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Query: EC:3.1.1.7 (acetylcholinesterase)
 28,390 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The phospholipid, cholesterol, SH-group content and acethylcholinesterase activity in ultrasonic sarcolenima fragments precipitating at 105000 g were much greater than in fragments precipitating at 3000 g. On the basis of the results obtained it is suggested that the 3000 g sediment consists largely of network of collagen fibrils, while the 105 000 g sediment is mainly composed by the plasma membrane fragments. It is shown that the amount of SH-groups in intact sarcolemma available for 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNG) in the presence of sodium dodecylsulphate was about twice as much as that in the absence of sodium dodecylsulphate. As to the sonicated fractions of sarcolemma (sediment and supernatant at 105 000 g) and high ionic strength extract of sarcolemma the amount of SH-groups available for DTNB in the presence and in the absence of sodium dodecylsulphate was similar. A decrease was observed in acetylcholinesterase stability after sonication (sediment and supernatant at 105 000 g). The normal Michaelis kinetics was found for the acetylcholinesterase of sarcolemma fractions solubilized by sonication.
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PMID:[Acetylcholinesterase activity, phospholipids, cholesterol and sulphydryl group content in sarcolemma fragments]. 55 57

Comparative kinetic studies of membrane-bound and solubilized sarcolemmal acetylcholinesterase reveal some difference in concentration-activity curves. A deviation from normal Michaelis-Menten kinetics is found in case of membrane-bound acetylcholinesterase. The solubilization of sarcolemma by a solution of high ionic strength or by sonication normalizes the reaction kinetics. It is shown that the amount of SH-groups in intact sarcolemma available for DTNB in the presence of sodium dodecyl sulphate, is about twice of that in the absence of sodium dodecyl sulphate. In case of the solubilized fraction of sarcolemma (by a solution of high ionic strength or by sonication), the amount of SH-groups available for DTNB in the presence and in the absence of sodium dodecyl sulphate is similar.
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PMID:[Comparative study of solubilized and membrane-bound acetylcholinesterase of sarcolemma]. 102 94

Rapid enzyme microassays for the detection of resistance due to organophosphate and carbamate in individual field-collected strains of Culex quinquefasciatus adults were conducted. These tests allowed accurate differentiation by eye, on the basis of color changes of susceptible and resistant individuals. Two separate tests were conducted for the biochemical assays. In the insensitive acetylcholinesterase (AChE) test, acetylthiocholine iodide (ACTH) and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) were used as substrate and coupling agent respectively. The resulting yellow chromophore indicated AChE activity. Test results showed that the color intensity decreased as increasing concentrations of propoxur were added, thereby confirming the susceptibility of the enzyme to inhibitor. Assay of non-specific esterase however, indicated elevated levels which were correlated with degree of malathion resistance. Electrophoretic data revealed the presence of 2 esterase bands in all strains. It was concluded that such a pattern was not contributory to malathion resistance in adults.
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PMID:Determination of insecticide susceptibility in Culex quinquefasciatus Say adults by rapid enzyme microassays. 148 1

The reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) with thiols is sensitive to daylight, in particular to ultraviolet radiation at wavelengths around 325 nm. Exposure to light at the absorbance maximum of the yellow product (the thionitrobenzoate ion) at 410 nm had no effect on the reaction. The light-sensitive species is apparently the DTNB, because a spectral-irradiation experiment showed that the wavelength of light that produced the maximum rate of absorbance change coincided with the peak absorbance of DTNB, and it was well separated from the thionitrobenzoate absorbance peak. Ascorbate is ineffective as a stabilizer and can produce an apparent increase in the rate of DTNB destruction. In a practical example we found the light interference to be severe when hydrolysis of propionylthiocholine by plasma cholinesterase (EC 3.1.1.8) was measured after a 20-min incubation. The apparent cholinesterase activity in clear glass or plastic tubes exposed to diffuse daylight could be decreased to 25% of the value obtained for samples in light-excluded tubes. We recommend the reaction be carried out in artificial room light, with total elimination of daylight, because window glass does not sufficiently attenuate 325-nm wavelength irradiation.
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PMID:Effect of daylight on the reaction of thiols with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid). 366 50

It has been found that epinephrine reversibly inhibits in vitro the acetylcholinesterase activity of intact sarcolemma and partially purified preparation of sarcolemmal acetylcholinesterase. The kinetics analysis has demonstrated a non-competitive type of inhibition with the apparent Ki values of about 4 . 10(-4) M. An increase in the ionic strength intensities the epinephrine-induced inhibition, the effect of CaCl2 being essentially more noticeable than that of NaCl. The number of SH-groups of the sarcolemma available for DTNB decreases in the presence of epinephrine and increases in the presence of CaCl2 or NaCl. The sulfhydryl reagents (e. g. DTNB, NEM) have no effect n the acetylcholinesterase activity of sarcolemma. It is assumed that the inhibiting effect of epinephrine may be due to the conformational changes in the acetylcholinesterase molecules.
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PMID:[Inhibiting effect of adrenaline on the acetylcholinesterase activity of skeletal muscle sarcolemma]. 729 19

Rat cholinesterase (ChE) activities were measured by DTNB-method after an oral administration of fenitrothion, and the following facts were observed. The inhibited plasma ChE (pseudo ChE) obtained within several hours after the administration was spontaneously reactivated at 10 degrees C or over, whereas no reactivation was observed at 1 degree C. Neither red blood cell nor brain ChE (true ChE) was spontaneously reactivated. In vitro, the spontaneous reactivation was also observed in rat plasma ChE inhibited by oxon-type of fenitrothion. In case the activity of plasma ChE obtained 30 min after administration was determined by delta pH-method, the activity was higher than the actual value, because of the spontaneous reactivation taking place during an incubation for 1 h at 37 degrees C. It is suggested from these results that an utilization of delta pH-method is unsuitable for the measurement of the activity of inhibited ChE which is spontaneously reactivated.
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PMID:Difference in fenitrothion-inhibited rat plasma cholinesterase activities determined by delta pH-method and DTNB-method, due to spontaneous reactivation. 729 16

Inhibited cholinesterase in tissues of animals exposed to carbamate pesticides is known to reactivate readily, presenting considerable problems in the accurate assessment of cholinesterase activity in these tissues. Decarbamylation of cholinesterase is favored when the tissue samples are diluted and/or are incubated for an extended time. The present study was performed to identify modifications of the commonly used spectrophotometric assay for cholinesterase activity that would minimize spontaneous reactivation of enzyme activity. Those modifications included preincubation of concentrated tissue with concentrated chromogen (i.e., DTNB), dilution to final reaction volume immediately before measurement, and measurement of cholinesterase over a short period of time (5-10 min). The Ellman assay with and without modifications was performed using a microtiter plate reader on tissues from carbaryl-treated rats:undiluted plasma, diluted erythrocytes (1:25), minimally diluted erythrocytes (1:2), diluted brain (1:100), or minimally diluted brain (1:2). The results were compared to cholinesterase activities obtained using a radiometric method which employs minimally diluted tissue and short incubation times. The degree of cholinesterase inhibition for undiluted or minimally diluted tissue assayed by the modified method agreed with those obtained using the radiometric method. Even if the tissues were diluted immediately before assay, however, significant reactivation occurred by the time the first measurements were made by the conventional method. Furthermore, significant spontaneous reactivation may still occur using the modified method if the assay is run for more than 10 min. Use of this modified Ellman method will enable more accurate estimation of in vivo cholinesterase activity in animals treated with carbamates.
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PMID:A modified spectrophotometric method appropriate for measuring cholinesterase activity in tissue from carbaryl-treated animals. 840 82

An automated method using 2,2'-dithiodipyridine (2-PDS) as chromophore for determination of whole-blood cholinesterase activity was developed. Assay procedures, optimal concentrations of chromophore and substrate, detection limit, precision, backgrounds, and sensitivity of the method were compared with those of an earlier automated method based on the Ellman method and using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as chromophore. The new method has the advantages of automation (resulting in increase throughput rate and decrease in amount of reagents used) and good precision and sensitivity. Sample dilutions also are reduced in the new method because hemoglobin interference is less.
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PMID:Automated spectrophotometric method using 2,2'-dithiodipyridine acid for determination of cholinesterase in whole blood. 863 43

The Ellman method for cholinesterase determination is a spectrophotometric method which entails the use of 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB) as a chromogen and records the level of cholinesterase activity as the change in absorbance at 412 nm. Although this procedure commonly poses no problem, an exception arises when analyzing tissues rich in hemoglobin, because hemoglobin also optimally absorbs light at 400-430 nm. Use of 6,6'-dithiodinicotinic acid (DTNA) might be a solution because, like DTNB, it also is a chromogen for sulfhydryl groups, but with an optimal absorption wavelength of 340 nm (ie removed from the hemoglobin absorbance maximum). Our validation studies indicate that although DTNA is a slightly less efficient indicator of sulfhydryl group concentration, DTNA yields similar activity and degree of enzyme inhibition in tissues from control and treated animals. Moreover, because the assay is read at 340 nm instead of 412 nm, the DTNA assay is markedly more sensitive for determining cholinesterase activity in hemoglobin-rich tissues. Since the advantages of the DTNA method far outweigh the disadvantages, it should be regarded as a sensitive and convenient procedure for determining cholinesterase activity, especially in hemoglobin-rich tissues.
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PMID:Validation of the use of 6,6'-dithiodinicotinic acid as a chromogen in the Ellman method for cholinesterase determinations. 882 40

A simple and effective method was set up to purify acetylcholinesterase (AChE, EC3.1.1.7) from the cotton aphid, Aphis gossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamide affinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100- and 33,680-fold with a yield of 30.3 and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDS-PAGE. However, three bands resolved on PAGE gel electrophoresis, leading to the inference that native AChE exists in three forms. The optimum conditions for measuring the activity of purified AChE with kinetic method were 0.02M phosphate buffer, pH7.2, 0.02 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and 25 degrees C. Investigation also revealed that crude extract and purified AChE had different kinetic characteristics and inhibitory properties. They responded differently to varied DTNB, ATChI, and phosphate buffer ion concentrations, as well as pH, temperature, and inhibitors. The purified AChE was more sensitive to eserine, methamidophos, and pirimicarb. Especially for resistant aphids, the sensitivity of purified AChE to methamidophos and pirimicarb was enhanced 6.43 and 11.73 times, respectively. We infer that one or more factors in the crude extract from the resistance strain have more influence on AChE sensitivity. Further study is needed to investigate the basis of these observations.
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PMID:Purification and characterization of acetylcholinesterase from cotton aphid (Aphis gossypii Glover). 1221 Sep 59


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