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1.
Enzyme-linked immunosorbent assays for acetylcholinesterase (AChE) and for butyrylcholinesterase (BuChE) were markedly more specific than conventional assays using selective enzyme inhibitors. The new assays were used with blood and brain samples containing traces of one enzyme dominated by large amounts of the other. The results showed that human plasma does contain AChE (8 ng/ml), even though its major cholinesterase is BuChE (3,300 ng/ml). BuChE immunoreactivity was not detected in human red blood cells but occurred in all brain regions. The cerebellum was the richest region tested (540 ng of BuChE/g of tissue), whereas the cerebral cortex was the poorest (240 ng of BuChE/g). However, because of the small local AChE content (99 ng/g), BuChE was the major cortical cholinesterase. The picture was reversed in the putamen, where BuChE immunoreactivity (340 ng/g) was far outweighed by that of AChE (6,100 ng/g).  相似文献   

2.
The differences in the inhibition activity of organophosphorus agents are a manifestation of different molecular properties of the inhibitors involved in the interaction with the active site of enzyme. We were interested in comparing the inhibition potency of four known synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl2, constituting organophosphorus compounds, where R = CCl3 (1), CHCl2 (2), CH2Cl (3) and CF3 (4), and four new ones with the general formula RC(O)NHP(O)(R')2, where R' = morpholine and R = CCl3 (5), CHCl2 (6), CH2Cl (7), CF3 (8), on AChE and BuChE activities. In addition, in vitro activities of all eight compounds on BuChE were determined. Besides, in vivo inhibition potency of compounds 2 and 6, which had the highest inhibition potency among the tested compounds, was studied. The data demonstrated that compound 2 from the compound series 1 to 4 and compound 6 from the compound series 5 to 8 are the most sensitive as AChE and BuChE inhibitors, respectively. Comparing the IC50 values of these compounds, it was clear that the inhibition potency of these compounds for AChE are 2- to 100-fold greater than for BuChE inhibition. Comparison of the kinetics (IC50, Ki, kp, KA and KD) of AChE and BuChE inactivation by these compounds resulted in no significant difference for the measured variables except for compounds 2 and 6, which appeared to be more sensitive to AChE and BuChE by significantly higher kp and Ki values and a lower IC50 value in comparison with the other compounds. The LD50 value of compounds 2 and 6, after oral administration, and the changes of erythrocyte AChE and plasma BuChE activities in albino mice were studied. The in vivo experiments, similar to the in vitro results, showed that compound 2 is a stronger AChE and BuChE inhibitor than the other synthesized carbacylamidophosphates. Furthermore, in this study, the importance of electropositivity of the phosphorus atom, steric hindrance and leaving group specificity were reinforced as important determinants of inhibition activity.  相似文献   

3.
The role of acetylcholinesterase (AChE) in the termination of the cholinergic response through acetylcholine (ACh) hydrolysis and the involvement of plasma butyrylcholinesterase (BuChE), mainly of hepatic origin, in the metabolism of xenobiotics with ester bonds is well known. Besides, BuChE has a crucial role in ACh hydrolysis, especially when selective anticholinesterases inhibit AChE. Herein, we analyzed the gender-related differences and the circadian changes of rat plasma cholinesterases. Plasma and liver cholinesterase activities were evaluated in control or 2–30-day castrated adult male and female rats. Plasma and liver AChE activities did not differ between genders and were not influenced by sex hormone deprivation. BuChE plasma activity was 7 times greater in female, reflecting gender differences in liver enzyme expression. Castration increased liver and plasma BuChE activity in male, while reduced it in female, abolishing gender differences in enzyme activity. Interestingly, female AChE and BuChE plasma activities varied throughout the day, reaching values 27% and 42% lower, respectively, between 2 p.m. and 6 p.m. when compared to the morning peaks at 8 a.m. Castration attenuated daily female BuChE oscillation. On the other hand, male plasma enzymes remained constant throughout the day. In summary, our results show that liver and plasma BuChE, but not AChE, expression is influenced by sex hormones, leading to high levels of blood BuChE in females. The fluctuation of female plasma BuChE during the day should be taken into account to adjust the bioavailability and the therapeutic effects of cholinesterase inhibitors used in cholinergic-based conditions such Alzheimer's disease.  相似文献   

4.
The uptake (Jd) of fatty acids (FA), fatty alcohols (Alc) and cholesterol (C) into the jejunum of rats (R, Rattus norvegicus), rabbits (RAB, Oryctolagus cuniculus), guinea pigs (GB, Cavia porcellus), and hamsters (H, Mesocricetus auratus) was assessed in vitro. Using jejunal discs the Jd of Alc was H greater than R = GP greater than RAB, the Jd of FA was H-RAB greater than R greater than GP, but the Jd of C was R greater than H greater than RAB greater than GP. The Jd of FA was quantitatively and qualitatively different when using jejunal biopsies; in man the Jd of FA into biopsies was greater than in the other animal species, but there was no difference in Jd of FA into normal human jejunal biopsies and those showing severe abnormalities in villus architecture. There are marked species differences in the passive permeability properties of the jejunum and in the effective resistance of the overlying unstirred water layer, but these differences do not explain the species variations in the uptake of cholesterol.  相似文献   

5.
Testicular cytochrome c (cyt ct) was isolated from testes of sexually mature, rat, mouse, rabbit, and bull, among which rat testis is highly rich in cyt ct. By fusion of NS-1 myeloma cells and spleen cells of mice immunized with rat cyt ct, 11 stable mouse hybridoma cell lines were established. Using an enzyme-linked immunosorbent assay, it was determined that 4 of the 11 anti-rat cyt ct monoclonal antibodies (McAb) did not bind to somatic cyt c (cyt cs) of vertebrates nor to cyt ct of mouse, rabbit, and bull. Four other McAb showed no binding to cyt cs but showed different patterns of cross-reactivity with these four cyt ct. Therefore, these McAb appear to be very sensitive and useful probes for the discrimination or identification of extremely similar isocytochromes c. Although the primary amino acid sequences between cyt cs of rat and mouse are identical, the antigenic structure of cyt ct of rat and mouse are clearly distinct with regard to cross-reactivity with some anti-rat cyt ct McAb. Furthermore, these McAb also reveal that the primary amino acid sequences of cyt ct, which reflect differences in the surface conformation of the molecule, are probably species specific.  相似文献   

6.
Synopsis Sections of ovary from plains viscacha, cat, ferret, rabbit, rat, guinea-pig and roe deer have been histochemically processed to demonstrate acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in nervous and non-nervous tissue. The effects of different reproductive states on enzyme activity were observed in some animals. AChE-containing nerves were sparse in rabbit and rat but plentiful in cat and roe deer. Nerves containing BuChE were not detectable in ferret or guinea-pig and were rare in cat. Species variations in the activity and type of enzyme were also found in non-neuronal tissues. Some blood vessels in the ovaries of guinea-pig and viscacha contained AChE. No other species showed a reaction for AChE in non-neuronal stromal tissue but BuChE was present at this site in all animals except rat. Granulosa cells reacted for AChE only in cat and rabbit while luteal cells were reactive in cat, rabbit and roe deer. Some BuChE activity was present in granulosa and or luteal cells in all species except roe deer. In rat, BuChE activity in luteal cells increased during oregnaney and the early phase of pseudopregnancy. The difficulty of assigning a function to ovarian cholinesterases is discussed.  相似文献   

7.
To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.  相似文献   

8.
A new method for obtaining stable butyrylcholinesterase (BuChE) samples based on the enzyme immobilization in starch and gelatin gels followed by drying is proposed. Coimmobilization of BuChE with the thiol group indicator 5,5'-dithiobis(2-nitrobenzoic) acid did not reduce the activity of BuChE, which allowed us to simplify the procedure and reduce the time of analysis of organophosphorus pesticides. The resulting immobilized samples retained activity for at least 300 days. BuChE samples based on the starch gel showed a greater sensitivity in the determination of pesticides as compared to the samples based on the gelatin gel.  相似文献   

9.
10.
Apart from its esterase activity, butyrylcholinesterase (BuChE) displays aryl acylamidase (AAA) activity able to hydrolyze o-nitroacetanilide (ONA) and its trifluoro-derivative (F-ONA). We report here that, despite amidase and esterase sites residing in the same protein, in human samples depleted of acetylcholinesterase the ratio of amidase to esterase activity varied depending on the source of BuChE. The much faster degradation of ONA and F-ONA by BuChE monomers (G1) of colon and kidney than by the tetramers (G4) suggests aggregation-driven differences in the AAA site between single and polymerized subunits. The similar ratio of F-ONAto butyrylthiocholine hydrolysis by serum G1 and G4 forms support structural differences in the amidase site according to the source of BuChE. The changing ratios of amidase to esterase activities in the human sources probably arise from post-translational modifications in BuChE subunits, the specific proportion of monomers and oligomers and the variable capacity of the tetramers for degrading ONA and F-ONA. The elevated amidase activity of BuChE monomers and the scant activity of the tetramers justify the occurrence of single BuChE subunits in cells as a means to sustain the AAA activity of BuChE which otherwise could be lost by tetramerization.  相似文献   

11.
Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) display both esterase and aryl acylamidase (AAA) activities. Their AAA activity can be measured using o-nitroacetanilide (ONA). In human samples depleted of acetylcholinesterase, we noticed that the ratio of amidase to esterase activities varied depending on the source, despite both activities being due to BuChE. Searching for an explanation, we compared the activities of BuChE molecular forms in samples of human colon, kidney and serum, and observed that BuChE monomers (G(1)) hydrolyzed o-nitroacetanilide much faster than tetramers (G(4)). This fact suggested that association might cause differences in the AAA site between single and polymerized subunits. This and other post-translational modifications in BuChE subunits probably determine their level of AAA activity. The higher amidase activity of monomers could justify the presence of single BuChE subunits in cells as a way to preserve the AAA activity of BuChE, which could be lost by oligomerization.  相似文献   

12.
Catalase electrophoretic patterns and enzymatic activities were measured in four organs of two anuran species, Rana ridibunda perezii and Discoglossus pictus. The D. pictus enzyme appeared as two distinguishable bands, whereas R. ridibunda catalase was monomorphic. Electrophoretic mobility of the major D. pictus catalase band was greater than that of R. ridibunda. Enzymes from both species showed slower mobility than that from bovine liver. Catalase activities did not show significant differences according to sex in any of the organs tested in R. ridibunda. Enzyme activities were similar in liver, kidney and brain when both species were compared. Only the heart showed much higher activity in D. pictus than in R. ridibunda. The catalase activity levels followed the order: liver greater than kidney greater than heart in both species. The heart showed higher activity than the brain in D. pictus but not in R. ridibunda.  相似文献   

13.
Organophosphate (OP) nerve agents are known as the most toxic chemical warfare agents that act by inhibiting the enzyme acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Because BuChE is present at a level of about 3,900 times higher than AChE in plasma, most OP agents first react with BuChE in plasma, suggesting that OP-inactivated BuChE (OP-iBuChE) may act as a biomarker of OP exposure. In this study, we generated an anti-BuChE monoclonal antibody (mAb) that has reduced binding activity to VX-inactivated BuChE compared to native BuChE by phage display. We performed subtractive biopanning of a synthetic human Fab library against native BuChE and soman-iBuChE or VX-iBuChE. As the results, we isolated four Fab clones that showed differential binding activities to VX-iBuChE and native BuChE in ELISAs. To confirm the antigen-binding specificity of the selected clones, the Fabs were converted to IgG1s, and the IgG antibodies were expressed in HEK293F cells and purified. One of them (A2) showed approximately 30% reduced binding activity to VX-iBuChE compared to BuChE in a dose-dependent manner, whereas the other three antibodies showed almost the same binding activities to VX-iBuChE and BuChE. In addition, the A2 antibody did not show reduced binding activity to sarin-iBuChE or soman-iBuChE compared to native BuChE. The results indicate that A2 antibody shows reduced binding activity only to VX-iBuChE. A2 antibody may be applied to specific diagnosis of VX exposure.  相似文献   

14.
Monoclonal antibodies (McAb) 2AH10, specifically reacting with protein preparations having mol. wt. of 18 and 38 kD and not interacting with brucellar lipopolysaccharide (LPS) and protein-polysaccharide antigen, have been obtained. As shown with the use of McAb 2AH10, Brucella spp. and Yersinia enterocolitica O:9 possess common antigenic determinants, localized not only in the area of their LPS, which is generally known, but also in the area of their outer cell-wall proteins with mol. wt. 18 and 38 kD. The sensitivity of the solid-phase enzyme immunoassay and the latex agglutination test with the use of McAb 2AH10 is essentially higher in the detection of B. abortus and B. suis, than B. melitensis and B. rangiferi, as well as Y. enterocolitica O:9. Essential differences observed in the detected concentrations of different Brucella species and Y. enterocolitica O:9 are seemingly linked with different expression of specific antigenic determinants, detected with the use of McAb 2AH10 in the corpuscular antigens under study.  相似文献   

15.
The change in the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in neoplastic colon and lung prompted us to study the possible effect of cancer on the expression of cholinesterases (ChEs) in kidney. Samples of papillary renal cell carcinoma (pRCC), conventional RCC (cRCC), chromophobe RCC (chRCC) and renal oncocytoma (RON), beside adjacent non-cancerous tissues, were analyzed. In pRCC both AChE and BuChE activities were statistically increased; in cRCC and chRCC only AChE activity increased and in RON neither AChE nor BuChE activities were affected. Abundant amphiphilic AChE dimers (G(2)(A)) and fewer monomers (G(1)(A)) were identified in healthy kidney as well as in all tumour classes. Incubation with PIPLC revealed glycosylphosphatidylinositol in AChE forms. BuChE is distributed between principal G(4)(H), fewer G(1)(H), and much fewer G(4)(A) and G(1)(A) species. RT-PCR showed similar amounts of AChE-H, AChE-T and BuChE mRNAs in healthy kidney. Their levels increased in pRCC but not in the other tumour types. The data support the idea that, as in lung tumours, in renal carcinomas expression of ChE mRNAs, biosynthesis of molecular components and level of enzyme activity change according to the specific kind of cell from which tumours arise.  相似文献   

16.
Passive immunization against murine malaria with an IgG3 monoclonal antibody   总被引:31,自引:0,他引:31  
Spleen cells of BALB/c mice that were immune to the 17X strain of P. yoelii were fused with P3X63Ag8 myeloma cells. Two hundred fifty-three of 1053 hybrid cells produced antibodies reactive with disrupted 17X parasites in a solid phase radioimmunoassay. One of these antibodies, McAb 302, reacted with the merozoites of the 17X (nonlethal) and 17XL (lethal) variants of P. yoelii. Of greater significance, McAb 302 passively protected mice against challenge infection with the lethal variant. Mice treated with this antibody before infection developed low-grade parasitemia (less than 0.3%) of short duration when challenged with P. yoelii 17XL . In contrast, control mice that had been untreated or injected with ascites fluid lacking McAb 302 uniformly died with fulminating malaria upon challenge with the same parasite. In other experiments, McAb 302 was shown capable of controlling blood parasite levels when administered to mice with patent P. yoelii 17XL infections. Although all control mice died, mice protected with a single dose of McAb 302 ultimately cleared their infections. Regardless of how passive immunization was performed, mice given McAb 302 were resistant to subsequent challenge with P. yoelii 17XL , indicating they had developed significant immunity during their initial controlled infections. McAb 302 also showed pronounced passive protective activity against the nonlethal 17X strain of P. yoelii, which is a parasite of reticulocytes. The protection afforded by McAb 302 was specific, because mice passively immunized with this antibody died when challenged with the unrelated P. vinckei. McAb 302 was shown to possess the IgG3 isotype and precipitated a 230-kd protein plus several smaller polypeptides from metabolically labeled parasite antigen preparation derived from both variants of P. yoelii. It did not react with similar preparations of other murine plasmodial species.  相似文献   

17.
The activities of acetylcholinesterase (ACHE) and butyrylcholinesterase (BuChE) in rat brain capillaries were measured as a function of temperature. Arrhenius plots of the data revealed that AChE exhibits a biphasic Arrhenius plot with a distinct break (transition temperature) at about 15.2 kcal/mol. In contrast, BuChE did not show evidence of discontinuity. BuChE showed an activation energy higher than that of AChE in the physiological range of temperature. These data suggest a lack of lipid-protein interaction in the case of BuChE. Although the possibility exists that BuChE is weakly anchored to the membranes, our results indicate that BuChE is not bound, at least significantly, to cellular membranes in brain capillaries as is ACHE.  相似文献   

18.
Abstract An in vitro assay involving the adherence of Bordetella pertussis to Vero cells was used to quantify the inhibitory effects of monoclonal antibodies (McAb) on adherence. McAbs to agglutinogens (fimbriae), filamentous haemagglutinin (FHA), lymphocytosis promoting factor (LPF) and to an X-mode specific outer membrane protein were tested. X-mode cells adhered to the Vero cells to a greater extent than C-mode cells. McAb to agglutinogen 2 inhibited the adherence of B. pertussis bearing homologous agglutinogen. McAbs to LPF, FHA, agglutinogen 3 and X-mode specific outer membrane protein also partially inhibited the adherence of B. pertussis to Vero cells.  相似文献   

19.
Butyrylcholinesterase (BuChE) occurs in a multiple molecular forms whose catalytic activity depends on tissue distribution and species. The hypothesis led us to the study of BuChE catalytic properties focused on the inter-tissue and inter-species level with benzoylcholine and N-alkyl derivates of benzoylcholine (BCHn) as substrates. These compounds are soft disinfectants easily biodegradable to biologically inactive hydrolytic products, substituted choline and benzoic acid. Different sources of BuChE were used: rabbit and rat liver microsomal fraction (membrane-anchored enzyme) and serum (soluble form). Hydrolytic activity of both these BuChE forms was compared to rat recombinant BuChE (rBuChE). Hydrolytic product (benzoic acid) formation was recorded as function of time, and hydrolytic rate was determined. Tissue distribution of BuChE plays an important role in hydrolysis of BCHn. High BuChE activity was observed in a serum of both studied species rat and rabbit and was significantly dependent on a structure of substrates. Activity of soluble serum forms was the same as that for the rBuChE. Significant change of BuChE activity was recorded on the inter-species level in the microsomal fractions. It is because the rabbit microsomal BuChE activity had absolutely different course for all substrates as compared to rat microsomes. Inhibitory studies of BCHn enzymatic hydrolysis of all BuChE forms were performed to determine the level of BuChE participation in BCHn hydrolysis. It can be concluded that short-chain BCHn substrates are exclusively hydrolyzed by BuChE from all studied sources except for the rabbit liver microsomal fraction. Rabbit seems to have different enzymes involved in the hydrolysis of all studied BCHn compounds.  相似文献   

20.
The laminin-alpha2 chain, referred to as merosin, forms part of the laminin-2 heterotrimer (alpha2beta1gamma1), which is principally expressed in the basement membrane of muscle. Nearly half of patients suffering from congenital muscular dystrophy (CMD) have abnormalities in the laminin-alpha2 chain (LAMA2) gene, and the merosin-deficient Lama2dy mouse shows CMD. The expression of merosin in thymus, the abnormalities in the gland of Lama2dy mice, and the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in thymus prompted us to study the possible effects of the deficiency of merosin on thymus BuChE. We found that, while AChE activity decreased by approximately 50% in merosin-deficient thymus, the deficiency had little effect on BuChE activity. About 65% of thymus BuChE activity was extracted with a saline buffer and 30% with 1% Triton X-100. Sedimentation analyses and phenyl-agarose chromatography showed that thymus contained amphiphilic BuChE monomers (G(1)(A),44%) and dimers (G(2)(A),33%), and hydrophilic tetramers (G(4)(H),23%). Binding assays with various plant lectins revealed differences between the oligoglycans linked to BuChE tetramers and lighter components. The deficiency of merosin had no effect on the biosynthesis of thymus BuChE as judged by the lack of major changes between control and Lama2dy mice thymuses in the distribution of BuChE molecules and the level of lectin binding. The detoxifying action of BuChE, its role as a backup to AChE, and the relevance of the cholinergic dialogue between T cells and stromal cells for T lymphocyte proliferation, maturation and survival support a physiological function for BuChE in thymus.  相似文献   

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