Kinetic behaviour of acetylcholinesterase from muscle microsomal membranes

In order to determine whether catalytic hydrolysis of acetylcholine, observed in muscle microsomes enriched in sarcoplasmic reticulum membranes, was carried out by true acetylcholinesterase we studied the substrate specificity of this enzyme, its kinetic behaviour and its sensitivity against several reversible inhibitors. The results showed that the enzyme from muscle microsomes had acetylcholine (or acetylthiocholine) as the preferent substrate and was also able to hydrolyze acetyl-beta-methylcholine. The enzyme had a Km of 100-120 microM, being inhibited by a high substrate concentration. Acetylcholinesterase in this source was competitively inhibited by BW-284-c-51, eserine and decamethonium with ki values of 0.025 microM, 0.021 microM and 65 microM, respectively. The enzyme was poorly inhibited by the pseudocholinesterase inhibitor ethopropazine. The results show that the hydrolytic enzyme is indeed acetylcholinesterase.     

Reciprocal regulation of acetylcholinesterase and butyrylcholinesterase in mammalian skeletal muscle

Developmental regulation, from the fetal period to 11 months of age, and the influence of denervation on the appearance and disappearance of the molecular forms of acetylcholinesterase (AchE) and butyrylcholinesterase (BuchE) in rat skeletal muscle were examined. The enzyme forms were extracted from anterior tibialis in 0.01 M sodium phosphate buffer, pH 7.0, containing 1 N NaCl, 0.01 M EGTA, 1% Triton X-100, and a cocktail of antiproteases, and analyzed by velocity sedimentation on 5-20% linear sucrose gradients. Three principal forms, denoted by sedimentation coefficients of 4, 10.8, and 16 S, were observed in muscle from all age groups. The amounts of each of the molecular forms of AchE and BuchE in skeletal muscle exhibited distinct and reciprocal patterns of appearance and disappearance during pre- and postnatal development. In tissue derived from animals less than 2 weeks of age, BuchE represented the predominant component of activity in the 4 S form, was present equally with AchE in the 10.8 S form, and was subordinate to AchE in the 16 S form. Between 1 and 2 weeks of age a progressive increase in AchE activities coincident with a reduction in BuchE activities resulted in inversion in the amounts of the two enzymes present in adult muscle. Denervation of muscle caused a dramatic reduction in the presence of AchE molecular forms with no discernable influence on the presence of BuchE molecular forms. These results indicate that biosynthesis of BuchE is strictly regulated in a reciprocal manner with that of AchE, and that BuchE metabolism is independent of the state of muscle innervation. Increased synthesis of AchE and either reduced synthesis or increased degradation of BuchE can account for the reciprocal regulation of these enzymes. These characteristics of mammalian muscle contrast sharply with characteristics deduced for avian tissue (Silman et al. (1979) Nature (London) 280, 160-162). The innervation-independent metabolism of BuchE and the diverse modes of its regulation in different tissue from different species signify that BuchE function may be unrelated to cholinergic neurotransmission.     

Association of tailed acetylcholinesterase to lipidic membranes in mammalian skeletal muscle

Tailed acetylcholinesterase (AChE) was studied in three subcellular membrane fractions of mouse skeletal muscle: a fraction enriched in isolated motor endplates (C), an extrasynaptic membrane fraction (A) and a microsomal fraction (S). In the (C) fraction, tailed asymmetric 16S AChE required high salt conditions to be extracted, while in (A) and (S) microsomal membranes, a collagenase sensitive 16S form, was extracted by detergent alone. This apparent “hydrophobic” property suggests that there is a pool of 16S AChE which is probably bound to lipidic membranes. The detergent extractable (DE) 16S AChE was not concentrated in motor endplate-rich regions and differential inhibition of external and internal AChE demonstrated that it could have both intra- and extracellular locations in the adult differentiated muscle fibres.     

Influence of substrate surface loading on the kinetic behaviour of aerobic granules

In the aerobic granular sludge reactor, the substrate loading is related to the size of the aerobic granules cultivated. This study investigated the influence of substrate surface loading on the growth and substrate-utilization kinetics of aerobic granules. Results showed that microbial surface growth rate and surface biodegradation rate are fairly related to the substrate surface loading by the Monod-type equation. In this study, both the theoretical maximum growth yield and the Pirt maintenance coefficient were determined. It was found that the estimated theoretical maximum growth yield of aerobic granules was as low as 0.2 g biomass g^–1 chemical oxygen demand (COD) and 10–40% of input substrate-COD was consumed through the maintenance metabolism, while experimental results further showed that the unit oxygen uptake by aerobic granules was 0.68 g oxygen g^–1 COD, which was much higher than that reported in activated sludge processes. Based on the growth yield and unit oxygen uptake determined, an oxidative assimilation equation of acetate-fed aerobic granules was derived; and this was confirmed by respirometric tests. In aerobic granular culture, about 74% of the input substrate-carbon was converted to carbon dioxide. The growth yield of aerobic granules was three times lower than that of activated sludge. It is likely that high carbon dioxide production is the main cause of the low growth yield of aerobic granules, indicating a possible energy uncoupling in aerobic granular culture.     

Inorganic ion effects on the kinetic parameters of acetylcholinesterase

EARLY work on the effects of inorganic ions on the activity of acetylcholine acetyl-hydrolase (EC 3.1.1.7; AChE) from various sources has been summarized by COHEN and OOSTERBAAN (1963) and many other reports have been published subsequently (CHANGEUX, 1966; CRONE, 1973; HELLER and HANAHAN, 1972; IVANOVA, 1967; KITZ, BRASWELL and GINSBURG, 1970; ROUF-CALIS and QUIST, 1972; ROUFOGALIS and THOMAS, 1968; WINS, SCHOFFENIELS and FOIDART, 1970). Despite this work, no comprehensive study has yet been made to determine whether the observed effects are specific to particular ions or dependent only on the ionic strength of the medium (CHANGEUX, 1966). In some cases, specific ion effects have been observed (CHANGEUX, 1966; HELLER and HANAHAN, 1972; ROUFOGALIS and QUIST, 1972; ROUFOGALIS and THOMAS, 1968) at salt concentrations from 600 mM to below 1 mM, but the studies were not detailed enough and in some cases the total ionic strength was not rigidly controlled, so that no general deductions can be drawn. We have studied the hydrolysis of acetylcholine (ACh) by bovine erythrocyte AChE at subinhibitory substrate concentrations, and now present our results on the effect of inorganic salts at varying ionic strength on the kinetic parameters K_m, and V_max. The present work shows that this hydrolysis follows simple Michaelis kinetics very closely, and therefore these two constants suffice to define the complete pattern of initial reaction velocity as a function of substrate concentration (ATKINSON, 1966).     

Neurotrophic control of 16S acetylcholinesterase from mammalian skeletal muscle in organ culture

The effects of rat obturator nerve extracts on total and 16S acetylcholinesterase (AChE) activity were studied in endplate regions of denervated anterior gracilis muscles maintained in organ culture for 48 hr. The decrease of total AChE activity in cultured muscles was similar to that observed in denervated muscles in vivo. This decrease in activity was partly prevented by addition of either 100 or 200 μl nerve extract (2.7 mg/ml protein) to the nutrient medium. Nerve extract treatment also decreased the release of AChE activity from the muscle into the bathing medium. Conversely, rat serum (20 μl; 90 mg/ml protein) had no effect on total AChE activity in muscle endplates, nor on release of the enzyme by the muscle. The 16S form of AChE was confined to motor endplate muscle regions and its activity was drastically decreased by denervation in both organ culture and in vivo preparations in a comparable manner. Nerve-extract supplemented cultures contained a significantly (p ? 0.001) larger amount of endplate 16S AChE activity (140–145%) than the corresponding controls (100-). Our results suggest that some nerve soluble substance, other than serum contaminants or 16S AChE itself, affects the maintenance of 16S AChE at the neuromuscular junction.     

Influence of experimental diabetes on the kinetic behaviour of renal cortex hexose monophosphate dehydrogenases

1. Short term (1-2 hr) and long-term (2 days) effects of experimental alloxan induced diabetes on the kinetics of the renal hexose monophosphate shunt dehydrogenases are reported. 2. Alloxan diabetes for 2 days significantly increased kidney weight (16%) adding about 80 mg/day per g of kidney. No significant changes were found in renal growth 1-2 hr after alloxan injection. 3. Under these experimental conditions, the activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase significantly increased (103 and 33% respectively) at all substrate concentrations, without affecting the KmS of either enzyme. 4. There was no effect of alloxan on the activity of these enzymes at 1-2 hr. Saturation curves show that all enzymes exhibited a M-M kinetic without evidence of sigmoidicity. 5. The results suggest that increased renal hexose monophosphate dehydrogenases activities are due to increased concentrations of the rate limiting proteins. 6. The relationship between these changes and renal hypertrophy is also discussed.     

Immunochemical studies of mammalian brain acetylcholinesterase.

Abstract— Specific antibodies were raised in rabbits to acetylcholinesterase (AChE) from bovine caudate nucleus and the‘native’(14S + 18S) and globular (11S) forms of AChE from eel electric tissue. All AChE preparations were purified by affinity chromatography to a specific activity of 100–400 mmol acetylthiocholine hydrolyzed/mg protein/h. Antigenic specificities of the different enzyme forms were studied by immunodiffusion, Immunoelectrophoresis and micro-complement fixation. Minor differences in antigenic determinants were observed between the different molecular forms of electric tissue AChE. In crossover experiments using both eel AChE and bovine caudate AChE antisera there was complete absence of cross reactivity between the mammalian brain AChE and the different molecular forms of the electric tissue enzyme. Brain AChE activity was inhibited up to 50% in the presence of its antiserum.     

Influence of the water structure on the acetylcholinesterase efficiency

We have studied the catalytic efficiency of acetylcholinesterase (AChE) in various solutions with ion-disturbed water structure to explore the role that the water structure plays in the substrate-enzyme encounter. The extent of water structuring in the different aqueous solutions was determined by near-infrared spectroscopy. The influence of water structure on the degree of solvation and on the intramolecular mobility of AChE was investigated for different aqueous ionic solutions by small-angle x-ray scattering technique and depolarization fluorescence spectroscopy. It was found that the encounter process between AChE and acetylthiocholine was promoted in solutions with less structured water. In these solutions it was also found that AChE is less solvated coinciding with higher intramolecular mobility. The found experimental results suggest that the water structure may influence the substrate-enzyme encounter process by diminishing the AChE solvation shell and may help diffusion of the substrate through the gorge by enhancing the intramolecular mobility of AChE.     

Influence of gait velocity on gastrocnemius muscle fascicle behaviour during stair negotiation

The gastrocnemius medialis (GM) muscle plays an important role in stair negotiation. The aim of the study was to investigate the influence of cadence on GM muscle fascicle behaviour during stair ascent and descent. Ten male subjects (young adults) walked up and down a four-step staircase (with forceplates embedded in the steps) at three velocities (63, 88 and 116 steps/min). GM muscle fascicle length was measured using ultrasonography. In addition, kinematic and kinetic data of the lower legs, and GM electromyography (EMG) were measured. For both ascent and descent, the amount of fascicular shortening, shortening velocity, knee moment, ground reaction force and EMG activity increased monotonically with gait velocity. The ankle moment increased up to 88 steps/min where it reached a plateau. The lack of increase in ankle moment coinciding with further shortening of the fascicles can be explained by an increased shortening of the GM musculotendon complex (MTC), as calculated from the knee and ankle angle changes, between 88 and 116 steps/min only. For descent, the relative instant of maximum shortening, which occurred during touch down, was delayed at higher gait velocities, even to the extent that this event shifted from the double support to the single support phase.     

Molecular forms of acetylcholinesterase in mammalian smooth muscles

A comparative study of the molecular forms of acetylcholinesterase (AChE) was made in various smooth muscles (intestine, vas deferens, ciliary body, iris, nictitating membrane retractor, ureter, arteries, anococcygeus muscles) of some mammals (cat, guinea-pig, rat, rabbit, mouse), seeking for a correlation between the presence of 16 S (asymmetric, tailed) form of AChE in smooth muscles and their type of innervation defined by morphological criteria, as well as by the nature of the main neurotransmitters involved in their neuroeffector junctions. Contrary to previous assertions, many smooth muscles contain 16 S AChE, although all those examined here exhibited a proportion clearly less than that of striated muscles. There are large species-specific and individual variations in the percentage of 16 S AChE. The highest percentages of 16 S AChE were found in ciliary and iris muscles, which are provided with an individual (= multiunit) cholinergic innervation. The vas deferens muscles, which are also individually, but noradrenergically innervated contain practically no 16 S AChE. In the muscles having a fascicular (= unitary) innervation, the differences are striking: 16 S AChE is in rather high amount in intestine muscle layers, whereas it is very low or virtually absent in ureter or arterial muscles. Thus, the type of innervation is not clearly involved in the amount of 16 S AChE present in smooth muscles. As for the nature of neurotransmitter a clear correlation exists only in the case of individual innervation, in which only one neurotransmitter is involved or largely predominant.     

Structural and kinetic effects of mobile phone microwaves on acetylcholinesterase activity

The present study provides evidence that "in vitro" simple exposure of an aqueous solution of electric eel acetylcholinesterase (EeAChE; EC 3.1.1.7.) to cellular phone emission alters its enzymatic activity. This paper demonstrates, by combining different experimental techniques, that radio frequency (RF) radiations irreversibly affect the structural and biochemical characteristics of an important CNS enzyme. These results were obtained by using a commercial cellular phone to reproduce the reality of the human exposition. This experimental procedure provided surprising effects collected practically without experimental errors because they were obtained comparing native and irradiated sample of the same enzyme solution. Although these results cannot be used to conclude whether exposure to RF during the use of cellular phone can lead to any hazardous health effect, they may be a significant first step towards further verification of these effects on other "ex vivo" or "in vivo" biological systems.     

The effect of temperature on the acetylcholinesterase activity of muscle microsomes

Membrane vesicles which constitute the sarcotubular system were separated and the fraction enriched in T-tubules purified by a calcium loading procedure. The preparations of unfractioned microsomes and T-tubules have been analyzed for their relative content of enzyme markers and acetylcholinesterase. The amount of this enzyme in the T-tubule fraction was higher than in mixed microsomes but less than two-fold the value of vesicles derived from sarcoplasmic reticulum. Arrhenius plots of membrane-bound and soluble acetylcholinesterase from either mixed microsomes or fractions enriched in T-tubules show an anomalous behaviour as two break points were obtained. The first discontinuity was found at about 17 degrees C for membrane-bound, and 12-14 degrees C for soluble acetylcholinesterase. The second one being at about 25 degrees C for both particulate and detergent-solubilized enzyme. The changes in activity with temperature suggest that lipid-protein, detergent-protein and protein-protein interactions might be involved in the stabilization of the enzyme both in the natural membrane and in the soluble state.     

Active-site determinations on forms of mammalian brain and eel acetylcholinesterase.

Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.