Genome of the small hive beetle (Aethina tumida, Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory.

Background: The small hive beetle (Aethina tumida; ATUMI) is an invasive parasite of bee colonies. ATUMI feeds on both fruits and bee nest products, facilitating its spread and increasing its impact on honey bees and other pollinators. We have sequenced and annotated the ATUMI genome, providing the first genomic resources for this species and for the Nitidulidae, a beetle family that is closely related to the extraordinarily species-rich clade of beetles known as the Phytophaga. ATUMI thus provides a contrasting view as a neighbor for one of the most successful known animal groups. Results: We present a robust genome assembly and a gene set possessing 97.5% of the core proteins known from the holometabolous insects. The ATUMI genome encodes fewer enzymes for plant digestion than the genomes of wood-feeding beetles but nonetheless shows signs of broad metabolic plasticity. Gustatory receptors are few in number compared to other beetles, especially receptors with known sensitivity (in other beetles) to bitter substances. In contrast, several gene families implicated in detoxification of insecticides and adaptation to diverse dietary resources show increased copy numbers. The presence and diversity of homologs involved in detoxification differ substantially from the bee hosts of ATUMI. Conclusions: Our results provide new insights into the genomic basis for local adaption and invasiveness in ATUMI and a blueprint for control strategies that target this pest without harming their honey bee hosts. A minimal set of gustatory receptors is consistent with the observation that, once a host colony is invaded, food resources are predictable. Unique detoxification pathways and pathway members can help identify which treatments might control this species even in the presence of honey bees, which are notoriously sensitive to pesticides.

Using comparative genomics to decode the genetics of acaricide resistance.

The availability of genome assemblies and other genomic resources is facilitating investigations of complex genetic traits for several species of ticks. Understanding the genetics of acaricide resistance is a priority for tick and tick-borne disease control. The synaptic enzyme acetylcholinesterase (ACE) is recognized as the target of organophosphates (OPs) and carbamates, and mutations in ACE have been tied to resistance. Multiple studies support three ACE (ace) loci in R. microplus but the molecular basis of OP-resistance in this tick remains elusive. Here, we exploited the genome assembly of the black-legged tick Ixodes scapularis and comparative genomic analyses to explore the complement of tick ACEs and their potential roles in OP resistance. We identified eight putative ace loci (IscaACE1a, 1b, 2a-c, 3a-c) in I. scapularis. Molecular analyses and homology modeling suggest ACE activity for IscaACE1a. Our analyses reveal the molecular complexity of the I. scapularis ace gene family, highlight the need for functional studies of ACEs in species of the Ixodidae, and reveal potential challenges to management of OP resistance in ticks.

Molecular biology of tick acetylcholinesterases.

Ticks vector many pathogens with major health and economic impacts and have developed resistance to most acaricides used for tick control. Organophosphate (OP) acaricides target acetylcholinesterase (AChE) critical to tick central nervous system function. Mutations producing tick AChEs resistant to OPs were characterized; but tick OP-resistance is not fully elucidated, due to remarkable complexity of tick cholinergic systems. Three paralogous tick AChEs exhibiting differences in primary structure and biochemical kinetics are encoded by amplified genes with developmentally regulated expression. Gene silencing data suggest tick AChEs are functional complements in vivo, and transcriptomic and genomic data suggest existence of additional tick AChEs. Cholinergic systems are crucial in neural transmission and are also regulators of vertebrate immune function. Ticks exhibit prolonged intimate host contact, suggesting adaptive functions for tick cholinergic system complexity. AChE was recently reported in tick saliva and a role in manipulation of host immune responses was hypothesized. Physiological roles and genetic control of multiple tick AChEs requires further elucidation and may provide unique opportunities to understand and manipulate cholinergic involvement in biological systems.

ACE: an efficient and sensitive tool to detect insecticide resistance-associated mutations in insect acetylcholinesterase from RNA-Seq data.

BACKGROUND: Insecticide resistance is a substantial problem in controlling agricultural and medical pests. Detecting target site mutations is crucial to manage insecticide resistance. Though PCR-based methods have been widely used in this field, they are time-consuming and inefficient, and typically have a high false positive rate. Acetylcholinesterases (Ace) is the neural target of the widely used organophosphate (OP) and carbamate insecticides. However, there is not any software available to detect insecticide resistance associated mutations in RNA-Seq data at present. RESULTS: A computational pipeline ACE was developed to detect resistance mutations of ace in insect RNA-Seq data. Known ace resistance mutations were collected and used as a reference. We constructed a Web server for ACE, and the standalone software in both Linux and Windows versions is available for download. ACE was used to analyse 971 RNA-Seq data from 136 studies in 7 insect pests. The mutation frequency of each RNA-Seq dataset was calculated. The results indicated that the resistance frequency was 30%-44% in an eastern Ugandan Anopheles population, thus suggesting this resistance-conferring mutation has reached high frequency in these mosquitoes in Uganda. Analyses of RNA-Seq data from the diamondback moth Plutella xylostella indicated that the G227A mutation was positively related with resistance levels to organophosphate or carbamate insecticides. The wasp Nasonia vitripennis had a low frequency of resistant reads (<5%), but the agricultural pests Chilo suppressalis and Bemisia tabaci had a high resistance frequency. All ace reads in the 30 B. tabaci RNA-Seq data were resistant reads, suggesting that insecticide resistance has spread to very high frequency in B. tabaci. CONCLUSIONS: To the best of our knowledge, the ACE pipeline is the first tool to detect resistance mutations from RNA-Seq data, and it facilitates the full utilization of large-scale genetic data obtained by using next-generation sequencing.

Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

The observation of acetylcholinesterase (AChE) type H (AChEH), which is the predominant AChE variant in visceral organs and immune cells, in lipid rafts of muscle supports functional reasons for the raft targeting of glypiated AChEH The search for these reasons revealed that liver AChE activity is mostly confined to rafts and that the liver is able to make N-extended AChE variants and target them to rafts. These results prompted us to test whether AChE and muscarinic receptors existed in the same raft. Isolation of flotillin-2-rich raft fractions by their buoyancy in sucrose gradients, followed by immunoadsorption and matrix-assisted laser desorption ionization-time of flight-mass spectrometry application, gave the following results: 1) most hepatic AChE activity emanates from AChE-H mRNA, and its product, glypiated AChEH, accumulates in rafts; 2) N-extended N-AChE readthrough variant, nonglypiated N-AChEH, and N-AChE tailed variant were all identified in liver rafts; and 3) M3 AChRs were observed in rafts, and coprecipitation of raft-confined N-AChE and M3 receptors by using anti-M3 antibodies showed that enzyme and receptor reside in the same raft unit. A raft domain that harbors tightly packed muscarinic receptor and AChE may represent a molecular device that, by means of which, the intensity and duration of cholinergic inputs are regulated.-Montenegro, M. F., Cabezas-Herrera, J., Campoy, F. J., Muñoz-Delgado, E., Vidal, C. J. Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

The activity of detoxifying enzymes in the infective juveniles of Heterorhabditis bacteriophora strains: Purification and characterization of two acetylcholinesterases.

The infectivity and detoxifying enzyme activities including glutathione-S-transferase (GST), acetylcholinesterase (AChE) and carboxylesterase (CaE) are investigated in the infective juveniles (IJs) of six different strains of Heterorhabditis bacteriophora as a biocontrol agent against insect pests. The specific activities ranged from 10.8-29.8 and 50-220units/mg protein for GST and AChE, respectively; and from 24.7-129 and 22.6-77.3units/mg protein for CaE as estimated by P-nitrophenyl and α-naphthyl acetates, respectively. H. bacteriophora EM2 strain has the highest infectivity and the highest enzymatic activities as well. AChE is the predominant detoxifying enzyme that might imply its major role in the detoxification of insecticide(s). The isoenzyme pattern demonstrated two major slow-moving isoforms in all EPN strains examined. Purification of two AChE isoforms, AChEAII and AChEBI, from H. bacteriophora EM2 strain is performed by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and chromatography on DEAE-Sepharose. AChEAII and AChEBII have specific activities of 1207 and 1560unit/mg protein, native molecular weights of 180 and 68kDa, and are found in dimeric and monomeric forms, respectively. Both isoforms showed optimum activity at pH8.5 and 35°C. AChEBI exhibited higher thermal stability and higher activation energy than AChEAII. The enzymatic activities of purified AChEs are completely inhibited by Hg(+2) and Ni(+2) and greatly enhanced by Mn(+2). The substrate specificity, the relative efficiency of substrates hydrolysis, substrate inhibition and inhibition by BW284C51, but not by iso-OMPA, clearly indicated that they are true AChEs; their properties are compared with those recorded for insects as target hosts for H. bacteriophora EM2.

A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.

Through the use of molecular and biochemical experiments and bioinformatic tools, this work demonstrates that the PA4921 gene of the Pseudomonas aeruginosa PAO1 genome is a gene responsible for cholinesterase (ChoE) activity. Similar to the acetylcholinesterase (AchE) of Zea mays, this ChoE belongs to the SGNH hydrolase family. In mature ChoE, i.e., without a signal peptide, (18)Ser, (78)Gly, (127)N, and (268)H are conserved aminoacyl residues. Acetylthiocholine (ATC) and propionylthiocholine (PTC) are substrates of this enzyme, but butyrylcholine is an inhibitor. The enzyme also catalyzes the hydrolysis of the artificial esters p-nitrophenyl propionate (pNPP) and p-nitrophenyl butyrate (pNPB) but with lower catalytic efficiency with respect to ATC or PTC. The second difference is that pNPP and pNPB did not produce inhibition at high substrate concentrations, as occurred with ATC and PTC. These differences plus preliminary biochemical and kinetic studies with alkylammonium compounds led us to propose that this enzyme is an acetylcholinesterase (AchE) or propionylcholinesterase. Studies performed with the purified recombinant enzyme indicated that the substrate saturation curves and the catalytic mechanism are similar to those properties described for mammalian AchEs. Therefore, the results of this work suggest that the P. aeruginosa ChoE is an AchE that may also be found in Pseudomonas fluorescens.

Metabolism of captopril carboxyl ester derivatives for percutaneous absorption.

OBJECTIVES: To determine the metabolism of captopril n-carboxyl derivatives and how this may impact on their use as transdermal prodrugs. The pharmacological activity of the ester derivatives was also characterised in order to compare the angiotensin converting enzyme inhibitory potency of the derivatives compared with the parent drug, captopril. METHODS: The metabolism rates of the ester derivatives were determined in vitro (using porcine liver esterase and porcine ear skin) and in silico (using molecular modelling to investigate the potential to predict metabolism). KEY FINDINGS: Relatively slow pseudo first-order metabolism of the prodrugs was observed, with the ethyl ester displaying the highest rate of metabolism. A strong relationship was established between in-vitro methods, while in-silico methods support the use of in-vitro methods and highlight the potential of in-silico techniques to predict metabolism. All the prodrugs behaved as angiotensin converting enzyme inhibitors, with the methyl ester displaying optimum inhibition. CONCLUSIONS: In-vitro porcine liver esterase metabolism rates inform in-vitro skin rates well, and in-silico interaction energies relate well to both. Thus, in-silico methods may be developed that include interaction energies to predict metabolism rates.

Characterization of trimeric acetylcholinesterase from a legume plant, Macroptilium atropurpureum Urb.

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41-42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs.

Comparative investigation between acetylcholinesterase obtained from commercial sources and genetically modified Drosophila melanogaster: application in amperometric biosensors for methamidophos pesticide detection.

Genetically modified acetylcholinesterase (AChE) from Drosophila melanogaster (dm) and from commercial sources, Electric eel (ee), Bovine erythrocites (be) and Human erythrocites (he), were investigated as biological receptors for the detection of methamidophos pesticide based on inhibition studies. Most engineered variant of AChE from dm showed enhanced sensitivity toward methamidophos pesticide. Among 24 dmAChE variants tested, 12 presented a sensitivity comparable to the commercially available eeAChE, but higher than AChEs from be and he. Four were found more sensitive and six others were insensitive to methamidophos insecticide. The D375G,Y370F,Y374A,F376L mutant was the most sensitive, with a ki value of 2.2 X 10(6) mol(-1) L min(-1), three orders of magnitude higher than eeAChE (1.1 X 10(3) mol(-1) L min(-1)). The sensor constructed with genetically modified enzyme showed better characteristics with respect to detection limit and sensitivity compared with those using commercial eeAChE. Differential pulse polarography and chronoamperometry were used as electrochemical techniques to characterize the AChE biosensors. The lower detection limit of 1 ppb was obtained with D375G,Y370F,Y374A,F376L mutant of dmAChE, compared to 90 ppb for the commercial eeAChE. This study may stimulate scientists to develop more sensitive and selective procedures for organophosphorus insecticides detection by using engineered variant of dmAChE.

Blood-cell-specific acetylcholinesterase splice variations under changing stimuli.

Developmental and trauma-induced mechanism(s) that modify inflammation and immune responses in blood cells were recently found to be regulated by acetylcholine. Here, we report corresponding blood cell-specific changes in acetylcholinesterase splice variants. Plasmon resonance and flow cytometry using acetylcholinesterase variant-specific antibody probes, revealed a progressive increase in myeloid cell fractions expressing the apoptosis-related acetylcholinesterase-S variant from newborns to adult controls and post-delivery mothers. Hematopoietic cell fractions positive for the myeloproliferative acetylcholinesterase-R variant, were similarly high in post-partum blood, both intracellular and on the cell surface. Moreover, intracellular acetylcholinesterase-S protein amounts as reflected by fluorescence intensity measurements remained unchanged in myeloid cells from post-partum mothers as compared with matched controls. Unlike brain neurons, which over-express intracellular acetylcholinesterase-R under stress, lymphocytes from post-partum mothers presented increased surface acetylcholinesterase-S and pronounced decreases in both the expression and contents of surface acetylcholinesterase-R. Peripheral stimuli-induced modulations in acetylcholine regulation may hence reflect blood cell lineage-dependent acetylcholinesterase splice variations.

Mutation of the feh-1 gene, the Caenorhabditis elegans orthologue of mammalian Fe65, decreases the expression of two acetylcholinesterase genes.

The molecular adaptor Fe65 is one of the cytosolic ligands of the Alzheimer's beta-amyloid precursor protein (APP), and this complex is believed to play important roles in mammalian cells. Upon cleavage of APP by specific processing activities, the complex between Fe65 and the APP intracellular domain (AICD) translocates to the nucleus. Experimental evidence suggests that the Fe65-AICD complex regulates gene transcription. In Caenorhabditis elegans the orthologue of the Fe65 gene, feh-1, regulates pharyngeal activity. In fact, the rate of pharyngeal contraction is increased following transient or stable suppression of the feh-1 gene expression. Here we show that the increased contraction rate of the pharynx in feh-1 mutant worms is associated to decreased acetylcholinesterase activity. The decreased activity is accompanied by reduced expression of ace-1 and ace-2 transcripts, coding for the two major acetylcholinesterase activities in the nematode. These results indicate a target of the regulatory mechanisms based on the Fe65-APP complex that could be relevant for the pathogenesis of Alzheimer's disease.

An amino acid substitution on the second acetylcholinesterase in the pirimicarb-resistant strains of the peach potato aphid, Myzus persicae.

cDNAs encoding two acetylcholinesterases (AChEs) were isolated from the peach potato aphid, Myzus persicae. MpAChE1 was orthologous and MpAChE2 was paralogous with the ace of Drosophila melanogaster. The deduced amino acid sequence of MpAChE1 cDNA was identical between the pirimicarb susceptible and resistant strains. However, a single amino acid substitution of Ser431Phe on MpAchE2 was found in the pirimicarb resistant strains. This substitution was located in the acyl pocket of the enzyme and was thought to alter the ligand specificity.

Molecular cloning and characterization of a greenbug (Schizaphis graminum) cDNA encoding acetylcholinesterase possibly evolved from a duplicate gene lineage.

An acetylcholinesterase (AChE, EC 3.1.1.7) cDNA was cloned and characterized from a greenbug (Schizaphis graminum (Rondani)) cDNA library. The complete cDNA (3283 bp) contains a 2028-bp open reading frame encoding 676 amino acid residues. The putative AChE preproenzyme has a 17 amino acid signal peptide, a 78 amino acid activation peptide and a mature enzyme of 581 amino acid residues. The first nine amino acid residues (YTSDDPLII) that were determined by sequencing the N-terminus of a 72-kDa AChE purified from the greenbug matched the nine residues deduced from the cDNA. The key amino acid residues, including the three residues Ser206 (200 in Torpedo), Glu332 (327) and His446 (440) forming a catalytic triad, three pairs of cysteine putatively forming intrachain disulfide bonds, and 10 out of the 14 aromatic residues lining the active site gorge of the Torpedo AChE, are conserved. However, Ser336 (Phe331) in the greenbug substituted an aromatic amino acid residue that is conserved in all other known AChEs. Northern blot analysis of mRNA revealed a 3.7-kb transcript, and Southern blot analysis suggested a single copy of this gene in the greenbug. The deduced amino acid sequence is most similar to AChE1 of the nematodes Caenorhabditis briggsae and C. elegans with 43% identity. Phylogenetic analysis showed that the greenbug AChE formed a cluster with those of nematodes, a squid and ticks, and grouped out of the insect cluster. This result suggests that the cloned gene evolved from a different duplicate gene lineage of insect AChEs.

An acetylcholinesterase purified from the greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics.

An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the greenbug, Schizaphis graminum (Rondani). The maximum velocities (Vmax) for hydrolyzing acetylthiocholine (ATC), acetyl-(beta-methyl) thiocholine (AbetaMTC), propionylthiocholine, and S-butyrylthiocholine were 78.0, 67.0, 37.4, and 2.3 micromol/min/mg, and the Michaelis constants (Km) were 57.6, 60.6, 31.3, and 33.4 microM, respectively. More than 98% of AChE activity was inhibited by 10 microM eserine or BW284C51, but only 7% of the activity was inhibited by ethopropazine at the same concentration. Based on the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nondenaturing polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing of the purified AChE revealed three molecular forms. The isoelectric points were 7.3 for the major form and 6.3 and 7.1 for two minor forms. The major form of purified AChE showed molecular masses of 129 kDa for its native protein and 72 kDa for its subunits on SDS-PAGE. However, the purified AChE exhibited some distinctive characteristics including: (1) lack of affinity to the affinity ligand 3-(carboxyphenyl) ethyldimethyl ammonium, which has been used widely in purification of AChE from various insect species; and (2) 20-200-fold higher substrate-inhibition thresholds for ATC and AbetaMTC than AChE from other insect species. These biochemical properties may reflect structural differences of AChE purified from the greenbug compared with that from other insect species.

A major portion of synaptic basal lamina acetylcholinesterase is detached by high salt- and heparin-containing buffers from rat diaphragm muscle and Torpedo electric organ.

Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from synaptic basal lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle. Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers. In the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers, although some non-extractable AChE activity remains associated with the junctional regions.

Characterization of acetylcholinesterase molecular forms of the root-knot nematode, Meloidogyne.

Multiple molecular forms of acetylcholinesterase have been isolated and characterized from the root-knot nematodes Meloidogyne arenaria and Meloidogyne incognita. The forms of enzyme present in these 2 species are similar but not identical to those that occur in the free-living nematode Caenorhabditis elegans. The 5 enzyme forms exhibit differential solubilities and can be classified into 3 classes, A, B, and C, based on substrate affinity, inhibitor and detergent sensitivity, and thermal inactivation profiles. An unusual class of acetylcholinesterase has been isolated from Meloidogyne which has very high affinity for acetylcholine, but is highly resistant to carbamate and organophosphate inhibitors. The potential roles of the molecular forms in nematode behavior and sensitivity to nematicides are discussed.

Selective increase of tetrameric (G4) acetylcholinesterase activity in rat hindlimb skeletal muscle following short-term denervation.

Acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in gracilis muscles from adult Sprague-Dawley rats were studied 24-96 h after obturator nerve transection. Results show a selective denervation-induced increase in the globular G4 isoform, which is predominantly associated with the plasmalemma. This enzymatic increase was (a) transient (occurring between 24 and 60 h) and accompanied by declines in all other identifiable AChE isoforms; (b) observed after concurrent denervation and inactivation of the enzyme with diisopropylfluorophosphate, but not following treatment with cycloheximide; and (c) more prominent in the extracellular compartment of muscle endplate regions. Aside from this transient change, G4 activity did not fall below control levels, indicating that at least the short-term maintenance of G4 AChE (i.e., at both normal and temporarily elevated levels) does not critically depend on the presence of the motor nerve. In addition, this isoform's activity increases in response to perturbations of the neuromuscular system that are known to produce elevated levels of acetylcholine (ACh), such as short-term denervation and exercise-induced enhancement of motor activity. The present study is consistent with the hypothesis that individual AChE isoforms in gracilis muscle are subject to distinct modes of neural regulation and suggests a role for ACh in modulating the activity of G4 AChE at the motor endplate.

Localization of the pool of G4 acetylcholinesterase characterizing fast muscles and its alteration in murine muscular dystrophy.

The distributions of acetylcholinesterase and its molecular forms within muscles of normal and dystrophic 129/ReJ mice were established by a concomitant cytochemical and biochemical study performed on 1-mm serial sections of three predominantly fast muscles, i.e., anterior tibialis, extensor digitorum longus, and sternomastoid, as well as the slow-twitch soleus. This comparative study showed the following main findings. 1) In every muscle of both normal and dystrophic mice a) the three asymmetric forms were confined to the motor zone where they systematically codistributed with the endplates, and b) all globular forms, including G4, were concentrated at the motor zone from which they extended over the entire muscle length along a concentration gradient. 2) In the normal muscles, the perijunctional sarcoplasmic cytochemical reaction exhibited by individual fibers was grouped into a well-defined cojunctional acetylcholinesterase compartment in which the endplates were embedded. The overall intensity of the cojunctional cytochemical reaction was either high or low according to whether the muscle was predominantly fast or slow. 3) This cojunctional acetylcholinesterase compartment varied in close parallelism with G4 and thus appeared as the cytochemical correlate of the G4 molecules concentrated around the endplates. In particular, as the shape of the motor zone progressively increased in complexity along with the intricacy of the muscle fiber organization, from sternomastoid to extensor digitorum longus to anterior tibialis, so did both the relative volume occupied by the cojunctional acetylcholinesterase compartment and the proportion of G4. 4) The motor zone of the normal fast-twitch muscles characteristically differed from that of the soleus by the presence of a G4-rich environment around the endplates, which was cooperatively provided by the surrounding fibers. 5) In dystrophic muscles, this cojunctional G4-rich compartment was lost: the cojunctional cytochemical compartment was no longer discernable, while G4 was reduced to a minimal low level similar to that characteristic of the normal soleus.

Generation of subunit-specific antibody probes for Torpedo acetylcholinesterase: cross-species reactivity and use in cell-free translations.

The assembly of the collagen tailed A12 form of acetylcholinesterase (AChE) is regulated by muscle contraction. To begin to study this regulation, we derived antibody probes for the three subunits (100 kd, catalytic, and collagen tail) of AChE purified from Torpedo californica electric tissue. These included a polyclonal antiserum that recognizes all 3 subunits and 19 monoclonal antibodies; 16 of the monoclonals recognized the catalytic subunit, 2 recognized the tail subunit, and 1 recognized the 100 kd subunit on Western blots. We used immunohistochemical procedures to show that several of the anticatalytic and one of the antitail monoclonals cross-reacted with frog muscle AChE and Western blotting to show that several of the anticatalytic monoclonals cross-react with rat brain AChE. These antibodies were then used to immunoprecipitate AChE precursors from a cell-free translation system. There were generally three primary translation products, corresponding to the three enzyme subunits. Therefore, each subunit is probably derived from a separate mRNA. Occasionally there were two translation products corresponding to the catalytic subunit alone. The catalytic subunit was glycosylated following addition of canine microsomal membranes to the translation mix. The mRNA coding for this subunit appeared to be present in the poly(A)- RNA pool.