Snake neurotoxins: effect of disulfide reduction on interaction with electroplax.

Reduction of disulfide bonds of cobra neurotoxin by dithiothreitol results in decreased activity on the electroplax preparation. Activity is restored completely after reoxidation by 3,3'-dithiobis[6-nitrobenzoic acid]. Reduction of the disulfide bonds in the vicinity of the receptor does not decrease the effect of cobrotoxin.

Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction.

The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the neuromuscular junction (NMJ). This enzyme form consists of catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail (ColQ). This synaptic form of the enzyme is tightly attached to the basal lamina associated with the glycosaminoglycan perlecan. Fasciculin-2 is a snake toxin that binds tightly to AChE. Localization of junctional AChE on frozen sections of muscle with fluorescent Fasciculin-2 shows that the labeled toxin dissociates with a half-life of about 36 h. The fluorescent toxin can subsequently be taken up by the muscle fibers by endocytosis giving the appearance of enzyme recycling. Newly synthesized AChE molecules undergo a lengthy series of processing events before final transport to the cell surface and association with the synaptic basal lamina. Following co-translational glycosylation the catalytic subunit polypeptide chain interacts with several molecular chaperones, glycosidases and glycosyltransferases to produce a catalytically active enzyme that can subsequently bind to one of two non-catalytic subunits. These molecular chaperones can be rate limiting steps in the assembly process. Treatment of muscle cells with a synthetic peptide containing the PRAD attachment sequence and a KDEL retention signal results in a large increase in assembled and exportable AChE, providing an additional level of post-translational control. Finally, we have found that Pumilio2, a member of the PUF family of RNA-binding proteins, is highly concentrated at the vertebrate neuromuscular junction where it plays an important role in regulating AChE translation through binding to a highly conserved NANOS response element in the 3'-UTR. Together, these studies define several new levels of AChE regulation in electrically excitable cells.

Replacement of the glycoinositol phospholipid anchor of Drosophila acetylcholinesterase with a transmembrane domain does not alter sorting in neurons and epithelia but results in behavioral defects.

Drosophila has a single glycoinositol phospholipid (GPI)-anchored form of acetylcholinesterase (AChE) encoded by the Ace locus. To assess the role that GPI plays in the physiology, of AChE, we have replaced the wild-type GPI-AChE with a chimeric transmembrane form (TM-AChE) in the nervous system of the fly. Ace null alleles provided a genetic background completely lacking in endogenous GPI-AChE, and Ace minigene P transposon constructs were used to express both GPI- and TM-AChE forms in the tissues where AChE is normally expressed. Control experiments with the GPI-AChE minigene demonstrated a threshold between 9 and 12% of normal AChE activity for adult viability. Ace mutant flies were rescued by GPI-AChE minigene lines that expressed 12-40% of normal activity and were essentially unchanged from wild-type flies in behavior. TM-AChE minigene lines were able to rescue Ace null alleles, although with a slightly higher threshold than that for GPI-AChE. Although rescued flies expressing GPI-AChE at a level of 12% of normal activity were viable, flies expressing 13-16% of normal activity from the TM-AChE transgene died shortly after eclosion. Flies expressing TM-AChE at about 30% of normal levels were essentially unchanged from wild-type flies in gross behavior but had a reduced lifespan secondary to subtle coordination defects. These flies also showed reduced locomotor activity and performed poorly in a grooming assay. However, light level and electron microscopic immunocytochemistry showed no differences in the localization of GPI- and TM-AChE. Furthermore, endogenous and ectopic-induced expression of both AChEs in epithelial tissues of the adult and embryo, respectively, showed that they were sorted identically. Most epithelial cells sorted GPI- and TM-AChE to the apical surface, but cuticle-secreting epithelia sorted both proteins basolaterally. Our data suggest that rather than having a primary role in protein sorting, the GPI anchor or AChE plays some other more subtle cellular role in neuronal physiology.

Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring.

Despite advances in understanding the cell biology of glycoinositol phospholipid (GPI)-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.

Functional identity of catalytic subunits of acetylcholinesterase.

11 S acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the electric eel Electrophorus electricus essentially consists of four catalytic subunits which appear to be identical structurally but to be assembled with slight asymmetry. During isolation and storage of the enzyme, proteolysis cleaves a portion of the subunits into major fragments containing the active site and minor fragments containing no active sites without change in the enzyme molecular weight. A previous report (Gentinetta, R. and Brodbeck, U. (1976) Biochim. Biophys. Acta 438 437--448) indicated that the intact and the fragmented subunits reacted with diisopropylfluorophosphate at different rates and that the reaction rate in the presence of excess phosphorylating agent was not strictly first order. Those findings could not be reproduced in this report. Intact and fragmented subunits were observed to react at the same rate with diisopropylfluorophosphate. In addition, the overall reaction kinetics both of 11 S and 18 S plus 14 S acetylcholinesterase were found to be strictly first order in the presence of an excess of diisopropylfluorophosphate throughout the course of reaction. These results are consistent with several previous reports that only one type of active site can be detected in acetylcholinesterase. The proteolysis which fragments a portion of the catalytic subunit has no apparent effect on the catalytic properties of the enzyme.

Inositol glycan phosphate derived from human erythrocyte acetylcholinesterase glycolipid anchor and inositol cyclic 1,2-phosphate antagonize glucagon activation of glycogen phosphorylase.

In this study we examine the hypothesis that an inositol glycan phosphate can act similarly to insulin on intact cells. The inositol glycan phosphate used in this study (glycan alpha) was isolated previously from the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase and was shown to have the structure glycine-ethanolamine-PO4-Man-Man-(N,N-dimethylethanolamine-PO4)Man- (N,N-dimethyl)GlcN-inositol-PO4. The cellular response investigated was the glucagon-stimulated activation of glycogen phosphorylase in rat hepatocytes. When hepatocytes were incubated with 20 nM glucagon for 4 min, the ratio of phosphorylase a activity to total phosphorylase increased from a basal value of 0.49 +/- 0.02 to 0.82 +/- 0.03 (mean +/- SE, n = 15). Inclusion of either 100 nM insulin or 3-10 microM glycan alpha during the glucagon incubation significantly decreased the glucagon-stimulated activity ratio to 0.74 +/- 0.03 for either agent. Furthermore, hepatocyte preparations differed in their response to insulin and were divided into insulin-responsive and -resistant groups. Glycan alpha had a significant effect only in the insulin-responsive group for which the observed activity ratio for 10 microM glycan alpha plus glucagon (0.68 +/- 0.05) compared closely with that for insulin plus glucagon (0.70 +/- 0.04). For the insulin-resistant group, the activity ratio in the presence of 10 microM glycan alpha was 0.81 +/- 0.03, unchanged from the control with glucagon alone. Because glycan alpha contains an inositol phosphate group, the effect of inositol cyclic 1,2-phosphate on the glucagon-stimulated activity ratio was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors.

We have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4-tetrahydro-N-(phenylmethyl)-9-acridinamine and 1,2,3,4-tetrahydro-N-(3-iodophenyl-methyl)-9-acridinamine--all three at 2.7 A resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active-site gorge, but some of the surface loops deviate by up to 8 A from their position in the vertebrate structures, and the C-terminal helix is shifted substantially. The active-site gorge of the insect enzyme is significantly narrower than that of Torpedo californica AChE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is approximately 50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active-site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three-dimensional structures.

The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231S mutant.

The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). At 30 degrees C or 37 degrees C, inactivation rates with 0.3 mM DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate increased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.

The collagen-like subunits of acetylcholinesterase from the eel electrophorus electricus.

Pepsin-resistant fragments of the tail subunits of 14S and 18S acetylcholinesterase from eel electric organ have been isolated and characterized. The native fragments are composed of three 24,000 molecular weight polypeptides linked by intersubunit disulfide bonds in a collagen-like triple helix. Intact tail subunits have also been isolated. These subunits appear to contain both the triple-helical domain and a noncollagenous domain that is linked to catalytic subunits by disulfide bonds.

Ionic strength dependence of the inhibition of acetylcholinesterase activity by Al3+.

Inhibition of acetylcholinesterase activity by Al3+ has been examined by initial velocity kinetics and by a first-order kinetic method. Both methods yield an inhibition constant of approx. 1.7 mM at 0.1 M ionic strength. The initial velocity study indicates a noncompetitive mechanism of inhibition by Al3+. Inhibition at 10 mM ionic strength shows a Ki of 0.03 mM. Evaluation of the ionic strength dependence concurs with the results of Nolte et al. (Biochemistry 19 (1980) 3705). An effective charge in the binding site of -9 predicts the ratio of inhibition constants at high and low ionic strength. Extrapolation to zero ionic strength gives a Ki0 = 0.34 microM.

A steric blockade model for inhibition of acetylcholinesterase by peripheral site ligands and substrate.

The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated that small peripheral site ligands like propidium can inhibit substrate hydrolysis simply by decreasing the substrate association and dissociation rate constants without altering the equilibrium constant for substrate binding to the acylation site. We now employ our nonequilibrium kinetic analysis to extend this model to include blockade of the dissociation of substrate hydrolysis products by bound peripheral site ligand. We also report here that acetylthiocholine can bind to the AChE peripheral site with an equilibrium dissociation constant K(S) of about 1 mM. This value was determined from the effect of the acetylthiocholine concentration on the rate at which fasciculin associates with the peripheral site. When substrate binding to the peripheral site is incorporated into our steric blockade model, hydrolysis rates at low substrate concentration appear to be accelerated while substrate inhibition of hydrolysis occurs at high substrate concentration. The model predicts that hydrolysis rates for substrates which equilibrate with the acylation site prior to the acylation step should not be inhibited by bound peripheral site ligand. Organophosphates equilibrate with AChE prior to phosphorylating the active site serine residue, and as predicted propidium had little effect on the phosphorylation rate constants for the fluorogenic organophosphate ethylmethyl-phosphonylcoumarin (EMPC). The 2nd-order phosphorylation rate constant kOP/K(OP) was decreased 3-fold by a high concentration of propidium and the 1st-order rate constant kOP increased somewhat. In contrast to propidium, when the neurotoxin fasciculin bound to the AChE peripheral site both a steric blockade and a conformational change in the acylation site appeared to occur. With saturating fasciculin, kOP/K(OP) decreased by a factor of more than 750 and kOP decreased 300-fold. These data suggest that new peripheral site ligands may be designed to have selective effects on AChE phosphorylation.

A pseudo-first-order kinetic approach to measurement of acetylcholine hydrolysis by acetylcholinesterase.

A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 3.1.1.7) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H+ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88-95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of KI for edrophonium, decamethonium and Al3+.

Large scale isolation of human erythrocyte membranes by high volume molecular filtration.

A molecular filtration procedure for preparing large quantities of human erythrocyte ghost membranes is presented. Hemolysate ghost membranes are rapidly cycled in the retentate channel of the filtration apparatus, while hemoglobin is removed as it passes through Pellicon filters into the filtrate channel. Several-liter quantities of washed packed erythrocytes can be processed in a few hours with this system, and the filtration procedure does not appear to alter intact erythrocyte or ghost membranes. Intact erythrocytes in isotonic solution can be circulated through the retentate channel for 16 h with only 3% hemolysis and with preservation of their original morphology in scanning electron microscopy. Ghost membranes isolated by the procedure are virtually identical in morphology, polypeptide composition and acetylcholinesterase content to membranes isolated by conventional centrifugation techniques.

Metabolism of GPIs in mammalian cells.

In Trypanosoma brucei, glycosylphosphatidylinositol (GPI) anchors of proteins and free GPIs with identical structures have been characterized. This identity provides strong presumptive evidence that the free GPIs are in fact precursors of the GPI anchors on proteins. In mammalian tissues, however, rather consistent differences in the structures of free GPIs and GPI anchors are observed. The terminal GPIs produced by the mammalian biosynthetic pathway differ from GPI anchors in being almost exclusively fatty acid acylated on the inositol residue, having a greater number of phosphoethanolamine residues, and perhaps in containing a greater percentage of diacylglycerol components. While in principle these differences could be reconciled by remodeling reactions before or after attachment of GPI anchors, it is possible that some of the mammalian free GPIs play cellular roles other than as anchor precursors. We have approached this question by studying the lifetimes of the last three GPIs on the biosynthetic pathway, denoted H6, H7 and H8, in K562 cells and in a K562 mutant designated class K that is devoid of GPI-anchored proteins. Pulse-chase metabolic labeling with [3H]-mannose indicated that H6 was a precursor of H7 and H8 and that the H8 lifetime was more than one hour in the parental cells and even longer in the mutant. Preliminary data indicated that the majority of each of the three GPIs was localized in the plasma membrane fraction rather than the endoplasmic reticulum. These observations argue that mammalian GPIs are not utilized exclusively as GPI anchor precursors.

Catalysis by acetylcholinesterase: evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis.

Inferences about the catalytic mechanism of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) are frequently made on the basis of a presumed analogy with chymotrypsin, EC 3.4.21.1. Although both enzymes are serine hydrolases, several differences in the steady-state kinetic properties of the two have been observed. In this report particular attention is focused on the second-order reaction constant, kcat/Kapp. While the reported pH dependence and deuterium oxide isotope effect associated with this parameter for chymotrypsin are generally consistent with simple models involving rate-limiting general acid-base catalysis, this study finds a more complicated situation with acetylcholinesterase. The apparent pKa of kcat/Kapp for acetylcholinesterase varies between 5.5 and 6.3 for neutral substrates and involves nonlinear inhibition by [H+]. Deuterium oxide isotope effects for kcat/Kapp range from 1.1 for acetylcholine to 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit step is proposed to account for apparent pKa values and low deuterium oxide isotope effects associated with low concentrations of phenyl acetate and isoamyl acetate.

Quantitative simulation of endplate currents at neuromuscular junctions based on the reaction of acetylcholine with acetylcholine receptor and acetylcholinesterase.

Two kinetic models are introduced which predict amplitudes and time-courses of endplate currents and miniature endplate currents at neuromuscular junctions, at both normal and acetylcholinesterase-inhibited endplates. Appropriate differential rate equations reflecting interactions of acetylcholine with acetylcholine receptor and with esterase, diffusion of acetylcholine both within and from the synaptic cleft, and cooperativity between receptor site occupancy and ion channel opening are solved. Acetylcholine release into the cleft is assumed to be instantaneous. The simpler homogeneous reaction space model accurately predicts decay phase time constants are inaccurate. The two-reaction space model predicts amplitudes and time constants within a factor of two of those observed experimentally. The simulations indicate that the amplitudes and time-courses are primarily determined by the chemical reaction rates that characterize acetylcholine interactions with receptor and esterase and that these interactions occur under nonequilibrium conditions. Approximately 50% of the total ion channels in the initial reaction space are predicted to be opened at the peak endplate current. The cooperative opening of ion channels by acetylcholine requires that acetylcholine be introduced into the cleft in discrete, concentrated elements. Virtually all the open channels are confined to the initial reaction space, although acetylcholine-bound receptor sites can be much more widely distributed.