The effect of temperature on the activity of acetylcholinesterase preparations from rat brain

Homogenization of rat brain with dilute buffer shows that about 15% of the acetylcholinesterase is soluble while the remaining 85% is present in a membrane-bound form which can be brought into solution by extraction with Triton X-100. The effect of temperature on the values of V_max and K_m of the buffer-soluble, the membrane-bound and the Triton-soluble forms of acetylcholinesterase have been compared and the results discussed in terms of possible changes in the conformation, dissociation or aggregation of the enzyme molecule.Gradient-gel electrophoresis of the soluble preparations carried out at 4°C or 37°C suggest that the normal tetrameric structure present at 4°C dissociates into monomers and forms some higher molecular weight species at 37°C.The effect of prior storage of the brains in toluene on these properties is also considered.     

Inhibitory effect of verapamil on the acetylcholinesterase activity of skeletal muscle sarcolemma

It has been found that verapamil reversibly inhibits "in vitro" the activity of membrane--bound and solubilized sarcolemmal acetylcholinesterase. The kinetic analysis has demonstrated a competitive type of inhibition at verapamil concentrations less than 100 mkM and a mixed one at higher verapamil concentrations. The apparent Ki values are similar to or approximately 5,0.10(-5) M and similar to or approximately 3,0.10(-4) M for both types of inhibition, respectively. The effect of vereapamil and Ca2+ on acetylcholinesterase is independent and non-competitive. An increase in the ionic strength leads to a decrease of the verapamil-induced inhibition of acetylcholinesterase. It is suggested that verapamil interacts with the anionic groups of both free and acylated enzyme.     

Thermal stability of molecular forms of acetylcholinesterase from muscle microsomes

To establish if the predominant form of acetylcholinesterase in muscle microsomes (4.8S) corresponded to the monomeric or dimeric form of the enzyme we studied the sensitivity to heating of Triton X-100 solubilized extract and that of 4.8S, 10-11S and 13.5S species of the enzyme. Inactivation of soluble acetylcholinesterase began at 45-47 degrees C and was almost complete at 60 degrees C. Sedimentation analysis revealed that the partial loss of activity was due to inactivation of the 4.8S form, although by heating the 13.5S was converted into the 10S enzyme. Inactivation of the 4.8S form began at 45 degrees C, whereas the larger forms required higher temperature. The 4.8S component follows a time course of inactivation which could be fitted by a double exponential equation (when heated at 52 degrees C, almost 83% of the activity showed a short half-life). The 10-11S species was also inactivated following a two step process while the 13.5S enzyme was fairly stable at 52 degrees C. The results show that the lightest component behaves as a monomeric form of acetylcholinesterase.     

The effect of temperature on canine papillary muscle

Measurements of the total duration,t _Aof the action potential for canine papillary muscle in the temperature range 25–45t_A = texp(Q/k_B T),t_A = \tau \exp (Q/k_B T),     

The effect of galactose metabolic disorders on rat brain acetylcholinesterase activity

To evaluate whether in classical galactosemia galactose (Gal), galactose-1-phosphate (Gal-1-P) and galactitol (Galtol) affect brain acetylcholinesterase (AChE) activity, various concentrations (1-16 mM) of these compounds were preincubated with brain homogenates of suckling rats as well as with pure eel Electroforus electricus AChE at 37 degrees C for 1 h. Initially, Galtol (up to 2.0 mM) increased (25%) AChE activity which decreased. thereafter, reaching the control value in high Galtol concentrations. Gal-1-P decreased gradually the enzyme activity reaching a plateau (38%), when incubated with 8-16 mM. However, when the usually found 2 mM of Galtol and 2 mM of Gal-1-P, concentrations in galactosemia were added in the incubation mixture simultaneously, brain AChE was stimulated (16%). Galtol or Gal-1-P modulated brain AChE as well as enzyme activity of E.electricus in the same way. Gal, Glucose (Glu) and glucose-1-phosphate (Glu-1-P) had no effect on AChE activity. It is suggested that Galtol as well as Gal-1-P can affect acetylcholine degradation acting directly on AChE molecule. Consequently the direct action of these substances on the enzyme might explain the brain cholinergic dysfunction in untreated galactosemia patients.     

Alkaline treatment of muscle microsomes releases amphiphilic and hydrophilic forms of acetylcholinesterase.

To obtain information about the mode of attachment of amphiphilic monomers of acetylcholinesterase (AChE) in sarcoplasmic reticulum (SR) of skeletal muscle, attempts were made to release the enzyme by alkaline hydroxylamine. About half of the activity measured in microsomes preincubated with 0.5% (w/v) Triton X-100 is detached by incubation of SR with bicarbonate buffer (pH 10.5). Addition of 1 M hydroxylamine to the alkaline buffer did not improve enzyme solubilization. Molecular forms of 16S (A12), 10.5S (G4) and 4.0S (G1) are separated by sedimentation analyses of Triton X-100 or bicarbonate-solubilized AChE. Monomeric AChE, released under alkaline conditions (G1A), displays amphiphilic properties. G1A, but not G4 and A12, forms are retained in a phenyl-Sepharose column and this allows its separation from hydrophilic forms. Isolated monomers extracted with Triton X-100 (G1D) or alkaline buffer showed identical kinetic behaviour. The two forms reacted with lectins in a similar manner. However, thermal inactivation experiments revealed that about 90 and 40% of the activity in the G1D and G1A forms were lost by heating at 50 degrees C, following the same rate constant (k = 0.130 min-1). Addition of Triton X-100 to the G1A form leads to an increase of its thermal sensitivity, the enzyme being fully inactivated very rapidly (k = 0.230 min-1). The results suggest that the hydrophobic moiety of the enzyme might be exposed or hidden depending on the environmental hydrophobicity. Changes in the composition of the solvent will determine the final conformational state of the protein.     

Alanine reverses the inhibitory effect of phenylalanine on acetylcholinesterase activity

The aim of this work was to evaluate, in vitro, the effect of L-alanine (Ala) on suckling rat brain acetylcholinesterase (AChE) and on eel Electrophorus electricus pure AChE inhibited by L-phenylalanine (Phe) as well as to investigate whether Phe or Ala is a competitive inhibitor or an effector of the enzyme. AChE activity was determined in brain homogenates and in the pure enzyme after 1 h preincubation with 1.2 mM of Phe or Ala as well as with Phe plus Ala. The activity of the pure AChE was also determined using as a substrate different amounts of acetylthiocholine. Ala reversed completely the inhibited AChE by Phe (18-20% in 500-600 microM substrate, p<0.01). Lineweaver-Burk plots showed that Vmax remained unchanged. However, Km was found increased with Phe (150%, p<0.001), decreased with Ala alone (50%, p<0.001) and unaltered with Phe plus Ala. It is suggested that: a) Phe presents a competitive inhibitory action with the substrate whereas Ala a competitive activation; b) Ala competition with Phe might unbind the latter from AChE molecule inducing the enzyme stimulation; c) Ala might reverse the inhibitory effect of Phe on brain AChE in phenylketonuric patients, if these results are extended into the in vivo reality.     

Effect of heat shock on acetylcholinesterase activity in chick muscle cultures

The effect of heat shock was studied on the acetylcholinesterase activity of chick muscle primary cultures. In cultures transferred from 37 degrees C to 45 degrees C, a sharp drop in activity was followed by rapid spontaneous recovery. The time of onset of recovery resembled the time needed for expression of heat shock proteins. In cultures exposed to heat shock at 45 degrees C and allowed to recover at 37 degrees C, reappearance of acetylcholinesterase activity did not involve de novo protein synthesis since it was not prevented by cycloheximide. Our data raise the possibility of a role for heat shock proteins as molecular chaperones in rescuing heat-denaturing acetylcholinesterase.     

The effect of some amine oxides and disulphide compounds on the activity of acetylcholinesterase.

Three amine oxides, two aliphatic and one aromatic, and 2,2'-dithiodipyridine were found to be weak reversible inhibitors of acetylcholinesterase. 5,5'-Dithiobis-(2-nitro-benzoic acid) had no effect on the enzyme under the conditions of the Ellman spectrophotometric assay.     

The neuropeptide TRH has a minor effect on the enzymatic activity of acetylcholinesterase in vitro

The neuropeptide thyrotropin-releasing hormone (TRH) elicits a variety of physiological effects of which some are due to cholinergic mechanisms. TRH modulates in vivo the effects of compounds affecting acetylcholinesterase (AChE). In the present study the in vitro effects of TRH on the activity of AChE were explored. TRH has no effect at physiologically relevant concentrations. At unphysiologically high concentrations (>5 mM) a slight inhibition was found. This was noticed also when the enzyme was exposed to the amide-free tripeptide analog p-Glu-His-Pro. We conclude that any cholinergic effect of TRH observed in vivo is unlikely to be due to a direct interaction of the peptide with AChE.     

Inhibition effect of graphene oxide on the catalytic activity of acetylcholinesterase enzyme

Variations in the enzyme activity of acetylcholinesterase (AChE) in the presence of the nano‐material, graphene oxide (GO), were investigated with the use of molecular spectroscopy UV‐visible and fluorescence methods. From these studies, important kinetic parameters of the enzyme were extracted; these were the maximum reaction rate, V_m, and the Michaelis constant, K_m. A comparison of these parameters indicated that GO inhibited the catalytic activity of the AChE because of the presence of the AChE–GO complex. The formation of this complex was confirmed with the use of fluorescence data, which was resolved with the use of the MCR‐ALS chemometrics method. Furthermore, it was found that the resonance light‐scattering (RLS) intensity of AChE changed in the presence of GO. On this basis, it was demonstrated that the relationship between AChE and GO was linear and such models were used for quantitative analyses of GO. Copyright © 2015 John Wiley & Sons, Ltd.     

The inhibitory effect of tetramethylethylene diamine on water soluble and membrane bound acetylcholinesterase activity

• 1.1. The inhibitory effect of N,N,N′,N′-tetramethylethylene diamine (TEMED) on water soluble (WSAChE) and membrane bound (MBAChE) acetylcholinesterase was investigated.
• 2.2. TEMED (0.5–4.0 mM) reversibly inhibited WSAChE activity (18–62%) and MBAChE (20–61%) in a concentration dependent manner.
• 3.3. The IC₅₀ being about 2.8 mM for WSAChE and 2.6 mM for MBAChE.
• 4.4. Lineweaver-Burk plots indicated that the nature of inhibition is noncompetitive for both water soluble and membrane bound acetylcholinesterase, with K_m values 68 μM and 123 μM respectively.
• 5.5. An Arrhenius plot showed that the transition temperature (TT) is unaffected in the presence of TEMED.
• 6.6. The activation energy was increased below and above TT in the case of WSAChE only.
• 7.7. On the basis of this behaviour of TEMED with AChE. it can be proposed that it can be used as an eluting agent for the bounded AChE to affinity ligand and may have beneficial action on the reactivatability of irreversibly-inhibited AChE due to its structure.
• 8.8. Moreover there is a possibility that it can be used as a therapeutic agent for the treatment of Alzheimer's disease, myasthenia gravia and glaucoma like some other inhibitors of AChE.

     

The effect of temperature on potassium equilibria in chick embryo muscle

The effect of temperature upon the exchange rates between intra- and extracellular potassium in chick embryo muscle was determined by the use of radioactive potassium. The temperature coefficient of at least four-fifths of the cell potassium is large. At temperatures below 15 degrees C., potassium is lost from the cell and is regained on warming. The results suggest the possibility that 20 per cent or less of the cell potassium may differ from the rest by being more rapidly exchangeable with the medium.     

The effect of temperature on conduction velocity in human muscle fibers

The effects of variation of intramuscular temperature (T) on conduction velocity (CV) of the action potential along single human muscle fibers of the biceps brachii was studied in situ in 15 normal volunteers (mean age 39 years, range 21–62 years). Cooling was obtained by direct application of ice over a rectangular skin region including the stimulating and recording area. The intramuscular T was monitored by a needle thermocouple (copperconstantane). In all the 24 muscle fibers studied, a linear relationship was observed between CV and T. The slopes of the regression lines, ranging between 0.190 and 0.079 m/s, were positively correlated with the starting CV at 36°C ranging between 2.2 and 5.2 m/s. If conduction changes are expressed as a percentage of the basal CV at 36°C, the CV/T coefficient is the same for all the fibers and independent of the individual CV: 3.4% of CV/°C.     

The effect of repetitive ankle perturbations on muscle reaction time and muscle activity

The use of a tilt platform to simulate a lateral ankle sprain and record muscle reaction time is a well-established procedure. However, a potential caveat is that repetitive ankle perturbation may cause a natural attenuation of the reflex latency and amplitude. This is an important area to investigate as many researchers examine the effect of an intervention on muscle reaction time. Muscle reaction time, peak and average amplitude of the peroneus longus and tibialis anterior in response to a simulated lateral ankle sprain (combined inversion and plantar flexion movement) were calculated in twenty-two physically active participants. The 40 perturbations were divided into 4 even groups of 10 dominant limb perturbations. Within-participants repeated measures analysis of variance (ANOVA) tests were conducted to assess the effect of habituation over time for each variable. There was a significant reduction in the peroneus longus average amplitude between the aggregated first and last 10 consecutive ankle perturbations (F_2.15,45.09 = 3.90, P = 0.03, ɳ_p² = 0.16). Authors should implement no more than a maximum of 30 consecutive ankle perturbations (inclusive of practice perturbations) in future protocols simulating a lateral ankle sprain in an effort to avoid significant attenuation of muscle activity.     

Ambient temperature effects on brain and spinal cord acetylcholinesterase activity of two poikilotherms

The changes in AChE activity and protein content following cold or heat exposure and heat death were determined in the brain and spinal cord of both Rana ridibunda and Chalcides ocellatus. Cold exposure (10 degrees C) caused a decrease in the enzyme activity and protein content of both animals. Exposure to heat (36-40 degrees C) increased markedly the AChE activity and the amount of protein in the two experimental animals. Heat death was found to be associated with a prominent decrease in enzyme activity and the protein level of the brain and spinal cord of the two poikilotherms.