE and APP secretion was shown to possess substantial similarity with the secretion of TNF-α. In the present study, we have attempted to demonstrate that a metalloproteinase might be involved in the shedding of another membrane-bound protein—acetylcholinesterase (AChE). Secretion of AChE by human neuroblastoma SH-SY5Y cells was found to be inhibited by a selective hydroxamate metalloproteinase inhibitor batimastat (20 μM), and stimulated by carbachol (20 μM), which have previously been shown to regulate the activity of APP α-secretase in a similar manner. The role of ADAMs proteins in the shedding of molecules from the cell surface is discussed.  相似文献   

19.
Abstract: We have observed that cultured neurons from chick spinal cord and the neuroblastoma hybrid line 108CC15 released lower amounts of acetylcholinesterase (AChE) when compared with the parental line, N18TG2. AChE activity extracted by hypotonic buffer, which can be regarded as the source of the released enzyme, was considerably higher in the parental than in the hybrid 108CC15 (respectively, ∼80% and ∼40% of cellular activity). On the other hand, evaluation of ectocellular, with respect to total, AChE activity showed that in N18TG2 cells only 7% of AChE was localized on the plasmalemma, whereas in the hybrid line the percentage of ectocellular activity was 3.7 times higher than in the parental line. We have also examined the effect of cytochalasin B and nocodazole. In the N18TG2 line, the former did not affect AChE release, which was significantly reduced by the latter. High K+ level in the culture medium, of both N18TG2 and hybrid 108CC15 cultures, induced an increase in AChE secretion; Ca2+ presence was required for high K+-induced release. Muscle extracts increased AChE secretion in both the hybrid 108CC15 and the spinal cord neurons. The present data suggest that AChE secretion during neuronal development is modulated by depolarizing stimuli and by soluble factors produced by target cells and may be involved in the control of neuronal differentiation.  相似文献   

20.
Brain tissue was analyzed for acetylcholinesterase (AChE) activity from 24 American pronghorn antelope (Antilocapra americana) collected on the US Army Dugway Proving Ground (DPG) (latitude 40°13' 52" N, longitude 112°45' 01" W), Tooele County, Utah. Pronghorn antelope from DPG were evaluated against 26 pronghorn antelope collected in Wyoming. The mean AChE activity was significantly greater (P < 0.001) in the Wyoming control group (4.612 ± 0.193 μM/gm brain tissue/min) relative to DPG (4.032 ± 0.621 μM/gm brain tissue/min). The DPG database exhibited a fourfold greater coefficient of variation, a tenfold greater variance, and a threefold increase in the standard deviation when compared to control AChE activity. Furthermore, the 95% confidence interval for the control and for the DPG data were not overlapping; the entire control data set was greater than the mean DPG AChE activity. A post hoc sequential Bonferroni statistical procedure showed two significantly (P < 0.001) distinct subsets in the DPG data. Mean DPG Subset I AChE activity (4.528 ± 0.347 μM/gm brain tissue/min) was indistinct from the mean control AChE value (4.612 ± 0.193 μM/gm brain tissue/min). The mean DPG Subset II AChE activity (3.537 ± 0.387 μM/gm brain tissue/min) differed significantly (P < 0.001) from the mean control AChE activity. The sum of resulting α values from the multiple statistical tests did not exceed the selected α value of P < 0.05, validating the post hoc sequential Bonferroni statistical procedure. Pronghorn antelope represented by Subset II, experienced a 23.3% mean loss of AChE activity suggesting sub-lethal organophosphate (OP) exposure rather than a low level chronic environmental influence was experienced by a population subset of the DPG pronghorn antelope herd. The origin of the DPG sublethal OP exposure and its long-term effects are speculative.  相似文献   

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