Functional consequences of ligand-dependent conformational changes in trypsin-solubilized and in membrane particle constrained-acetylcholinesterase.

Pattison, Scott E.; Bernhard, Sidney A.

doi:10.1073/pnas.75.8.3613

Cited by 12 publications

(15 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The acetylthiocholine turnover numbers determined from active site titration with M7C (Rosenberry & Bernhard, 1971) are the same for the phospholipid-associated dimer, the detergent-solubilized dimer (3.0 X 105 min™1, reported herein), and the trypsin-solubilized tetramer reported previously (Pattison & Bernhard, 1978). The specific activity of the purified enzymes is 4000 units/mg for the dimer, 4300 units/mg for the trypsin-solubilized enzyme, and 3400 units/mg for the tailed enzyme.…”

Section: Resultssupporting

confidence: 59%

“…(5) Acetylcholinesterase-enriched suspension ("low-density fraction") was obtained by the following flotation method. Eight milliliters of protein suspension in buffer IV was prepared according to Pattison & Bernhard (1978). The suspension was brought to >40% w/v sucrose by the addition of 80% w/v sucrose.…”

Section: Methodsmentioning

confidence: 99%

“…Analogous "in situ" studies have usually been carried out with electroplax tissue homogenates of either Electrophorus (Robaire & Kato, 1974, 1975 or Torpedo (Foidart & Gridelet, 1974). Some differences in the catalytic and regulatory properties of the in situ vs. the solubilized enzyme have been reported (Robaire & Kato, 1974, 1975, whereas others have found no difference in function (Pattison & Bernhard, 1978). Such comparative studies are difficult to interpret due both to the problem of acquiring quantitative isothermal data in heterogeneous solution and to the possible existence of a variety of specific ligand binding sites, other than those of the enzyme, in the heterogeneous mixture.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Major component of acetylcholinesterase in Torpedo electroplax is not basal lamina associated

Viratelle¹,

Bernhard²

1980

Biochemistry

View full text Add to dashboard Cite

Electroplax tissue from Torpedo californica contains two major structural forms of the enzyme acetylcholinesterase. One form, composed of tetrameric protomers which are further aggregated by interactions among associated collagenous "tail fibers", has been well characterized previously. This form is associated in situ with the basal lamina. The other form is described and characterized herein. This latter form accounts for at least 50% of the acetylcholinesterase activity of the tissue. This enzyme associated with the tissue phospholipids. It aggregates in aqueous solution but readily dissociates to dimers in 1% sodium cholate solution, a solvent in which it is both soluble and catalytically fully active. The same dimer is obtained in sodium dodecyl sulfate solution where the enzyme is denatured. Denaturation in the presence of the reductant dithiothreitol results in the formation of a single 80000-dalton subunit. The purified enzyme contains no collagenous component. It is not derivable from the collagenous "tailed-enzyme" form in the tissue homogenate. However, the two enzymes have similar molecular weight catalytic subunits and the same substrate-dependent turnover numbers (per active site) for a variety of choline esters which are generally utilized to distinguish specific esterase function. In the tissue homogenate each form of the enzyme is associated with a characteristic structural component (phospholipid or collagen). By implication, acetylcholinesterase function is localized in situ in the phospholipid membrane as well as at the basal lamina.

show abstract

Section: Resultssupporting

confidence: 59%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Major component of acetylcholinesterase in Torpedo electroplax is not basal lamina associated

Viratelle¹,

Bernhard²

1980

Biochemistry

View full text Add to dashboard Cite

show abstract

“…A similar scenario appears to exist for AChE. Results from several groups indicate that this enzyme is composed of two discrete forms in slow conformational equilibrium and that ligands of the PAS bind only to one form [23,24].…”

mentioning

confidence: 85%

Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate

Masson

Goldstein

Debouzy³

et al. 2003

European Journal of Biochemistry

View full text Add to dashboard Cite

Steady-state kinetics for the hydrolysis of benzoylcholine (BzCh) and benzoylthiocholine (BzSCh) by wild-type human butyrylcholinesterase (BuChE) and by the peripheral anionic site mutant D70G were compared. k cat /K m for the hydrolysis of BzSCh was 17-fold and 32-fold lower than that for hydrolysis of BzCh by wild-type and D70G, respectively. The rate-limiting step for hydrolysis of BzCh was deacylation, whereas acylation was rate-limiting for hydrolysis of BzSCh. Wild-type enzyme and the D70G mutant were found to reach steady-state velocity slowly with BzCh as the substrate. At pH 6, the approach to steady-state for both enzymes consisted of a mono-exponential acceleration upon which a set of damped oscillations was superimposed. From pH 7 to 8.5, the approach to steady-state consisted of a simple exponential acceleration. The damped oscillations were analyzed by both a numerical approximation and simulation based on a theoretical model. BuChE-catalyzed hydrolysis of the thiocholine analogue of BzCh showed neither lags nor oscillations, under the same conditions. The frequency and amplitude of the damped oscillations decreased as the BzCh concentration increased. The apparent induction time for the exponential portion of the lag was calculated from the envelope of the damped oscillations or from the smooth lag. Wild-type BuChE showed a hyperbolic increase in induction time as the BzCh concentration increased (s max ¼ 210 s at pH 6.0). However, the induction time for D70G was constant over the whole range of BzCh concentrations (s max ¼ 60 s at pH 6.0). Thus, the induction time does not conform to a simple hysteretic model in which there is a slow conformational transition of the enzyme from an inactive form E to an active form E¢. No pH-dependence of the induction time was found between pH 6.0 and 8.5 in sodium phosphate buffers of various concentrations (from 1 mM to 1 M). However, increasing the pH tended to abolish the oscillations (increase the damping factor). This effect was more pronounced for D70G than for wild-type. Although the lyotropic properties of phosphate change from chaotropic at pH 6.0 to kosmotropic at pH > 8.0, no effect of phosphate concentration on the oscillations was noticed at the different pH values, suggesting that the oscillations are not related to a pH-dependent Hofmeister effect of phosphate ions. Simulation and theoretical analysis of the oscillatory behaviour of the approach to the steady-state for BuChE led us to propose a model for the hysteresis of BuChE with BzCh. In this model, the substrate-free enzyme is present as an equilibrium mixture of two forms, E and E¢. Substrate binds to E and E¢, but only E¢S makes products. It is proposed that oscillations originate from a time-dependent change in the local concentration, solvation and/or conformation of substrate in the bulk solution. 1 H-NMR measurements provided evidence for a slow equilibrium between two BzCh conformers. Binding of the conformationally preferred substrate conformer leads to products.

show abstract

“…A particularly intriguing mode of AchE inhibition was recently uncovered by Pattison & Bernhard (1978), who observed that either rf-tubocurarine in high concentrations or transition metals effect a slow conversion of the enzyme to an inactive state. The use of a fluorescent carbamylating agent enabled these workers to distinguish between inhibition of the initial rate of substrate association and the conversion to nonreactive enzyme.…”

mentioning

confidence: 99%

Ligand-induced conformational changes in acetylcholinesterase investigated with fluorescent phosphonates

Epstein¹,

Berman²,

Taylor³

1979

Biochemistry

View full text Add to dashboard Cite

Steady-state kinetic studies have suggested that the catalytic activity of acetylcholinesterase is altered upon binding of certain ligands at a locus removed from the active center. In this study fluorescent phosphonates which conjugate with the active-center serine are employed to demonstrate directly that ligands which associate with the peripheral site alter active-site conformation. The fluorescent reagent [1-(dimethylamino)naphthalene-5-sulfonamido]pentyl methylphosphonofluoridate, when conjugated to the enzyme, proves to be a sensitive probe of active-site conformation. Displacement of propidium, a reversible fluorescent ligand of the fFrom the

show abstract

Functional consequences of ligand-dependent conformational changes in trypsin-solubilized and in membrane particle constrained-acetylcholinesterase.

Abstract: ABSIRACT The effect of a class of ligands on the catalytic activity of acetyicholinesterase (

Cited by 12 publications

References 28 publications

Major component of acetylcholinesterase in Torpedo electroplax is not basal lamina associated

Major component of acetylcholinesterase in Torpedo electroplax is not basal lamina associated

Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate

Ligand-induced conformational changes in acetylcholinesterase investigated with fluorescent phosphonates

Contact Info

Product

Resources

About