Presence of a Membrane-Bound Acetylcholinesterase Form in a Preparation of Nerve Endings from Torpedo marmorata Electric Organ

Abstract: We adapted a method, originally described by Israel et al. (1976) for the preparation of cholinergic nerve endings from Torpedo , to deal with a larger quantity of electric tissue. We followed the distribution of acetylcholine (ACh), ATP, acetylcholine receptor (AChR), choline acetyltransferase (ChAT), ouabainresistant and -sensitive ATPase, lactate dehydrogenase (LDH) and acetylcholinesterase (AChE) and obtained a nerve ending fraction, without detectable contamination by postsynaptic components. This preparation consisted of closed structures of 1–5 μm diameter, containing synaptic vesicles. It had the capacity to synthetize and release ACh. This preparation is therefore quite suitable for biochemical analysis of presynaptic elements. We particularly investigated its content of AChE: it consists exclusively of the 6S dimeric, hydrophobic form of the enzyme. This enzyme is enriched in the nerve ending preparation, by a factor higher than that obtained for ChAT. The yields obtained for the two enzymes suggest that the hydrophobic 6S AChE form may be mostly presynaptic in Torpedo electric organs. We characterized this form as a membrane-bound, externally active enzyme in the nerve ending preparation. It may thus participate in the hydrolysis of extracellularly liberated AChE and its abundance suggests that presynaptic AChE could play an essential role in cholinergic transmission in Torpedo electric organs and perhaps also in other cholinergic synapses.     

Inactive Monomeric Acetyleholinesterase in the Low-Salt-Soluble Extract of the Electric Organ from Torpedo marmorata

Proteolytic fragmentation of [3H]diisopropylfluorophosphate-labelled catalytic subunits of different molecular forms of acetylcholinesterase demonstrates that all forms extracted from the electric organ from Torpedo marmorata are true acetylcholinesterases. This is supported by immunochemical results showing that the radiolabelled polypeptides are readily recognized by specific anti-acetylcholinesterase antibodies. Although distinct structural differences exist, all forms contain a similar peptide carrying the serine hydroxyl of the esteratic subsite. Dimeric, detergent-soluble acetylcholinesterase is present in the low-salt-soluble extract (Mr of the catalytic subunit 66,000) together with a monomeric form (apparent Mr 76,000). This monomeric polypeptide is hydrophilic, enzymatically inactive, and might represent a precursor of the asymmetric forms of acetylcholinesterase.     

Characterization of aminopeptidase N from Torpedo marmorata kidney

Summary— A major antigen of the brush border membrane of Torpedo marmorata kidney was identified and purified by immunoprecipitation. The sequence of its 18 N terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase N (EC 3.4.11.2). Indeed aminopeptidase N activity was efficiently immunoprecipitated by monoclonal antibody 180K1. The purified antigen gives a broad band at 180 kDa after SDS-gel electrophoresis, which, after treatment by endoglycosidase F, is converted to a thinner band at 140 kDa. This antigen is therefore heavily glycosylated. Depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of 18S (2% CHAPS) or as a single peak of 7.8S (1% CHAPS plus 0.2 % C₁₂E₉), showing that Torpedo aminopeptidase N behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a 160 kDa monomer. The antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells (600 gold particles/μm² of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolatéral membranes, vacuoles or vesicles). Monoclonal antibodies prepared here will be useful tools for further functional and structural studies of Torpedo kidney aminopeptidase N.     

Polymorphism of Pseudocholinesterase in Torpedo marmorata Tissues: Comparative Study of the Catalytic and Molecular Properties of this Enzyme with Acetylcholinesterase

We report the existence, in Torpedo marmorata tissues, of a cholinesterase species (sensitive to 10(-5) M eserine) that differs from acetylcholinesterase (AChE, EC 3.1.1.7) in several respects: (a) The enzyme hydrolyzes butyrylthiocholine (BuSCh) at about 30% of the rate at which it hydrolyzes acetylthiocholine (AcSCh), whereas Torpedo AChE does not show any activity on BuSCh. (b) It is not inhibited by 10(-5) M BW 284C51, but rapidly inactivated by 10(-8) M diisopropylfluorophosphonate. (c) It does not exhibit inhibition by excess substrate up to 5 X 10(-3) M AcSCh. (d) It does not cross-react with anti-AChE antibodies raised against purified Torpedo AChE. This enzyme is obviously homologous to the "nonspecific" or pseudocholinesterase (pseudo-ChE, EC 3.1.1.8) that exists in other species, although it is closer to "true" AChE than classic pseudo-ChE in several respects. Thus, it shows the highest Vmax with acetyl-, and not propionyl- or butyrylthiocholine, and it is not specifically sensitive to ethopropazine. Pseudo-ChE is apparently absent from the electric organs, but represents the only cholinesterase species in the heart ventricle. Pseudo-ChE and AChE coexist in the spinal cord and in blood plasma, where they contribute to AcSCh hydrolysis in comparable proportions. Pseudo-ChE exists in several molecular forms, including collagen-tailed forms, which can be considered as homologous to those of AChE. In the heart the major component of pseudo-ChE appears to be a soluble monomeric form (G1). This form is inactivated by Triton X-100 within days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Calelectrin, a Ca2+-Binding Protein Isolated from the Electric Organ of Torpedo marmorata

We report a fast (less than 1 day) and efficient (2-3 mg protein/100 g tissue) isolation method for calelectrin, a protein of Mr 34,000 in the electric organ of Torpedo marmorata that binds to membranes in the presence of Ca2+. Purified protein was used to investigate the nature of its interaction with membranes and with Ca2+. Calelectrin binds to liposomes composed of total extractable lipids from the electric organ in a Ca2+-dependent and -specific manner with half-maximal binding between 3 and 7 microM free Ca2+. This binding is totally inhibited by 1 mM mercaptoethanol. It is also shown that calelectrin directly binds Ca2+ in solution by two techniques: at 1 and 10 microM Ca2+ it binds 45Ca2+ as measured by gel permeation chromatography, and it contains saturable Tb3+-binding sites that are Ca2+-displaceable. An investigation of the protein's endogenous fluorescence shows that although it contains both tryptophan and tyrosine, there is no change in the apparent quantum yield as a function of Ca2+. Ca2+-dependent hydrophobic affinity chromatography of the total soluble proteins from Torpedo electric organ shows that Torpedo calelectrin, like calmodulin and mammalian calelectrins, is specifically retained in the presence of Ca2+ and eluted by EGTA. Calelectrin also contains high-affinity sites for hydrophobic fluorescence probes such as N-phenyl-1-naphthylamine, 2-CP-toluidinylnaphthalene-6-sulfonic acid, and 1-anilinonaphthalene-8-sulfonic acid, which again unlike calmodulin, show no changes as a function of Ca2+. We conclude that calelectrin is a Ca2+-binding protein whose binding to the lipid moieties of membranes is regulated by physiological change in the Ca2+ concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and Expression of a Rat Acetylcholinesterase Subunit: Generation of Multiple Molecular Forms and Complementarity with a Torpedo Collagenic Subunit

Abstract: We obtained a cDNA clone encoding one type of catalytic subunit of acetylcholinesterase (AChE) from rat brain (T subunit). The coding sequence shows a high frequency of (G + C) at the third position of the codons (66%), as already noted for several AChEs, in contrast with mammalian butyrylcholinesterase. The predicted primary sequence of rat AChE presents only 11 amino acid differences, including one in the signal peptide, from that of the mouse T subunit. In particular, four alanines in the mouse sequence are replaced by serine or threonine. In northern blots, a rat AChE probe indicates the presence of major 3.2-and 2.4-kb mRNAs, expressed in the CNS as well as in some peripheral tissues, including muscle and spleen. In vivo, we found that the proportions of G₁, G₂, and G₄ forms are highly variable in different brain areas. We did not observe any glycolipid-anchored G₂ form, which would be derived from an H subunit. We expressed the cloned rat AChE in COS cells: The transfected cells produce principally an amphiphilic G₁^a form, together with amphiphilic G₂^a and G₄^a forms, and a nonamphiphilic G₄^na form. The amphiphilic G₁^a and G₂^a forms correspond to type II forms, which are predominant in muscle and brain of higher vertebrates. The cells also release G₄^na, G₂^a, and G₁^a in the culture medium. These experiments show that all the forms observed in the CNS in vivo may be obtained from the T subunit. By cotransfecting COS cells with the rat T subunit and the Torpedo collagenic subunit, we obtained chimeric collagentailed forms. This cross-species complementarity demonstrates that the interaction domains of the catalytic and structural subunits are highly conserved during evolution.     

Selection and Characterization of the Choline Transport Mutation Suppressor from Torpedo Electric Lobe,CTL1

The presumptive choline transporter, CTL1, was initially identified through functional complementation of a triple yeast mutant (ctr ise URA3) with deficiencies in both choline transport and choline neosynthesis under selective conditions that cause perturbations in membrane synthesis and growth. After transformation of these yeasts with a heterologous yeast expression library made from Torpedo electric lobe cDNAs, several colonies showed increased growth but only one clone increased the accumulation of external choline. The corresponding full-length cDNA was isolated and encodes a protein with 10 transmembrane domains. Northern analysis of Torpedo mRNA indicates that CTL1 is expressed at high levels in the spinal cord and brain. In Xenopus oocytes, Torpedo CTL1 expression was associated with the appearance of sodium independent high-affinity choline uptake. We propose that CTL1 plays a role in providing choline for membrane synthesis in the nervous system.     

Bovine Nucleus Caudatus Acetylcholinesterase: Active Site Determination and Investigation of a Dimeric Form Obtained by Selective Proteolysis

Abstract: The number of catalytic subunits of purified bovine nucleus caudatus acetylcholinesterase (E.C. 3.1.1.7) has been determined by active site labelling with [³H]diisopropyl fluorophosphate ([³H]DFP). The 10.5 S, 16 S, and 20 S forms were estimated to contain two, four, and six active sites, respectively, per molecule. A 4.8 S form, which showed a weak amphiphile-dependent activity behavior, was obtained by selective proteolytic digestion with pronase. The inability of the purified 4.8 S form to aggregate after detergent removal, and the molecular mass in the range of 130-165 kD under nondenaturating conditions, indicate that this form is a dimeric form, lacking those hydrophobic regions responsible for aggregation.     

Purification and Characterization of a Peptidyl Dipeptidase Resembling Angiotensin Converting Enzyme from the Electric Organ of Torpedo marmorata

The electric organ of Torpedo marmorata contains a membrane-bound, captopril-sensitive metallopeptidase that resembles mammalian angiotensin converting enzyme (peptidyl dipeptidase A; EC 3.4.15.1). The Torpedo enzyme has now been purified to apparent homogeneity from electric organ by a procedure involving affinity chromatography using the selective inhibitor lisinopril immobilised to Sepharose via a 28-A spacer arm. The purified protein, like the mammalian enzyme, acted as a peptidyl dipeptidase in cleaving dipeptides from the C-terminus of a variety of peptide substrates, including angiotensin I, bradykinin, [Met5]enkephalin, [Leu5]enkephalin, and the model substrate hippuryl (benzoylglycyl; BzGly)-His-Leu. The hydrolysis of BzGly-His-Leu was activated by Cl-. Enzyme activity was inhibited by classical angiotensin converting enzyme inhibitors, including captopril, enalaprilat (MK422), and lisinopril (MK521). Torpedo angiotensin converting enzyme, like its mammalian counterpart, was also able to act as an endopeptidase in hydrolysing the amidated neuropeptide substance P. Hydrolysis of substance P occurred primarily at the Phe8-Gly9 bond with release of the C-terminal tripeptide, Gly-Leu-MetNH2, and this hydrolysis was blocked by selective inhibitors. The Torpedo enzyme was recognised by a polyclonal antibody to pig kidney angiotensin converting enzyme on immunoelectrophoretic (Western) blot analysis. Thus, on the basis of substrate specificity, inhibitor sensitivity, and immunological criteria, the Torpedo enzyme closely resembles mammalian angiotensin converting enzyme. However, the Torpedo enzyme appears somewhat larger (Mr = 190,000) than the pig kidney enzyme (Mr = 180,000) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The endogenous peptide substrate(s) for Torpedo electric organ angiotensin converting enzyme and the physiological role of the enzyme in this tissue remain to be evaluated.     

Acetylcholinesterase in Mouse Neuroblastoma NB2A Cells: Analysis of Production, Secretion, and Molecular Forms

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.     

An Immunoglobulin M Monoclonal Antibody, Recognizing a Subset of Acetylcholinesterase Molecules from Electric Organs of Electrophorus and Torpedo, Belongs to the HNK-1 Anti-Carbohydrate Family

An immunoglobulin M (IgM) monoclonal antibody (mAb Elec-39), obtained against asymmetric acetylcholinesterase (AChE) from Electrophorus electric organs, also reacts with a fraction of globular AChE (amphiphilic G2 form) from Torpedo electric organs. This antibody does not react with asymmetric AChE from Torpedo electric organs or with the enzyme from other tissues of Electrophorus or Torpedo. The corresponding epitope is removed by endoglycosidase F, showing that it is a carbohydrate. The subsets of Torpedo G2 that react or do not react with Elec-39 (Elec-39+ and Elec-39-) differ in their electrophoretic mobility under nondenaturing conditions; the Elec-39+ component also binds the lectins from Pisum sativum and Lens culinaris. Whereas the Elec-39- component is present at the earliest developmental stages examined, an Elec-39+ component becomes distinguishable only around the 70-mm stage. Its proportion increases progressively, but later than the rapid accumulation of the total G2 form. In immunoblots, mAb Elec-39 recognizes a number of proteins other than AChE from various tissues of several species. The specificity of Elec-39 resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1, NSP-4, as well as IgMs that occur in human neuropathies. Although some human neuropathy IgMs that recognize the myelin-associated glycoprotein did not react with Elec-39+ AChE, mAbs HNK-1, NC-1, and NSP-4 showed the same selectivity as Elec-39 for Torpedo G2 AChE, but differed in the formation of immune complexes.     

An immunohistochemical study of synaptogenesis in the electric organ of Torpedo marmorata by use of antisera to vesicular and presynaptic plasma membrane components

Summary Synaptogenesis has been studied in the electric organ of embryonic Torpedo marmorata by use of two antisera directed against components of synaptic vesicles (anti-SV) and presynaptic plasma membranes (ap-anti-TSM), respectively. The anti-SV serum was previously shown to recognize a proteoglycan specific for synaptic vesicles. The ap-anti-TSM serum was raised to plasma membranes of synaptosomes derived from the electromotor nerve terminals and affinity-purified on electric-organ gangliosides. The vesicular antigen was first detectable at the 81-mm stage of development, which is 1–2 weeks earlier than the formation of morphologically mature presynaptic terminals, but is coincident with a rise in choline acetyltransferase levels and the ability of the electric organ to generate discharges. The gangliosidic antigen recognized by the ap-anti-TSM was first detectable on the ventral electrocyte surface at the 93-mm stage of development. This indicates that specific carbohydrate epitopes, not present on the growth cones, are expressed during maturation of the nerve terminal. The nerve terminal components recognized by these sera arose pari passu with neurite coverage of the ventral surface of the electrocyte, reaching a maximum in the adult. In contrast, postsynaptic aggregates of acetylcholine receptor, rendered visible with rhodamine-labeled -bungarotoxin, arose previous to the presynaptic antigens, reaching a maximum surface density at 110 mm and then declining in the adult.     

Mammalian protein homologous to VAT-1 of Torpedo californica: Isolation from Ehrlich ascites tumor cells,biochemical characterization,and organization of its gene

Recently, interest has focused on the human gene encoding the putative protein homologous to VAT-1, the major protein of the synaptic vesicles of the electric organ of the Pacific electric ray Torpedo californica, after it has been localized on chromosome locus 17q21 in a region encompassing the breast cancer gene BRCA1. Chromosomal instability in this region is implicated in inherited predisposition for breast and ovarian cancer. Here we describe isolation and biochemical characterization of a mammalian 48 kDa protein homologous to the VAT-1 protein of Torpedo californica. This VAT-1 homolog was isolated from a murine breast cancer cell line (Ehrlich ascites tumor) and identified by sequencing of cleavage peptides. The isolated VAT-1 homolog protein displays an ATPase activity and exists in two isoforms with isoelectric points of 5.7 and 5.8. cDNA was prepared from Ehrlich ascites tumor cells, and the murine VAT-1 homolog sequence was amplified by polymerase chain reaction and partially sequenced. The known part of the murine and the human translated sequences share 97% identity. By Northern blots, the size of the VAT-1 homolog mRNA in both murine and human (T47D) breast cancer cells was determined to be 2.8 kb. Based on the presented data, a modified gene structure of the human VAT-1 homolog with an extended exon 1 is proposed. VAT-1 and the mammalian VAT-1 homolog form a subgroup within the protein superfamily of medium-chain dehydrogenases/reductases. J. Cell. Biochem. 69:304–315, 1998. © 1998 Wiley-Liss, Inc.     

Conversion of cyanide to formate and ammonia by a pseudomonad obtained from industrial wastewater

Summary A cyanide-degrading pseudomonad was isolated by selective enrichment in a chemostat inoculated with coke-plant activated sludge and maintained at a dilution rate of 0.042/h for 60 days with a feed of 10 mg/l cyanide. The isolate, a facultative methylotroph capable of growth on methanol and methylamine, degraded cyanide to formate and ammonia; it could utilize the released ammonia as a nitrogen source but did not further metabolize formate under the experimental conditions employed. Both cyanide-degrading enzyme activity and respiratory resistance to cyanide were inducible and were enhanced by repeated exposure to the compound. Cell-free extracts stoichiometrically converted cyanide to formate and ammonia in a reaction that did not require oxygen. Enzyme activity, lost upon dialysis, was restored by less than equimolar ratios of NAD(P)H or ascorbate to cyanide, indicating that the reductants did not function directly as co-enzymes.     

Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock–key system to protect Acacia food bodies from exploitation

Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two‐dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz‐type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin‐like and elastase‐like proteolytic activity in the guts of seed‐feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase‐like and to a lower degree chymotrypsin 1‐like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term ‘exclusive rewards’ can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.     

Inactivation of the Peptide-Sensitive Channel from the Yeast Mitochondrial Outer Membrane: Properties,Sensitivity to Trypsin and Modulation by a Basic Peptide

The yeast Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane closes with slow kinetics at potentials of either polarity. The properties of this inactivation closely resemble those of the Voltage-Dependent Anion Channel (VDAC) slow kinetics closures. Addition of trypsin to one compartment suppresses the inactivation observed when this compartment is made positive, but does not affect the inactivation observed at potentials of reverse polarity. Both sides of the channel are sensitive. The reduced form of the Mast Cell Degranulating peptide (rMCD) increases the rate of inactivation, but only when the polarity of the compartment to which it is added is positive. The effect is not reversed by washing the peptide out, but is suppressed by trypsin. The peptide can bind to both sides of the membrane. The effect of rMCD on PSC closely resembles that of the ``modulator' on VDAC. The similarities between PSC and VDAC suggest that the former might be a cationic porin of the mitochondrial outer membrane possessing a structure closely related to that of VDAC. Received: 2 February 1996/Revised: 18 October 1996     

A Low-Km 5'-Nucleotidase from Rat Brain Cytosolic Fraction: Purification, Kinetic Properties, and Description of Regulation by a Novel Factor that Increases Sensitivity to Inhibition by ATP and ADP

Abstract: A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000- g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low K _m for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by α,β-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of M_r = 54,300 ± 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- K _m"soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000- g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.