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1.
HETEROGENEITY OF ACETYLCHOLINESTERASE IN NEUROBLASTOMA 总被引:2,自引:2,他引:0
Abstract– Multiple forms of acetylcholine hydrolyzing activity have been observed in Triton X-100 treated homogenates of mouse neuroblastoma cells. All these forms appear to be the true acetylcholinesterase, AChE (EC 3.1.17): they are substrate-inhibited; hydrolyze acetylcholine > acetyl-β-methylcholine ≫ butrylcholine and are preferentially inhibited by specific AChE inhibitors. Almost all of the cell AChE activity is membrane associated, but readily 'solubilized' by Triton X-100 and as such appears free of membrane contamination. With the aid of affinity chromatography the 'solubilized' neuroblastoma AChE has been partially purified (490-fold) to a specific activity of 34,300 nmol/min/mg protein.
Four active neuroblastoma AChE species appear upon acrylamide gel electrophoresis (with MWs of 64,000; 116,000; 186,000 and 284,000) while three species (4S, 6S and 9.6S) have been found upon sucrose gradient sedimentation analysis. We have determined that the 4S form migrates on acrylamide as the 116,000 MW species and the 9.6S form contains, in equal amounts, the 186,000 and 284,000 MW acrylamide species. Numerous active AChE forms are seen on Sepharose 6B chromatography. From comparing the crude, 4S, 9.6S and partially purified AChEs on acrylamide gels, sucrose gradients and Sepharose, mouse neuroblastoma appears to contain active AChE units which are capable of multiple types of dissociation and reassociation. An attempt is made to correlate all the observed AChE forms as well as relate this data to that known about AChE obtained from other sources. 相似文献
Four active neuroblastoma AChE species appear upon acrylamide gel electrophoresis (with MWs of 64,000; 116,000; 186,000 and 284,000) while three species (4S, 6S and 9.6S) have been found upon sucrose gradient sedimentation analysis. We have determined that the 4S form migrates on acrylamide as the 116,000 MW species and the 9.6S form contains, in equal amounts, the 186,000 and 284,000 MW acrylamide species. Numerous active AChE forms are seen on Sepharose 6B chromatography. From comparing the crude, 4S, 9.6S and partially purified AChEs on acrylamide gels, sucrose gradients and Sepharose, mouse neuroblastoma appears to contain active AChE units which are capable of multiple types of dissociation and reassociation. An attempt is made to correlate all the observed AChE forms as well as relate this data to that known about AChE obtained from other sources. 相似文献
2.
J. W. Phillis 《Journal of neurochemistry》1968,15(7):691-698
Histochemical procedures for the localisation of AChE have been used on the cerebella of normal and operated cats to investigate the presence of cholinergic pathways. The evidence indicates that some mossy afferent fibres and deep nuclear cells are cholinergic. The presence of AChE in many cells in the granule cell layer suggests that some parallel fibres and association pathways in the cerebellum may also be cholinergic. The relationship of these results to studies on the pharmacology and distribution of choline acetyltransferase in the cerebellum is discussed. 相似文献
3.
ACETYLCHOLINESTERASE IN DEVELOPING CHICK EMBRYO BRAIN 总被引:1,自引:0,他引:1
–Acetylcholinesterase has been assayed at different stages of development to see whether changes in the activity of this enzyme are correlated in any way with the ontogenesis of electrical activity in the brain of growing chick embryo. The specific activity of the enzyme was highest in the synaptosomal fraction of the brain. The activity of the enzyme increased progressively with the age of the embryo. There were three isozymic forms of the enzyme in the 6-day-old embryo brain. A new isozyme appeared around the 9th day. The Km values of the enzyme for acetylthiocholine from 6- and 20-day-old embryo brains were 6.5 ± 10-5m and 3.3 ± 10-5m respectively. Enzyme preparations from 6-day-old embryos were found to lose 50 per cent of their activity when heated at 50°C for 10 min. Under similar conditions the loss in activity in 18-day-old embryo brain enzyme was 22 per cent. 相似文献
4.
THE DISTRIBUTION OF ACETYLCHOLINESTERASE IN PERIPHERAL NERVES 总被引:1,自引:1,他引:0
5.
BEHAVIOUR OF ACETYLCHOLINESTERASE IN ISOLATED NERVE SEGMENTS 总被引:1,自引:1,他引:0
6.
THE DETERMINATION OF ACETYLCHOLINESTERASE ACTIVITY OF BRAIN SLICES AND ITS SIGNIFICANCE IN STUDIES OF EXTRACELLULAR ACETYLCHOLINESTERASE 总被引:2,自引:1,他引:1
The AChE activity of single slices obtained from the surface of the temporoparietal region of rat brains was measured colorimetrically under anaerobic conditions with acetylthiocholine as substrate. In intact slices from untreated rats AChE activity was only a small proportion of that of homogenates made from the slices, but this proportion increased with the surface area to weight ratio of slices and with an increase in substrate concentration, intact slices not showing substrate inhibition. The inhibition of AChE determined in slices from rats treated with DFP or paraoxon was less than that in homogenates obtained from the slices. When the access of substrate was limited to the cut surface of a slice, the rate of hydrolysis was four times greater than that observed when access was solely from the uncut surface. It is concluded that under anaerobic conditions the substrate diffuses into slices to a depth which is not constant but a function of both substrate and enzyme concentration. Thus the AChE activity of slices cannot be used as a measure of extracellular AChE. 相似文献
7.
Abstract—The effect of pentylenetetrazol (PTZ) on acetylcholinesterase (E.C.3.1.1.7) was studied in vitro. The kinetics of the reaction were studied on AChE in crude homogenates of rat brain and in purified preparations from Electrophorus electricus. The Km for rat brain AChE was 1·22 × 10-4m, with a Vmax of 1·37 μmol/g/min whereas the K4 for competitive inhibition of the enzyme by PTZ was 4·7 × 10-3m. The commercially purified enzyme exhibited a Km of 1·73 × 10-4m and a Ki of 1·00 × 10-3m. 相似文献
8.
THE HALF-LIFE OF ACETYLCHOLINESTERASE IN MATURE RAT BRAIN 总被引:2,自引:2,他引:0
—The rate of degradation of acetylcholinesterase [EC 3.1.1.7] in mature rat cerebral cortex was determined from the time course of the label introduced into the protein by one intraventricular injection of l -leucine-l-14C. The half life of the enzyme was 2.84 ± 013 days. 相似文献
9.
Abstract— The properties of the enzyme acetylcholinesterase (EC 3.1.1.7) from mouse brain, erythrocytes and muscle were investigated. The enzymes were examined by gel filtration in Sephadex G-200, polyac-rylamide gel electrophoresis, immunological reactions, active-site labelling with tritiated di-isopropyl-phosphorofluoridate and also their kinetic properties were compared. All three enzymes appeared to have a single active small mol. wt. component of 80,000 to 82,000 which produced higher mol. wt. forms by aggregation. The partial purification of the enzyme from brain was achieved by affinity chromatography and this product was used to prepare antibodies. The purified immunoglobulin was shown to react with all three enzymes. 相似文献
10.
Abstract— The acetylcholinesterase (AChE) of intact iris, the so-called external AChE, differs in several respects from the AChE in an homogenate of iris, called the total AChE. Maximum enzyme activities of the external and total AChE were obtained with an ACh concentration of 10 and 1.3 m m , respectively. The total AChE exhibited substrate inhibition at high substrate concentrations, whereas the external enzyme did not exhibit substrate inhibition in the range studied. The external AChE activity, when measured at 1.3 m m -ACh. accounted only for 12% of the total enzyme activity. After irreversible inhibition of AChE with diisopropylfluorophosphate (DFP) or methylisocyclopentylfluorophosphate (soman) the external AChE recovered to almost normal values after 48 h, whereas only 30% of the total AChE recovered during this period. Pupillographic studies after inhibition with DFP demonstrated that pupillary diameter had reached normal size after 24 h.
Destruction of the cholinergic input to iris reduced the total AChE activity by 40%, but did not alter the external AChE activity nor its rate of recovery after DFP inhibition. The specific activities of total AChE and total choline acetyltransferase were significantly higher in the sphincter than in the dilator muscle. After such denervation of iris the greatest reduction in total AChE and choline acetyltransferase were found in the sphincter region. After treatment with DFP the total AChE was inhibited to the same extent and recovered at the same rate in both regions.
After extraction of AChE from iris with various salt solutions and detergents, the particulate enzyme recovered faster than the soluble enzyme from DFP inhibition. 相似文献
Destruction of the cholinergic input to iris reduced the total AChE activity by 40%, but did not alter the external AChE activity nor its rate of recovery after DFP inhibition. The specific activities of total AChE and total choline acetyltransferase were significantly higher in the sphincter than in the dilator muscle. After such denervation of iris the greatest reduction in total AChE and choline acetyltransferase were found in the sphincter region. After treatment with DFP the total AChE was inhibited to the same extent and recovered at the same rate in both regions.
After extraction of AChE from iris with various salt solutions and detergents, the particulate enzyme recovered faster than the soluble enzyme from DFP inhibition. 相似文献
11.
Abstract— AChE activity and protein content in chicken ALD and PLD muscles was studied during pre- and postnatal development. Protein content in both muscles increased whereas AChE activity increased in ALD and decreased in PLD during development. All studied values reached the steady-state 3 weeks after hatching.
Electrophoretic separation of the samples showed three molecular forms of AChE present in both adult ALD and PLD muscles. Two molecular forms in ALD muscle increased slowly, one form quickly. On the other hand, the activity of AChE forms in PLD muscle decreased with different rates. It appears from these results that the multiple molecular forms of AChE in muscles are not of the same physiological importance. 相似文献
Electrophoretic separation of the samples showed three molecular forms of AChE present in both adult ALD and PLD muscles. Two molecular forms in ALD muscle increased slowly, one form quickly. On the other hand, the activity of AChE forms in PLD muscle decreased with different rates. It appears from these results that the multiple molecular forms of AChE in muscles are not of the same physiological importance. 相似文献
12.
Abstract— Acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) is carried down mammalian nerve fibres by the fast axoplasmic transport system. This conclusion was derived from experiments involving the ligation of cat sciatic nerves at two sites placed 83.5 mm apart. The enzyme accumulated in segments of nerve proximal to the upper ligation in a linear fashion over a period of at least 20 h. At approximately 5 h the accumulation of enzyme ceased in the nerve segment proximal to the distal ligation within the isolated length of nerve, an observation indicating that the portion of AChE free to move within the isolated nerve had been depleted during this period of time. The freely moving fraction of AChE was estimated to be 15% of the total enzyme activity present in the nerve (10% in the proximo-distal direction and 5% in the retrograde direction). The rate of AChE downflow (as estimated from the intercept of the curve plotting accumulation with the line denoting when depletion started) was 431 mm/day within a 95% confidence interval of 357–543 mm/day. In view of the variability, our results demonstrated that AChE was being carried by the fast axoplasmic transport system, which in earlier studies was estimated to have a characteristic rate close to 410 mm/day.
An accumulation of AChE was also found on the distal side of the ligations that represented a movement of AChE in the distal-proximal direction in the fibres. This retrograde transport was smaller in amount (about one-half) than the proximo-distal rate of transport, or close to 220 mm/day. The rate of AChE transport was discussed in relation to the 'transport filament' hypothesis of fast axoplasmic transport. 相似文献
An accumulation of AChE was also found on the distal side of the ligations that represented a movement of AChE in the distal-proximal direction in the fibres. This retrograde transport was smaller in amount (about one-half) than the proximo-distal rate of transport, or close to 220 mm/day. The rate of AChE transport was discussed in relation to the 'transport filament' hypothesis of fast axoplasmic transport. 相似文献
13.
INCREASED RATE OF ACETYLCHOLINESTERASE SYNTHESIS IN DIFFERENTIATING NEUROBLASTOMA CELLS 总被引:3,自引:1,他引:2
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When neuroblastoma cells (N18) in vitro are maintained in the absence of serum, the specific activity of AChE begins to rise rapidly after an initial lag period of about 2–3 days, reaching a maximum level (10–20-fold increase) by 7 days after induction. In order to clarify the mechanism of induction, it was necessary to measure the rate of AChE synthesis and its sensitivity to metabolic inhibitors. Return of enzymatic activity after irreversible inhibition of AChE in "differentiated" cells was blocked by cycloheximide, but not by cordycepin or actinomycin D, suggesting that protein but not mRNA synthesis was required for replacement. By using the initial rate of this replacement as a measure of the rate of synthesis of the enzyme, it was shown that cells which had differentiated in the absence of serum synthesized AChE 50-fold faster on a specific activity basis than their undifferentiated counterparts. In contrast, cordycepin effectively blocked the increase in the rate of AChE synthesis that occurs as a result of serum deprivation, indicating that the induction process itself requires the synthesis of new mRNA. Axonation, another index of differentiation, was not completely blocked by inhibition of RNA or protein synthesis and presumably utilizes only pools of pre-existing structural proteins. 相似文献
14.
—By assay of acetylcholine hydrolysis to measure total cholinesterase activity and acetyl-β-methylcholine hydrolysis to measure acetylcholinesterase (E.C 3.1.1.7) activity, patterns of regeneration of enzyme activity were measured in seven areas of brain, cerebrospinal fluid and plasma of cats after administration of an irreversible inhibitor. Halftimes of recovery of total cholinesterase in the brain tissues ranged from 0·9 to 3·8 days (av = 2·5 days) and acetylcholinesterase recovery halftimes ranged from 1·2 to 5·3 days (av = 3·6 days). Regeneration of total cholinesterase was also followed in subcellular fractions of guinea-pig and rat brains after similar inhibition. In both species, the fastest recovery occurred in the soluble fraction with halftimes of 1·8 and 1·6 days, while the synaptosomal fractions exhibited the slowest recoveries with halftimes of 8·3 and 4·1 days. Regeneration of activity in plasma and CSF most nearly resembled that of the soluble brain fraction. 相似文献
15.
抗性小菜蛾的乙酰胆碱酯酶敏感性 总被引:23,自引:7,他引:23
用FAO推荐的点滴法测定了小菜蛾Plutella xylostella(Linn.)对马拉硫磷和西维因的抗药性,并对抗性?和敏感(S)小菜蛾幼虫的胆碱酯酶(ChE)的性质、活性、动力学和抑制作用进行了研究.广州天河、上海梅陇的小菜蛾对马拉硫磷的抗性分别是江西南昌种群的113.8和27.4倍;它们对西维因的抗性是》3.1倍.R幼虫ChE对乙酰胆碱(ATCh)、丙酰胆碱(PrTCh)和丁酰胆碱(BuTCh)的活性分别是S幼虫的0.3、0.7和9.9倍;它们的Vmax分别是0.4、1.4和7.0倍;Km分别是209.1,87和37.8倍.毒扁豆碱、西维因、对氧磷、敌敌畏和残杀威对R幼虫AChE的抑制中浓度I50,分别是S幼虫的333.3,66.7,17.2,12.5和10.0倍.对氧磷、敌敌畏、残杀威和西维因对R幼虫的双分子速率常数K1,分别是S幼虫的126.4,86.5,32.9和12.3倍.由此可见,AChE敏感度降低是小菜蛾对有机磷和氨基甲酸酯产生抗性的主要机理之一.本文中还讨论了防治抗性小菜蛾的策略. 相似文献
16.
棉铃虫乙酰胆碱酯酶的底物专一性和发育期变化(英) 总被引:2,自引:0,他引:2
通过对河北省邯郸地区和山东省冠县的棉铃虫(Helicoverpa armigera H(?)bner)乙酰胆碱酯酶(AChE)研究,结果表明,棉铃虫乙酰胆碱酯酶对乙酰硫代胆碱(ATCh)和乙酰-β-甲基硫代胆碱(MeTCh)的比活力以及米氏常数(K_(?))值随其发育阶段呈有规律的变化,AChE比活力在幼虫期呈现两个高峰,一个在三龄,另一个在化蛹前;在蛹期AChE比活力没有明显的变化,而且比活力值比较低;到成虫期第4天有一个明显的高峰,比活力值高于其它任何虫态。K_(?)和最大反应速度(V_(max))的变化趋势基本上与比活力一致。棉铃虫AChE的K_(?)和比活力随其生长发育阶段的周期性变化对于指导用有机磷和氨基甲酸酯类杀虫药剂的化学防治具有重要的意义。不同地点采集到的棉铃虫AChE对AICh和MeTCh水解的活化能有所不同,邯郸地区的棉铃虫AChE水解MeTCh的活化能,蛹和成虫是幼虫期的3.9-4.3倍,而冠县棉铃虫水解MeTCh的活化能则变化不大。AChE水解ATCh的活化能,邯郸地区棉铃虫的AChE不同虫态间变化不大,冠县棉铃虫AChE,幼虫和成虫期约是蛹期的4倍。这说明不同生长发育时期,棉铃虫AChE对底物的水解所消耗的能量是不同的。棉铃虫幼虫AChE的最适反应条件是酶量以重量计为50-100mg,反应时间为10-20min,反应温度为35℃,反应体系的pH值为8.0。 相似文献
17.
HIRONOBU OZAKI 《Development, growth & differentiation》1974,16(4):267-279
Acetylcholinesterase during the development of the sea urchin Pseudocentrotus depressus was examined by enzyme assay (the colorimetric method of E llman et al. ), histochemistry (a Cu-thiocholine method), polyacrylamide gel electrophoresis and DEAE-Sephadex ion exchange chromatography.
The enzyme activity is detected in the unfertilized egg, remains low during cleavage, elevates slightly through gastrulation, and then increases rapidly thereafter. The intense activity is localized in the mesenchyme cells associated with the larval skeleton of young pluteus larvae, and their cell membranes and nuclear envelops. Soluble enzyme accounts for 60% of the total activity. The additional 34% is extracted by 1% Triton X-100 from particulates. The soluble enzyme consists of two forms. Both are strongly acidic proteins which are similar in electric charge, but dissimilar in size, being 180,000 and 280,000 in molecular weights. The enzyme released from the membrane by the detergent possesses a component which is not present in the soluble complement of the enzyme. It is not a secondary product of the soluble enzyme interacting with the detergent.
Acetylcholinesterase serves as a marker of late differentiation and regional differentiation in the sea urchin embryo. 相似文献
The enzyme activity is detected in the unfertilized egg, remains low during cleavage, elevates slightly through gastrulation, and then increases rapidly thereafter. The intense activity is localized in the mesenchyme cells associated with the larval skeleton of young pluteus larvae, and their cell membranes and nuclear envelops. Soluble enzyme accounts for 60% of the total activity. The additional 34% is extracted by 1% Triton X-100 from particulates. The soluble enzyme consists of two forms. Both are strongly acidic proteins which are similar in electric charge, but dissimilar in size, being 180,000 and 280,000 in molecular weights. The enzyme released from the membrane by the detergent possesses a component which is not present in the soluble complement of the enzyme. It is not a secondary product of the soluble enzyme interacting with the detergent.
Acetylcholinesterase serves as a marker of late differentiation and regional differentiation in the sea urchin embryo. 相似文献
18.
SOLUBLE AND PARTICLE-BOUND ACETYLCHOLINESTERASE AND ITS ISOENZYMES IN PERIPHERAL NERVES 总被引:2,自引:2,他引:0
Abstract— The distribution of AChE (EC 3.1.1.7) in soluble and particulate fractions of the peripheral nerves of dogs, cats, rabbits and frogs was examined. About 20–30% of the total AChE activity was found in the supernatant fluid after centrifugation (100,000 g for 90 min) of iso-osmotic sucrose homogenates. The effect of different media on the extent of solubilization of the enzyme was studied and Triton X-100 (0.2%) was found to be the most effective. The electrophoretic pattern of AChE in peripheral nerves was also investigated. The 2–3 types of AChE observed previously were found in both particulate and soluble fractions, but the proportions of these forms were different. The most slowly migrating form of AChE is the most firmly bound to nerve membranes. A very small but consistent proportion (3%) of AChE escaped into the medium from surviving dog nerves kept in aerated Ringer solution. It was calculated that the possible contribution of blood AChE contained in the nerve is negligible. Electrophoretograms of AChE released during incubation into Ringer solution were similar in pattern to those found for the soluble fraction. 相似文献
19.
Abstract The resistance mechanism underlying phoxim resistance was investigated through biochemical assays. The results show that there are marked relations between the phosphatase activity and resistance to phoxim. Furthermore, there is a significant difference in the K i values of AChE between the two strains and it is inferred that the change in ability of binding to phoxim is a mechanism responsible for resistance to phoxim. The metabolic inhibitors Triphenyl Phosphate (TPP) and O,O-diethyl-O-phenyl-thiophosphate (SV1 ) displayed significant synergism of phoxim toxicity to the two strains. It implied that esterase might be involved in resistance. However, there is no marked correlation between resistance and CarE. 相似文献
20.