G4 Form Research Articles

A constitutive model for predicting shear modulus of metals using aluminum as the prototype

With a two-step approach，a pressure，P，and temperature，T，dependent constitutive model of shear modulus，G，applicable to metals was developed in this work. The goal of the first step is to find the relation of G with P along 0 K isotherm，i.e. the functional form of G1=G1（P，0 K），and the second one is to find the relation of G，starting from a given state of （P，0 K） with a value that was already determined in the first step，with T along the isobar of P，i.e. the functional form of G=G（P，T） or the constitutive model proposed by us. In both steps，results of supersonic measurement and first principles calculation were used. Aluminum，as a model material，was utilized to validate the rationality of this model. It is demonstrated that the predicted results of this model are in satisfactory agreement with the measured and numerically simulated G despite whether it evolves along shock compressed，isentropic，isothermal，or isobaric loci，thereby displaying the rationality and the universal nature of this constitutive model.

Reciprocal neural regulation of extrajunctional acetylcholinesterase and collagen Q in rat muscles—The role of calcineurin signaling

There are two main differences regarding acetylcholinesterase (AChE) expression in the extrajunctional regions of fast and slow rat muscles: (1) the activity of AChE catalytic subunits (G1 form) is much higher in fast than in slow muscles, and (2) the activity of the asymmetric forms of AChE (A 8 and A 12) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the absence of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. We showed that both differences are caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the respective levels of mRNA of both proteins. Whereas the changes in AChE mRNA levels in fast and slow muscles, as well as the levels of ColQ mRNA levels in slow muscles, observed in response to exposing either slow or fast muscles to different muscle activation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible. Calcineurin signaling pathway in muscles is activated by high-average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, typical for slow muscle fibers, but is inactive in fast muscle fibers, which are activated by infrequent high-frequency bursts of neural impulses. Application to rats of two inhibitors of calcineurin (tacrolimus-FK506 and cyclosporin A) demonstrated that the mRNA levels of both the AChE catalytic subunit and ColQ in the extrajunctional regions of the soleus muscle are regulated by the calcineurin signaling pathway, but in a reciprocal way. Under the conditions of low calcineurin activity, AChE expression is enhanced and that of ColQ is suppressed, and vice versa. Our results also indicated that different, calcineurin-independent regulatory pathways are responsible for the reduction of AChE expression during muscle denervation, and for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers.

Human butyrylcholinesterase components differ in aryl acylamidase activity

Apart from its esterase activity, butyrylcholinesterase (BuChE) displays aryl acylamidase (AAA) activity able to hydrolyze o-nitroacetanilide (ONA) and its trifluoro-derivative (F-ONA). We report here that, despite amidase and esterase sites residing in the same protein, in human samples depleted of acetylcholinesterase the ratio of amidase to esterase activity varied depending on the source of BuChE. The much faster degradation of ONA and F-ONA by BuChE monomers (G1) of colon and kidney than by the tetramers (G4) suggests aggregation-driven differences in the AAA site between single and polymerized subunits. The similar ratio of F-ONAto butyrylthiocholine hydrolysis by serum G1 and G4 forms support structural differences in the amidase site according to the source of BuChE. The changing ratios of amidase to esterase activities in the human sources probably arise from post-translational modifications in BuChE subunits, the specific proportion of monomers and oligomers and the variable capacity of the tetramers for degrading ONA and F-ONA. The elevated amidase activity of BuChE monomers and the scant activity of the tetramers justify the occurrence of single BuChE subunits in cells as a means to sustain the AAA activity of BuChE which otherwise could be lost by tetramerization.

Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide

To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.

Pho85, a multifunctional cyclin‐dependent protein kinase in budding yeast

Pho85 is a multifunctional cyclin-dependent kinase (Cdk) in Saccharomyces cerevisiae that has emerged as an important model for the role of Cdks in both cell cycle control and other processes. Pho85 is targeted to its substrates by 10 different cyclins or Pcls. Three of these Pcls have specific roles in G1 phase of the cell cycle, both in regulating G1-specific gene expression and in controlling polarized growth. Many known substrates of the G1 forms of Pho85 are also phosphorylated by the homologous Cdk Cln-Cdc28, suggesting parallel or overlapping roles. Most of the remaining Pcls function in signalling: Pho85 is generally active when environmental conditions are satisfactory, phosphorylating proteins involved in transcription and other regulatory events to keep the stress response and inappropriate activities turned off. Recently, genetic screens for synthetic lethality and synthetic dosage lethality, and proteomic screens for in vitro Pho85 substrates, have revealed more details about how Pho85 functions to regulate a variety of cellular processes.

Regularity of weak foliations for thermostats

Let M be a closed oriented surface endowed with a Riemannian metric g. We consider the flow ϕ determined by the motion of a particle under the influence of a magnetic field Ω and a thermostat with external field e. We show that if ϕ is Anosov, then it has weak stable and unstable foliations of class C1,1 if and only if the external field e has a global potential U, g1 ≔ e−2Ug has constant curvature and e−UΩ is a constant multiple of the area form of g1. We also give necessary and sufficient conditions for just one of the weak foliations to be of class C1,1 and we show that the combined effect of a thermostat and a magnetic field can produce an Anosov flow with a weak stable foliation of class C∞ and a weak unstable foliation which is not C1,1. Finally we study Anosov thermostats depending quadratically on the velocity and we characterize those with smooth weak foliations. In particular, we show that quasi-Fuchsian flows as defined by Ghys (1992 Ann. Inst. Fourier 42 209–47) can arise in this fashion.

Aspects of correlation function realizability

The pair-correlation function g2(r) describes short-range order in many-particle systems. It must obey two necessary conditions: (i) non-negativity for all distances r, and (ii) non-negativity of its associated structure factor S(k) for all k. For the elementary unit step-function g2 form, previous work [F. H. Stillinger, S. Torquato, J. M. Eroles, and T. M. Truskett, J. Phys. Chem. B 105, 6592 (2001)] indicates that (i) and (ii) could be formally satisfied, but only up to a terminal density at which the covering fraction of particle exclusion diameters equaled 2−d in d dimensions. To test whether the unit step g2 is actually achievable in many-particle systems up to the apparent terminal density, a stochastic optimization procedure has been used to shift particles in large test systems toward this target g2. Numerical calculations for d=1 and 2 confirm that the step function g2 is indeed realizable up to the terminal density, but with substantial deviation from the configurational preferences of equilibrium hard-rod and hard-disk models. We show that lineal statistical measures are particularly sensitive to this difference. Our results also illustrate the characteristics of “closest approach” to the step function g2 above the terminal density.

Theoretical Conformational Analysis for Neurotransmitters in the Gas Phase and in Aqueous Solution. Norepinephrine

The natural neurotransmitter (R)-norepinephrine takes the monocationic form in 93% abundance at the physiological tissue pH of 7.4. Ab initio and DFT/B3LYP calculations were performed for 12 protonated conformers of (R)-norepinephrine in the gas phase with geometry optimizations up to the MP2/6-311++G level, and with single-point calculations up to the QCISD(T) level at the HF/6-31G-optimized geometries. Four monohydrates were studied at the MP2/6-31G//HF/6-31G level. In the gas phase, the G1 conformer is the most stable with phenyl.NH(3)(+) gauche and HO(alc).NH(3)(+) gauche arrangements. A strained intramolecular hydrogen bond was found for conformers (G1 and T) with close NH(3)(+) and OH groups. Upon rotation of the NH(3)(+) group as a whole unit about the C(beta)-C(alpha) axis, a 3-fold potential was calculated with free energies for barriers of 3-12 kcal/mol at the HF/6-31G level. Only small deviations were found in MP2/6-311++G single-point calculations. A 2-fold potential was calculated for the phenyl rotation with free energies of 11-13 kcal/mol for the barriers at T = 310 K and p = 1 atm. A molecular mechanics docking study of (R)-norepinephrine in a model binding pocket of the beta-adrenergic receptor shows that the ligand takes a conformation close to the T(3) arrangement. The effect of aqueous solvation was considered by the free energy perturbation method implemented in Monte Carlo simulations. There are 4-5 strongly bound water molecules in hydrogen bonds to the conformers. Although hydration stabilizes mostly the G2 form with gauche phenyl.NH(3)(+) arrangement and a water-exposed NH(3)(+) group, the conformer population becomes T > G1 > G2, in agreement with the PMR spectroscopy measurements by Solmajer et al. (Z. Naturforsch. 1983, 38c, 758). Solvent effects reduce the free energies for barriers to 3-6 and 9-12 kcal/mol for rotations about the C(beta)-C(alpha) and the C(1)(ring)-C(beta) axes, respectively.

Effects of huperzine A on acetylcholinesterase isoforms in vitro: comparison with tacrine, donepezil, rivastigmine and physostigmine

Five inhibitors of acetylcholinesterase, huperzine A, donepezil, tacrine, rivastigmine and physostigmine, were compared with regard to their effects on different molecular forms of acetylcholinesterase in cerebral cortex, hippocampus, and striatum from the rat brain. In general, huperzine A preferentially inhibited tetrameric acetylcholinesterase (G4 form), while tacrine and rivastigmine preferentially inhibited monomeric acetylcholinesterase (G1 form). Donepezil showed pronounced selectivity for G1 acetylcholinesterase in striatum and hippocampus, but not in cortex. Physostigmine showed no form-selectivity in any brain region. In cortex, the most potent inhibitors of G4 acetylcholinesterase were huperzine A ( K i 7×10 −9 M) and donepezil ( K i 4×10 −9 M). The potent inhibitors of cortical G1 acetylcholinesterase were donepezil ( K i 3.5×10 −9 M) and tacrine ( K i 2.3×10 −8 M). In hippocampus, huperzine A and physostigmine were the most potent inhibitors of G4 acetylcholinesterase, while donepezil and tacrine were most potent against G1 acetylcholinesterase. In striatum, huperzine A and donepezil were the most potent against G4 acetylcholinesterase, while again donepezil was the most potent against G1. Although the inhibition constants ( K i) of these acetylcholinesterase inhibitors differed significantly from region to region, the nature of the inhibition did not vary. These results suggest that the use of acetylcholinesterase inhibitors in treatment of Alzheimer's disease must consider both form-specific and region-specific characteristics of acetylcholinesterase inhibition.

Use of immobilised glucoamylase G2 for separation of enantiomers

The aim of this study was to compare the isolated glucoamylase G2 domain as chiral selector with previously presented data obtained using immobilised native glucoamylase as chiral stationary phase. The glucoamylase G2 was isolated from a commercial preparation ofAspergillus niger glucoamylase and immobilised onto silica particles. Enantioselectivity was tested for several amino alcohols including the β-receptor blocking agents metoprolol and alprenolol. Separation factors greater than 2 were observed. Mobile phase pH was varied to optimise the enantioselective recognition and several different uncharged additives were included to examine their effect on retention. For some of the tested solutes increased retention times were observed when increasing the content of uncharged modifier. Increased mobile phase concentration of several of the tested organic modifiers resulted in increased separation factors. The effect of column temperature was studied. Retention and enantioselectivity increased at higher temperatures. Addition to the mobile phase of acarbose, an inhibitor that binds to the catalytic site of G2 with picomolar affinity, resulted in total loss of enantioselectivity. Comparison of the present results with those obtained previously with the glucoamylase G1 form also containing a starch binding domain, shows that the catalytic domain is essential for chiral recognition of amino alcohols.

Acetyl- and butyrylcholinesterase in normal and diabetic rat retina.

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinas. Tissues were sequentially extracted with saline (S1) and saline-detergent buffers (S2). 50% decrease in the amphiphilic G4 and G1 AChE molecular forms was observed in the diabetic retina compared to the controls. Less than 5% of the cholinesterase activity was due to BChE. 60% of the BChE activity in normal retina was brought into solution and evenly distributed between S1 and S2. In spite of the low BChE activity in the retina it was possible to detect globular forms (G(A)1, G(A)2, G(A)4, G(H)4) and a small proportion of an asymmetric form (A12) in the S1 extract. The G(A)4 and G(A)1 forms were found in the S2 extract. In the diabetic retina the activity of G(A)4 and G(A)1 BChE molecular forms was reduced 60% and 40% respectively. Our results indicate that diabetes caused a remarkable decrease in the activity of cholinesterase molecular forms in the retina. These decrease might participate in the alterations observed in the diabetic retina.

Zebrafish Acetylcholinesterase Is Encoded by a Single Gene Localized on Linkage Group 7: GENE STRUCTURE AND POLYMORPHISM; MOLECULAR FORMS AND EXPRESSION PATTERN DURING DEVELOPMENT

We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.

Effect of halothane administration on acetylcholinesterase activity in guinea-pig muscle and brain

The effect of halothane administration on the activity of acetylcholinesterase molecular forms was studied in diaphragm, extensor digitorum longus (EDL), and soleus muscles, and six regions of the brain (striatum, cerebellum, cortex, hippocampus, medulla-pons, midbrain) of guinea-pigs. Six days after the anaesthetic, the activity of the G4 form was significantly increased in all three muscles and the A12 form was significantly increased in EDL. The G1 precursor form was significantly decreased in soleus. The G4 form was significantly increased in medulla-pons, and the G1 form was significantly decreased in hippocampus and midbrain. These findings show that halothane can have prolonged effects on acetylcholinesterase activity in both muscle and brain, and may have important implications for the use of halothane and other volatile anaesthetics in studies of the cholinergic system.

Acetylcholinesterase from Schistosoma mansoni: interaction of globular species with heparin

In the cercarial and schistosomal stages of the life cycle of the trematode Schistosoma mansoni, acetylcholinesterase occurs as two principal molecular forms (both globular), present in approximately equal amounts, with sedimentation coefficients of 6.5 S and 8 S. The 6.5 S form is solubilized by bacterial phosphatidylinositol-specific phospholipase C from intact schistosomula. It is thus located on the outer surface of the schistosomal tegument and is most probably analogous to the glycosylphosphatidylinositol-anchored G2 form of acetylcholinesterase found in the electric organ of Torpedo, on the surface of mammalian erythrocytes, and elsewhere. Both forms are fully solubilized by the non-ionic detergent Triton X-100. Upon passing such a detergent extract over a heparin-Sepharose column, only the 8 S form was retained on the column. The bound acetylcholinesterase could be progressively eluted by increasing the salt concentration, with approx. 0.5-0.6 M NaCl being needed for complete elution. Selective inhibition experiments carried out on live parasites using the covalent acetylcholinesterase inhibitor echothiophate (phospholine), which does not penetrate the tegument, selectively inhibited the 6.5 S form, but not the 8 S form, suggesting an internal location for the latter. Monoclonal antibodies raised against S. mansoni acetylcholinesterase also distinguished between the two forms. Thus monoclonal antibody SA7 bound the 6.5 S form selectively, whereas SA57 recognized the 8 S form. The selective binding of the 8 S form to heparin suggests that, within the parasite, this form may be associated with the extracellular matrix of the musculature.

Acetylcholinesterase from Schistosoma mansoni: interaction of globular species with heparin

In the cercarial and schistosomal stages of the life cycle of the trematode Schistosoma mansoni, acetylcholinesterase occurs as two principal molecular forms (both globular), present in approximately equal amounts, with sedimentation coefficients of 6.5 S and 8 S. The 6.5 S form is solubilized by bacterial phosphatidylinositol-specific phospholipase C from intact schistosomula. It is thus located on the outer surface of the schistosomal tegument and is most probably analogous to the glycosylphosphatidylinositol-anchored G(2) form of acetylcholinesterase found in the electric organ of Torpedo, on the surface of mammalian erythrocytes, and elsewhere. Both forms are fully solubilized by the non-ionic detergent Triton X-100. Upon passing such a detergent extract over a heparin-Sepharose column, only the 8 S form was retained on the column. The bound acetylcholinesterase could be progressively eluted by increasing the salt concentration, with approx. 0.5-0.6 M NaCl being needed for complete elution. Selective inhibition experiments carried out on live parasites using the covalent acetylcholinesterase inhibitor echothiophate (phospholine), which does not penetrate the tegument, selectively inhibited the 6.5 S form, but not the 8 S form, suggesting an internal location for the latter. Monoclonal antibodies raised against S. mansoni acetylcholinesterase also distinguished between the two forms. Thus monoclonal antibody SA7 bound the 6.5 S form selectively, whereas SA57 recognized the 8 S form. The selective binding of the 8 S form to heparin suggests that, within the parasite, this form may be associated with the extracellular matrix of the musculature.

Transient association of calnexin and calreticulin with newly synthesized G1 and G2 glycoproteins of uukuniemi virus (family Bunyaviridae).

The membrane glycoproteins G1 and G2 of Uukuniemi virus, a member of the Bunyaviridae family, are cotranslationally cleaved from a common precursor in the endoplasmic reticulum (ER). Here, we show that newly made G1 and G2 associate transiently with calnexin and calreticulin, two lectins involved in glycoprotein folding in the ER. Stable complexes between G1-G2 and calnexin or calreticulin could be immunoprecipitated after solubilization of virus-infected BHK21 cells with the detergents digitonin or Triton X-100. In addition, G1-G2-calnexin complexes could be recovered after solubilization with CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), while G1-G2-calreticulin complexes were not readily detected by using this detergent. Only endoglycosidase H-sensitive forms of G1 were found complexed with calnexin. Pulse-chase experiments showed that G1 and G2 associated with both chaperones transiently for up to 120 min. Sequential immunoprecipitations with anticalreticulin and anticalnexin antisera indicated that about 50% of newly synthesized G1 and G2 was associated with either calnexin or calreticulin. Our previous results have shown that newly synthesized G1 and G2 transiently interact also with the ER chaperone BiP and with protein disulfide isomerase (R. Persson and R. F. Pettersson, J. Cell Biol. 112:257-266, 1991). Taking all of this into consideration, we conclude that the folding of G1 and G2 in the ER is catalyzed by at least four different folding factors.

Acetylcholinesterase activity of skeletal muscle in a non-immunogenic model for myasthenia gravis in rats.

Myasthenia gravis is caused by an autoimmune attack to acetylcholine receptors of skeletal muscle. Acetylcholine release from motor nerve terminals is upregulated in patients with myasthenia gravis and also in rat "myasthenic" models, dependent on the reduction of the number of acetylcholine receptors. This study addresses the question as to whether at "myasthenic" endplates there are changes in the activity of acetylcholinesterase. To this end we studied acetylcholinesterase activity in junctional and extrajunctional regions of dilator naris, extensor digitorum longus, and hemidiaphragm muscles from rats with alpha-bungarotoxin-induced myasthenia gravis. In all studied muscles from "myasthenic" rats there was no significant change of junctional acetylcholinesterase activity. In contrast, in dilator naris and extensor digitorum longus muscles, there was a 60% and 30% increase of extrajunctional acetylcholinesterase activity. There was no significant change in the extrajunctional activity in hemidiaphragm muscles. Velocity sedimentation analysis revealed that the increase in extrajunctional activity in extensor digitorum longus muscles could be attributed to an increase of the activity of the G4 form of acetylcholinesterase. Treatment of rats with 6.4 microgh(-1) neostigmine bromide for 29 days had no influence on junctional and extrajunctional acetylcholinesterase activity of extensor digitorum longus muscles from rats with alpha-bungarotoxin-induced myasthenia gravis.

The starch-binding domain from glucoamylase disrupts the structure of starch

The full-length glucoamylase from Aspergillus niger, G1, consists of an N-terminal catalytic domain followed by a semi-rigid linker (which together constitute the G2 form) and a C-terminal starch-binding domain (SBD). G1 and G2 both liberate glucose from insoluble corn starch, although G2 has a rate 80 times slower than G1. Following pre-incubation of the starch with SBD, the activity of G1 is uniformly reduced with increasing concentrations of SBD because of competition for binding sites. However, increasing concentrations of SBD produce an initial increase in the catalytic rate of G2, followed by a decrease at higher SBD concentrations. The results show that SBD has two functions: it binds to the starch, but it also disrupts the surface, thereby enhancing the amylolytic rate.

Selectivity of Cholinesterase Inhibition

The deterioration in cognitive function that is characteristic of Alzheimer’s disease is related to a reduction in cholinergic transmission in the basal forebrain, and the appearance of neurofibrillatory tangles and plaques containing β-amyloid (Aβ). Some plaques are neurotoxic and contain acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). AChE consists of different molecular forms: G1, G2 and G4. In the cortex and hippocampus, G4 is located presynaptically and comprises about 70 to 80% of the AChE present. The remainder is in the G1 form which is present in postsynaptic neurons. G4 is the predominant type of AChE that is reduced in Alzheimer’s disease. Skeletal muscle contains mainly G4 and G2. Eight cholinesterase inhibitors that reduce the inactivation of acetylcholine (ACh) have been tested in placebo-controlled trials in patients with Alzheimer’s disease. All these agents were found to improve cognitive function, or delay its rate of decline, in a significant proportion of patients. Tacrine, eptastigmine and rivastigmine also significantly improved activities of daily living. Furthermore, most of these drugs increased cerebral blood flow and glucose metabolism, parameters that are decreased in the basal forebrain of individuals with Alzheimer’s disease. This latter effect may contribute to the therapeutic effect of cholinesterase inhibitors and may delay the formation of Aβ. Currently available cholinesterase inhibitors differ in the incidence and severity of adverse effects produced at clinical doses. Reversible liver damage, seen with tacrine and velnacrine, and cholinergic hyperactivity, such as nausea, vomiting and muscle cramps, with these and other drugs can prevent the attainment of an optimum dose. The selectivity of cholinesterase inhibitors for AChE versus BuChE, and for the different molecular forms of AChE, may have an influence on both therapeutic and adverse effects. Donepezil and galantamine (galanthamine) are selective inhibitors of AChE, while the other agents also inhibit BuChE. The adverse effects associated with cholinergic hyperactivity are not due to blockade of BuChE as previously suggested, since they are seen with the AChE-selective inhibitors and not with those inhibitors that only inhibit BuChE. The advantage of nonselective inhibitors is that they may also increase ACh levels by inhibiting BuChE in glial cells. Selective inhibitors of the G1 form of AChE, such as rivastigmine, are more likely to elevate ACh levels in the brain of patients with Alzheimer’s disease and may be less likely to cause skeletal muscle cramps than nonselective inhibitors such as metrifonate.

CDNA cloning, in vitro expression, and biochemical characterization of cholinesterase 1 and cholinesterase 2 from amphioxus--comparison with cholinesterase 1 and cholinesterase 2 produced in vivo.

We have isolated cDNAs coding for the complete amino acid sequences of cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2) from amphioxus. Both ChE transcripts have the characteristics of H-type catalytic subunits, which are inserted in the membrane via an ethanolamine-glycan-phosphatidylinositol anchor. The members of the catalytic triad of ChEs, the three pairs of cysteine residues involved in intrachain disulfide bonding, a cysteine near the carboxy terminal of both sequences, which could mediate interchain disulfide bonding, and 11 of the 14 aromatic amino acids that line the catalytic gorge of AChE are conserved. A remarkable difference between the two enzymes is in the region of the acyl-binding pocket, which plays an important role in determining substrate specificity in cholinesterases. ChE2 contains a sequence that resembles the acyl pocket of invertebrate ChE, while the acyl-binding site of ChE1 is novel. There are also differences between the two enzymes in the peripheral anionic site, which mediates inhibition by certain ligands. In vitro expression in COS-7 cells demonstrates that ChE2 hydrolyzes acetylthiocholine almost exclusively, while ChE1 hydrolyzes both acetylthiocholine and butyrylthiocholine. Both enzymes are inhibited comparably by BW284c51, but ChE1 is considerably more resistant to inhibition by propidium, ethopropazine, and eserine than is ChE2. Velocity sedimentation indicates that ChE1 and ChE2 are present as amphiphilic and nonamphiphilic G2 forms in vivo and in vitro. Another molecular form, which sediments at 17 S, is also present in vivo. Nondenaturing gel electrophoresis in conjunction with digestion by phosphatidylinositol-specific phospholipase C demonstrates that the vast majority of ChE1 and ChE2 is present as ethanolamine-glycan-phosphatidylinositol-anchored G2 forms in vivo. ChE1 also possesses an ethanolamine-glycan-phosphatidylinositol-anchor in vitro; however, ChE2 produced in vitro could not be detected on nondenaturing gels.