Selective high-affinity transport of aspartate by a Drosophila homologue of the excitatory amino-acid transporters.

Excitatory amino-acid transporters (EAATs) are structurally related plasma membrane proteins that mediate the high-affinity uptake of the acidic amino acids glutamate and aspartate released at excitatory synapses, and maintain the extracellular concentrations of these neurotransmitters below excitotoxic levels [1] [2] [3] [4]. Several members of the EAAT family have been described previously. So far, all known EAATs have been reported to transport glutamate and aspartate with a similar affinity. Here, we report that dEAAT2 - a nervous tissue-specific EAAT homologue that we recently identified in the fruit fly Drosophila [5] - is a selective Na(+)-dependent high-affinity aspartate transporter (K(m) = 30 microM). We found that dEAAT2 can also transport L-glutamate but with a much lower affinity (K(m) = 185 microM) and a 10- to 15-fold lower relative efficacy (V(max)/K(m)). Competition experiments showed that the binding of glutamate to this transporter is much weaker than the binding of D- or L-aspartate. As dEAAT2 is the first known EAAT to show this substrate selectivity, it suggests that aspartate may play a specific role in the Drosophila nervous system.

A new brain dopamine-deficient Drosophila and its pharmacological and genetic rescue.

Dopamine (DA) is a neurotransmitter with conserved behavioral roles between invertebrate and vertebrate animals. In addition to its neural functions, in insects DA is a critical substrate for cuticle pigmentation and hardening. Drosophila tyrosine hydroxylase (DTH) is the rate limiting enzyme for DA biosynthesis. Viable brain DA-deficient flies were previously generated using tissue-selective GAL4-UAS binary expression rescue of a DTH null mutation and these flies show specific behavioral impairments. To circumvent the limitations of rescue via binary expression, here we achieve rescue utilizing genomically integrated mutant DTH. As expected, our DA-deficient flies have no detectable DTH or DA in the brain, and show reduced locomotor activity. This deficit can be rescued by l-DOPA/carbidopa feeding, similar to human Parkinson's disease treatment. Genetic rescue via GAL4/UAS-DTH was also successful, although this required the generation of a new UAS-DTH1 transgene devoid of most untranslated regions, as existing UAS-DTH transgenes express in the brain without a Gal4 driver via endogenous regulatory elements. A surprising finding of our newly constructed UAS-DTH1m is that it expresses DTH at an undetectable level when regulated by dopaminergic GAL4 drivers even when fully rescuing DA, indicating that DTH immunostaining is not necessarily a valid marker for DA expression. This finding necessitated optimizing DA immunohistochemistry, showing details of DA innervation to the mushroom body and the central complex. When DA rescue is limited to specific DA neurons, DA does not diffuse beyond the DTH-expressing terminals, such that DA signaling can be limited to very specific brain regions.

Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a 'proteostasis network' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Identification and structural characterization of two genes encoding glutamate transporter homologues differently expressed in the nervous system of Drosophila melanogaster.

In vertebrates, excitatory amino acid transporters (EAATs) are believed to mediate the removal of glutamate released at excitatory synapses and to maintain extracellular concentrations of this neurotransmitter below excitotoxic levels. Glutamate is also used in insects as an excitatory neurotransmitter at the neuromuscular junction and probably in the central nervous system where its role remains to be established. We report the molecular characterization and developmental expression pattern of two Drosophila cDNAs: dEAATI, which has recently been identified as a high affinity glutamate transporter [1], and dEAAT2, a novel protein sharing strong homology to dEAATI and to the mammalian EAAT protein family. The developmental expression pattern of the two Drosophila EAAT genes has been compared by Northern blot analysis and whole-mount in situ hybridizations. The two transporters are transcribed in distinct cell types of the nervous system and are strongly expressed in the adult visual system.

A 15 kDa proteolipid found in mediatophore preparations from Torpedo electric organ presents high sequence homology with the bovine chromaffin granule protonophore.

Upon SDS PAGE of isolated mediatophore, an acetylcholine-translocating protein, a doublet at 15 kDa was identified. Amino acid sequencing after CNBr cleavage gave a 17 residue-long peptide completely homologous with a sequence of the proton-translocating proteolipid from bovine chromaffin granules. A 51-mer oligodeoxynucleotide corresponding to this sequence was used to screen a library of electric lobe cDNAs constructed in lambda Zap II. A positive recombinant clone was isolated and found to encode the complete sequence of a 15.5 kDa protein highly homologous to the bovine chromaffin or yeast vacuolar ATPase proteolipid. In vitro translation of sense RNA transcripts of the clone indeed yielded a single 15 kDa proteolipid. Northern blot analysis showed that the 1.3 kb mRNA encoding this protein is significantly expressed in nervous tissues but not in electric organ or liver of Torpedo marmorata.

Regulation of hemicholinium-3 sensitive choline uptake in Xenopus laevis oocytes by the second C2 domain of synaptotagmin.

A size-fractionated torpedo electric lobe cDNA library was screened for the neuronal choline transporter by functional expression in oocytes. A clone, TLC2B, was isolated that induced a component of choline uptake that was hemicholinium-3 sensitive and inhibited by the substitution of lithium for sodium at low choline concentrations. However, [3H]choline uptake by both injected and non-injected oocytes were characterized by high affinity constants, suggesting that TLC2B could be affecting a native choline transporter. Indeed, hemicholinium-3 sensitive choline uptake could also be induced by preincubation of non-injected oocytes with a protein kinase C inhibitor, H-7. By sequence analysis and immuno-precipitation, the peptide produced by injection of TLC2B cRNA was identified as a soluble 24 kDa C-terminal fragment of the neuronal protein, synaptotagmin. Full length synaptotagmin was, however, ineffective in the functional test. The peptide encoded by TLC2B corresponds to the second protein kinase C-homologous domain of torpedo synaptotagmin, and like other soluble C2 domain peptides, was capable of calcium-dependent translocation to membranes. Its action on choline uptake in oocytes was, however, abolished by the addition of calcium in the presence of a calcium ionophore. These results suggest that the interaction of certain C2 domains, such as the C-terminal domain of synaptotagmin, with more specific targets may be anulled in the presence of calcium due to its absorption to membrane phospholipids.

Cyclic-amp dependent protein kinase in mouse striatal neurones and astrocytes in primary culture: development, subcellular distribution and stimulation of endogenous phosphorylation.

The cAMP-dependent protein kinase (cAMPdPK) activity and the endogenous cAMP-dependent phosphorylation of protein have been studied on pure populations of striatal neurones or astrocytes in primary culture originating from embryonic mouse brain. The appearance of cAMPdPK in cultured striatal neurones was rapid and paralleled neuritic outgrowth and cell maturation. Its highest value, reached between day 6 and 9 in culture, was comparable to that found in adult tissue. In cultured neurones as in adult striatum, cAMPdPK was found both in membrane and in cytoplasmic fractions. In astrocytes, cAMPdPK activity was low and only detectable in the cytoplasm. The presence of several cAMP-regulated phosphoproteins could be demonstrated in cultured cells, some of which being neurone-specific or astrocyte-specific. The phosphorylation of these striatal proteins was either enhanced, or, surprisingly, inhibited in the presence of cAMP. This study indicates that primary cultures of nerve cells provide valuable preparations for analysing protein phosphorylation processes induced by neurotransmitters.

The lipid requirements of mediatophore for acetylcholine release activity. Large-scale purification of this protein in a reactive form.

The mediatophore, a protein which translocates acetylcholine, has been recently purified from the plasma membrane of the Torpedo electric organ nerve terminals. The functional integrity of the mediatophore requires the presence of lipids. Removal of associated lipids led to an irreversible loss of activity when obtained by ether precipitation of the protein. A two-phase extraction procedure was developed to gently remove the lipids. Conditions were found to reactivate the protein by emulsifying it with electric organ lipids. A large-scale preparation procedure of the delipidified mediatophore in a reactive form is described.

Characterization of two ectocellularly oriented 67 K presynaptic proteins. Lack of effect of their removal on acetylcholine release.

A presynaptic plasma membrane fraction was purified after subfractionation of pure cholinergic synaptosomes prepared from Torpedo electric organ. Two 67 kdalton proteins were highly enriched in the synaptosomal plasma membrane (SPM): the hydrophobic form of AChE and a protein against which we raised a monoclonal antibody (C1-8). These two proteins exhibit similar biochemical properties: both exist as disulphide linked dimers with the same molecular weight; they are glycoproteins binding Concanavalin A; they are exposed on the external surface of the SPM and detached as almost entire molecules by Pronase. Nevertheless, using the C1-8 monoclonal antibody, it was possible to show that they are different proteins. The C1-8 binding protein appears to be specific for the SPM in Torpedo electric organ since it was not detected in plasma membranes from the electroplaque, the electric nerve trunks or the electric lobe. The hydrophobic AChE and the C1-8 binding protein appear therefore to be useful markers of the SPM. Pronase treatment of intact synaptosomes removes most of the ectocellularly exposed proteins of the SPM, which amount to 35% of the SPM protein. Presynaptic AChE and the C1-8 binding protein are detached. But ACh release can still be induced by depolarization of the Pronase treated synaptosomes. This demonstrates that the two 67 kdalton presynaptic proteins are not directly involved in the release of the neurotransmitter.

[Possible use of new corrosion-resistant steels in the manufacture of medical instruments]. / O vozmozhnosti primeneniia v proizvodstve meditsinskikh instrumentov novykh korrozionno-stoikikh stalei.

Demands on metals intended for the manufacture of medical instruments call for the use of corrosion-resisting grades of steel with high strength characteristics. Grades of steel of the martensite class reinforced by age hardening (martensive-aging) and mild grades of steel (chrome-manganese and chrome-nickel) have a number of valuable advantages over the steel of other classes. The possibility of obtaining high strength with low carbon dontent ensures great plasticity, toughness and brittle failure resistance along with corrosion-resistance and the possibility of achieving a substantial simplification in technology of making the instruments. Investigations of martensite-aging grades of steel--ep832,ochX11H9M2d2Tyu, O3X11HMd2yu and O3X13g3Md2 demostrated them to hold good promise for the manufacture of surgical and atraumatic needles, and rod-type stomatological instruments.

Involvement of the Drosophila taurine/aspartate transporter dEAAT2 in selective olfactory and gustatory perceptions.

Excitatory amino acid transporters (EAATs) are membrane proteins involved in the uptake of neurotransmitter amino acids in the nervous system. The Drosophila dEAAT2 gene was previously described to encode a taurine/aspartate transporter. To analyze further the expression pattern and physiological function of this protein, we generated transgenic flies containing either the dEAAT2 promoter region fused to GAL4 (dEAAT2-GAL4) or a transgene allowing expression of a dEAAT2::GFP fusion protein (UAS-dEAAT2::GFP). We observed that dEAAT2-GAL4 expresses green fluorescent protein (GFP) in neurons in central and peripheral structures of third-instar larvae and adult flies. Labeled neurons were found in olfactory and gustatory pathways, in which dEAAT2::GFP was detected from the dendrites of the sensory neurons up to the first- and second-order centers. dEAAT2-GAL4 is also expressed in mechanosensory neurons. We found that a viable piggyBac insertion strain disrupts dEAAT2 expression. This mutant appears morphologically normal and presents no locomotor or phototaxis impairments; however, its brain taurine level is significantly reduced compared with that of wild-type flies. The dEAAT2 mutant showed decreased avoidance behavior in the presence of high concentration of propionic acid compared with wild-type flies, but no modification of the avoidance response to benzaldehyde. In gustatory tests, both mutant and control flies were normally attracted to sucrose; however, the dEAAT2 mutant presented a higher salt sensitivity, being repulsed by low and high salt concentrations. Therefore, we conclude that dEAAT2 does function as a taurine transporter in vivo and that this protein is physiologically required for the sensory perception of specific environmental molecules.

Determination of acetylcholinesterase activity by a new chemiluminescence assay with the natural substrate.

A chemiluminescence method for determining acetylcholinesterase activity is described. It is an adaptation of the chemiluminescence assay of acetylcholine described by Israël & Lesbats [(1981) Neurochem. Int. 3, 81-90; (1981) J. Neurochem. 37, 1475-1483]. The acetylcholinesterase activity is measured by monitoring the increase in light emission produced by the accumulation of choline or by determining the amount of choline generated after a short interval. The assay is rapid and sensitive, and uses the natural substrate of the enzyme. Kinetic data obtained with this procedure for acetylcholinesterase from Torpedo and Electrophorus electric organs were comparable with those obtained by using the method of Ellman, Courtney, Andres & Featherstone [(1961) Biochem. Pharmacol. 7, 88-95]. In addition, it was shown that sodium deoxycholate totally inactivated Torpedo acetylcholinesterase but not the Electrophorus enzyme. Competitive inhibitors of acetylcholinesterase protected the enzyme from inactivation.

Blockade of the central generator of locomotor rhythm by noncompetitive NMDA receptor antagonists in Drosophila larvae.

The noncompetitive antagonists of the vertebrate N-methyl-D-aspartate (NMDA) receptor dizocilpine (MK 801) and phencyclidine (PCP), delivered in food, were found to induce a marked and reversible inhibition of locomotor activity in Drosophila melanogaster larvae. To determine the site of action of these antagonists, we used an in vitro preparation of the Drosophila third-instar larva, preserving the central nervous system and segmental nerves with their connections to muscle fibers of the body wall. Intracellular recordings were made from ventral muscle fibers 6 and 7 in the abdominal segments. In most larvae, long-lasting (>1 h) spontaneous rhythmic motor activities were recorded in the absence of pharmacological activation. After sectioning of the connections between the brain and abdominal ganglia, the rhythm disappeared, but it could be partially restored by perfusing the muscarinic agonist oxotremorine, indicating that the activity was generated in the ventral nerve cord. MK 801 and PCP rapidly and efficiently inhibited the locomotor rhythm in a dose-dependent manner, the rhythm being totally blocked in 2 min with doses over 0.1 mg/mL. In contrast, more hydrophilic competitive NMDA antagonists had no effect on the motor rhythm in this preparation. MK 801 did not affect neuromuscular glutamatergic transmission at similar doses, as demonstrated by monitoring the responses elicited by electrical stimulation of the motor nerve or pressure applied glutamate. The presence of oxotremorine did not prevent the blocking effect of MK 801. These results show that MK 801 and PCP specifically inhibit centrally generated rhythmic activity in Drosophila, and suggest a possible role for NMDA-like receptors in locomotor rhythm control in the insect CNS.

Transient release of acetylcholine from Torpedo synaptosomes in response to prolonged depolarization.

The release of ACh (acetylcholine) from purely cholinergic Torpedo synaptosomes was monitored continuously using a chemiluminescent assay. A maintained depolarization by high KCl in the presence of Ca2+ triggered only a transient ACh release. It was shown that neither depletion of the transmitter store nor an inhibition of the release mechanism itself were involved in this phasic response. The termination of release was probably caused by inactivation of voltage-dependent Ca2+ entry and rapid removal of intraterminal Ca2+ by a (Na+)0 dependent mechanism. It was found that exposure of the synaptosomes for a short period to low Ca2+-high K+ solutions greatly reduced the responses to Ca2+ reintroduction, as compared to the control release obtained when high K+ was applied in the presence of normal Ca2+. The response to Ca2+ reintroduction was measured following various times of preincubation with high K+ and low Ca2+; thus, an estimate of the time course of the inactivation of Ca2+ permeability during a depolarization could be made. A two component exponential kinetic was observed, with a rapid (tau = 3.6 s) and a slow phase (tau = 77 s). This inactivation was more pronounced when a higher KCl concentration was used to induce a greater depolarization. The presence of EGTA during the preincubation with high KCl greatly increased the response provoked by Ca2+ reintroduction, whereas increases in Ca2+ during the preincubation period caused proportional reduction in the subsequent response to Ca2+ reintroduction, indicating that the Ca2+ influx itself was involved in the inactivation process.

High-level synthesis and fate of acetylcholine in baculovirus-infected cells: characterization and purification of recombinant rat choline acetyltransferase.

Rat choline acetyltransferase (ChAT) has been expressed at a high level in Spodoptera frugiperda Sf9 cells using a baculovirus expression system. A cDNA containing the coding sequence for ChAT was inserted into the transfer vector pAcYM1 to yield the recombinant vector pAcYM1/ChAT. Sf9 cells were then coinfected with pAcYM1/ChAT and the wild-type Autographa californica virus. One recombinant virus particle, containing the cDNA for ChAT, was selected that expressed a protein of 68.5 kDa. Forty hours after infection of cells with the recombinant virus, the specific activity of ChAT in the cytosol was 190 nmol of acetylcholine/min/mg of protein, accounting for approximately 24% of the cell cytosolic proteins as being ChAT. The apparent Km values of the enzyme for choline and acetyl-CoA were 299 and 221 microM, respectively, whereas the respective Vmax values were 10.6 and 11.4 mumol of acetylcholine/min/mg of protein. In addition, analysis of the protein revealed that ChAT is phosphorylated in Sf9 cells. About 0.5 mg of ChAT was obtained from a one-step purification procedure starting with 10(8) infected Sf9 cells. Addition of choline to the incubation medium led to accumulation of high amounts of acetylcholine in the cytosol of the infected cells. The neurotransmitter was not released by Sf9 cells in response to membrane depolarization or on ionophore-mediated calcium entry. Some acetylcholine, which most likely originated from cell death inherent to viral infection, accumulated in the culture medium. The infected insect cells, which synthesize and store neurotransmitter, provide a new and convenient model for analyzing synaptic transmission at the molecular level.

Inactivation of acetylcholine release from Torpedo synaptosomes in response to prolonged depolarizations.

The release of acetylcholine (ACh) from purely cholinergic Torpedo synaptosomes was monitored continuously using a chemiluminescent assay (Israël & Lesbats, 1981 a, b). Upon prolonged K+ depolarization in the presence of Ca2+, the release of ACh was transient and returned to a steady low level in about 3 min. Addition of the Ca2+ ionophore A23187 triggered the release again, suggesting that neither depletion of the transmitter store nor an inhibition of the release mechanism itself were involved in this phasic response, but rather an inactivation of the Ca2+ entry. The release response evoked by adding Ca2+ back after exposure of the synaptosomes to high K+ (70 mM) and low Ca2+ (0.57 mM) solution inactivates as a function of the duration of the pre-depolarization with a two-component time course with rapid (tau = 5.5 s) and slow phases (tau = 143 s). This response to Ca2+ addition was more strikingly reduced as the level of depolarization during pre-treatment was increased. The inactivation was found to be dose dependent with respect to the amount of Ca2+ present during the pre-depolarization period (conditioning Ca2+). Moreover, the presence of EGTA during pre-treatment with high-K+ solutions increased the response to applied Ca2+. These observations suggest that Ca2+ entry itself was responsible for this inactivation. No inactivation was found when ACh release was induced by the depolarizing agent Gramicidin D, except when external Na+ was replaced by Li+. This result indicates that part of the Ca2+ influx promoted by Gramicidin D depends on a Na+ entry, and may be mediated by the Na-Ca exchange mechanism.

Solubilization and partial purification of a presynaptic membrane protein ensuring calcium-dependent acetylcholine release from proteoliposomes.

In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.

A novel and major isoform of tyrosine hydroxylase in Drosophila is generated by alternative RNA processing.

We report that two isoforms of Drosophila tyrosine hydroxylase protein are encoded via alternatively spliced exons. The major isoform (Type II) contains a novel acidic extension of 71 amino acids in the amino-terminal regulatory domain, which is likely to alter the regulatory properties of the tyrosine hydroxylase protein. The minor isoform (Type I) corresponds to the cDNA sequence reported previously. We also report the structure of the Drosophila tyrosine hydroxylase (DTH) gene and the diversity and tissue localization of its transcripts. At least three types of DTH mRNA are generated from a single primary transcript through alternative splicing and polyadenylation. Type II mRNA is the most abundant tyrosine hydroxylase transcript in Drosophila and is found predominantly in the hypoderm throughout all stages of development. Type I mRNA is present only in the CNS, where it is the primary form. The DTH transcripts detected in the CNS contain a longer 3'-untranslated region than the transcript expressed in the hypoderm, due to differential polyadenylation. In contrast, the same start site is used for DTH gene transcription in both tissues. These results show unexpected diversity in the DTH transcripts and point out possible mechanisms for differential regulation of tyrosine hydroxylase activity in the CNS and in the hypoderm.

Differential regulation of Drosophila tyrosine hydroxylase isoforms by dopamine binding and cAMP-dependent phosphorylation.

Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates. We have previously reported that tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II (Birman, S., Morgan, B., Anzivino, M., and Hirsh, J. (1994) J. Biol. Chem. 269, 26559-26567). Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nervous tissues like the epidermis. The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain. We show here that the enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH and that the isoform DTH II presents unique characteristics. The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography. The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate. However, the two enzymes differ in many of their properties. DTH II has a lower Km value for the co-substrate (6R)-tetrahydrobiopterin and requires a lower level of ferrous ion than DTH I to be activated. The two isoforms also have a different pH profile. As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels. However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition. The central nervous system isoform DTH I is activated through phosphorylation by cAMP-dependent protein kinase (PKA) in the absence of dopamine. In contrast, activation of DTH II by PKA is only manifest in the presence of dopamine. Site-directed mutagenesis of Ser32, a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA. We propose that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects.

Purification of a presynaptic membrane protein that mediates a calcium-dependent translocation of acetylcholine.

A protein, which we call "mediatophore," that mediates calcium-dependent release of acetylcholine from proteoliposomes has been purified from the presynaptic plasma membrane. About 250 micrograms of this material was obtained from 500 g of Torpedo marmorata electric organ. Precipitation of the protein and subsequent removal of associated lipids inactivated the protein, which then became water soluble; this permitted evaluation of its Stokes radius (52 A) and its sedimentation coefficient (9.8 +/- 0.75 S) and, hence, an approximate molecular mass of 210 +/- 16 kDa could be determined. PAGE analysis showed that the protein is made of 17-kDa subunits, not linked by disulfide bonds. When this material was observed by electron microscopy after negative staining, the apparently pentameric structures had an average diameter of about 7 nm.