Recruitment and diversification of an ecdysozoan family of neuropeptide hormones for black widow spider venom expression

Venoms have attracted enormous attention because of their potent physiological effects and dynamic evolution, including the convergent recruitment of homologous genes for venom expression. Here we provide novel evidence for the recruitment of genes from the Crustacean Hyperglycemic Hormone (CHH) and arthropod Ion Transport Peptide (ITP) superfamily for venom expression in black widow spiders. We characterized latrodectin peptides from venom gland cDNAs from the Western black widow spider (Latrodectus hesperus), the brown widow (Latrodectus geometricus) and cupboard spider (Steatoda grossa). Phylogenetic analyses of these sequences with homologs from other spider, scorpion and wasp venom cDNAs, as well as CHH/ITP neuropeptides, show latrodectins as derived members of the CHH/ITP superfamily. These analyses suggest that CHH/ITP homologs are more widespread in spider venoms, and were recruited for venom expression in two additional arthropod lineages. We also found that the latrodectin 2 gene and nearly all CHH/ITP genes include a phase 2 intron in the same position, supporting latrodectin's placement within the CHH/ITP superfamily. Evolutionary analyses of latrodectins suggest episodes of positive selection along some sequence lineages, and positive and purifying selection on specific codons, supporting its functional importance in widow venom. We consider how this improved understanding of latrodectin evolution informs functional hypotheses regarding its role in black widow venom as well as its potential convergent recruitment for venom expression across arthropods.     

Physiological and Biochemical Analysis to Reveal the Molecular Basis for Black Widow Spiderling Toxicity

The early research found that the spiderlings of black widow spider (Latrodectus tredecimguttatus) exhibited obvious toxicity to animals. The present work performed a systematical analysis of the aqueous extract of newborn black widow spiderlings. The extract was shown to contain 69.42% of proteins varying in molecular weights and isoelectric points. Abdominal injection of the extract into mice and cockroaches caused obvious poisoning symptoms as well as death, with LD₅₀ being 5.30 mg/kg in mice and 16.74 µg/g in Periplaneta americana. Electrophysiological experiments indicated that the extract at a concentration of 10 µg/mL could completely block the neuromuscular transmission in isolated mouse nerve‐hemidiaphragm preparations within 21 ± 1.5 min, and 100 µg/mL extract could inhibit a certain percentage of voltage‐activated Na⁺, K⁺, and Ca²⁺ channel currents in rat dorsal root ganglion neurons. These results demonstrate that the spiderlings are rich in neurotoxic components, which play important roles in the spiderling toxicity.     

Interactions of charatoxins and nereistoxin with the nicotinic acetylcholine receptors of insect cns and Torpedo electric organ

Interactions of charatoxin (4-methylthio-1,2-dithiolane; ChTX) and four openchain analogs as well as nereistoxin (NTX) with acetylcholine (ACh) receptors were studied using biochemical assays on the Torpedo electric organ and honey bee brain receptors and using electrophysiological assays on the response of the cell body of the fast coxal depressor motoneuron (D_f) of the cockroach Periplaneta americana to ACh. The actions of ChTXs were complex. Except for ChTX Xl, they all potentiated the ACh-induced current in Periplaneta neurons, but at higher concentrations all ChTXs, except for ChTX XII, caused voltage-dependent block of this current. All CHTXs inhibited binding of [³H]perhydrohistrionicotoxin in the presence of ACh to the highaffinity noncompetitive blocker site on the Torpedo receptor, but all, except for ChTX XI, potentiated its binding in absence of ACh. The actions of ChTXs on the honey bee brain receptor were quite different from those on the Torpedo receptor. They inhibited, or had no effect on, [¹²⁵I]α-bungarotoxin (α-BGT) binding to the Torpedo receptor, but all ChTXs, except for ChTX I, potentiated its binding to the honey bee receptor. It is suggested that the action of ChTXs on nicotinic ACh-receptors resulted from binding to lowaffinity noncompetitive blocker site. On the other hand, NTX was more potent than ChTXs on nicotinic ACh-receptors, and some similarities were noted between the actions of NTX on Torpedo and honey bee receptors NTX had a weak agonistlike effect in both cases and possibly bound to the ACh binding sites as well as the high-affinity noncompetitive blocker site. Thus the mechanisms of action of ChTXs and NTX on nicotinic ACh-receptors are different, and there are also differences in the responses to these toxins between receptors of insect central nervous system and Torpedo electric organ.     

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.     

The Polarity of Lipid-Exposed Residues Contributes to the Functional Differences between Torpedo and Muscle-Type Nicotinic Receptors

A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues αS287, αC412, βY441, γM299, γS460, δM293, δS297 and δN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [¹²⁵I]-α-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC₅₀), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC₅₀. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.     

Some properties of choline acetyltransferase isolated from the electric organ ofTorpedo marmorata: Specificity for choline analogues and inhibition by certain amines and amino acids

(1) Choline acetyltransferase ofTorpedo marmorata electric organ was studied by using soluble tissue extracts partially purified by (NH₄)₂SO₄ fractionation. (2) Linear enzymatic rates were observed at 30°C, in the presence of 350 M acetyl-CoA and 50 mM choline, over a 30–40 min incubation period. (3) A number of analogues of choline, including mono-, di-, and triethylcholine and pyrrolcholine were synthesized and theK _m (apparent) andV (maximum velocity) values determined. TheK _m (apparent) for choline (11.5 mM), with theTorpedo enzyme, was high in comparison to values reported for mammalian or invertebrate nervous tissue. TheTorpedo enzyme was also not so specific for choline in comparison with the other choline analogues (based onK _m values) as were other sources of the enzyme. TheV values for choline and mono-, di-, and triethylcholine with theTorpedo enzyme indicated a direct relationship between enzyme activity andN-alkyl substitution. (4) Several amines and amino acids inhibited choline acetyltransferase fromTorpedo. Histamine (15 mM) brought about a 60% inhibition and was found to be a noncompetitive inhibitor with respect to choline.     

Myelin lipids: A phylogenetic study

The lipid composition of CNS and PNS myelin was studied in rat,Xenopus, trout andTorpedo. The main difference lay in the proportion of cerebrosides, which decreased in the sequence rat > Xenopus > Torpedo > trout. In additionTorpedo CNS and PNS myelins were extremely rich in sulfatides. In some respects,Torpedo appeared closer to tetrapods than trout. Otherwise the proportion of the different lipid classes did not reveal any clear evolutionary trends.The presence of hydroxylated galactolipids in CNS myelin was investigated in several additional species. Considerable amounts were found inTorpedo, Polypterus, Protopterus, lizard, and chicken, with the highest values in rat and anurans. Only very small amounts of hydroxylated cerebrosides were detected in trout and in axolotl, while newt had none. This parameter appears therefore of doubtful usefulness for phylogenetic studies. In contrast to myelin proteins, myelin lipids are of limited value for establishing phylogenetic relationship among vertebrates.Abbreviations CH cholesterol - CNS central nervous system - FA fatty acids - GC cerebrosides - HPTLC high-performance thin-layer chromatography - PC phosphatidylcholine - PE phosphatidylethanolamine and ethanolamine plasmalogens - PI phosphatidylinositol - PLP proteolipid protein - PNS peripheral nervous system - PS phosphatidylserine - SM sphingomyelin - SU sulfatides     

Structural characterization and agonist binding to human α4β2 nicotinic receptors

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition – particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences.     

Cuticular permeability of the black widow spiderLatrodectus hesperus

Summary The water permeability of abdominal cuticle of the black widow spiderLatrodectus hesperus was examined using gravimetric, in situ and in vitro techniques. At 30 °C and 0% RH, water loss rates (WLR) of whole, living spiders were higher than in situ WLR measured with capsules secured to the cuticle surface, while in situ WLR of living spiders were greater than in situ WLR for dead spiders. Although these differences are not statistically significant, these results suggest that there may be important extracuticular water loss pathways in living animals, and that the living epidermis does not provide an active barrier to water efflux. In vitro WLR measured on excised pieces of cuticle, in contrast, were nearly 5 times greater than in situ WLR of dead spiders. Temperature/transpiration curves show that permeability increased exponentially with rising temperature, with rates increasing most rapidly between 45 and 51 °C. The WLR for spiders whose cuticle was scrubbed with chloroform: methanol was consistently and substantially (200x) higher than WLR for control or sham-treated spiders. Discussion focuses on the location and composition of the epicuticular lipid water barrier and the potential use of black widow cuticle for in vitro studies of lipid barrier effectiveness in arthropods in general.     

Frequent Failure of Male Monopolization Strategies as a Cost of Female Choice in the Black Widow Spider Latrodectus tredecimguttatus

In many mating systems, males strive for securing paternity through monopolizing females. As male monopolization attempts often contradict female interests, this conflict may fuel an evolutionary arms race. In the widow spider genus Latrodectus, females are commonly polyandrous, whereas males are monogynous, hence restricted to mate with a single female, making paternity protection particularly important. Potential mating plugs (specialized embolus sclerites of male copulatory organs) have been discovered in the complex female genital tracts of several Latrodectus species. In this study, we investigated mating strategies in the Mediterranean black widow spider Latrodectus tredecimguttatus and tested the adaptive value of female attacks against male monopolization efforts. In a double mating experiment, we manipulated the number of insertions (=copulations) for first and second males to assess female behaviour and male embolus sclerite placement success. Our results indicate that first males′ embolus sclerites inside the females′ sperm stores physically block sclerites of subsequent males. While female attacks did not affect the deposition of potential mating plugs, they significantly reduced copulation duration. Irrespective of female aggression, male sclerite placement failure occurred frequently, but large males were more successful than smaller competitors. We suggest that the complex genital morphology in both sexes may have co‐evolved antagonistically and female morphology could serve to favour large males for fertilization.     

α-Latrotoxin stimulates glutamate release from cortical astrocytes in cell culture

The mechanism responsible for the ability of bradykinin to cause calcium-dependent release of glutamate from astrocytes in vitro was investigated. The glutamate transport inhibitor, dihydrokainate, did not block bradykinin-induced glutamate release, and bradykinin did not cause cell swelling. These data exclude the involvement of glutamate transporters or swelling mechanisms as mediating glutamate release in response to bradykinin. α-Latrotoxin (3 nM), a component of black widow spider venom, stimulated calcium-independent glutamate release from astrocytes. Since α-latrotoxin induces vesicle fusion and calcium-independent neuronal neurotransmitter release, our data suggest that astrocytes may release neurotransmitter using a mechanism similar to the neuronal secretory process.     

Altered patterns ofN-linked glycosylation of theTorpedo acetylcholine receptor expressed inXenopus oocytes

Summary The nicotinic acetylcholine receptor (AChR) fromTorpedo electroplax is an oligomeric transmembrane glycoprotein made up of four highly homologous subunits in a stoichiometry of ₂. The role ofN-linked glycosylation of the AChR has been studied in several cell lines and these studies have suggested that the addition of carbohydrate may be important for receptor expression. WhileXenopus oocytes have proven to be an invaluable tool for studying the AChR, little is known aboutN-linked glycosylation of the oocyte-expressed receptor. The present report demonstrates that the oocyte-expressed AChR is glycosylated and contains the same number of oligosaccharide residues per subunit as the native receptor. However, unlike the nativeTorpedo receptor which contains both high mannose and complex oligosaccharides, the oocyte-expressed AChR contains only high mannose oligosaccharide modifications. However, as has been well documented, theTorpedo AChR expressed in oocytes is fully functional, demonstrating that the precise nature of the oligosaccharide modification is not critical for receptor function.The role of the oligosaccharide component of the AChR in receptor function was examined using tunicamycin (TM) to inhibitN-linked protein glycosylation. TM treatment resulted in a 70–80% inhibition of AChR expression in oocytes. Functional, unglycosylated receptors were not expressed; receptors expressed in TM-treated oocytes were functional wild-type, glycosylated AChR, formed only during the initial 12 hr of TM exposure. These data suggest that while glycosylation of the oocyte-expressedTorpedo AChR is required for assembly of subunits into a functional receptor, as has been demonstrated in other cells, oocyte modification of normalTorpedo glycosylation patterns does not affect receptor function or assembly.     

Homology Built Model of Acetylcholinesterase from Drosophila Melanogaster

Abstract

Acetylcholinesterases from Drosophila melanogaster and Torpedo marmorata possess 35% identical residues. We built a homology model of the Drosophila enzyme on the basis of the known three-dimensional structure of Torpedo acetylcholinesterase, which revealed an oval rim of the active site gorge with an additional hollow which could accept small charged ligands more firmly than the corresponding surface in the Torpedo enzyme. This difference at the peripheral site, together with the kinetics of W121A and W359L mutants, suggests coordinate action of important hydrophobic residues that form the active site gorge during the catalytic process. It may also account for the activation-inhibition kinetic pattern which is characteristic for the insect enzyme.     

A fluorescence decay study of parinaroyl-phosphatidylinositol incorporated into artificial and natural membranes

Phosphoinositide metabolism in the plasma membrane is linked to transmembrane signal transduction. In this study we have investigated some physical properties (e.g. molecular order and dynamics) of phosphatidylinositol (PI) in various membrane preparations by time-resolved fluorescence techniques, using a synthetic PI derivate with a cis-parinaroyl chain on the sn-2 position. Phospholipid vesicles, normal and denervated rat skeletal muscle sarcolemmal membranes, and acetylcholine receptor rich membrances from Torpedo marmorata were investigated both at 4°C and 20 °C. For comparison we have also included 2-parinaroyl-phosphatidylcholine (PC) in this study. The fluorescent lipids were incorporated into the membrane preparations by way of specific phospholipid transfer proteins, to ensure an efficient and non-perturbing insertion of the lipid-probes. In the Torpedo membranes the order parameters measured for the parinaroyl derivatives of both PC and PI were higher than in phospholipid vesicles. For the Torpedo membrane preparations the acyl chain order for the PI was lower than that for PC, whereas the opposite was true for the vesicles. This inversion strongly suggests that PI has different interactions with certain membrane components as compared to PC. This is also suggested by the significantly higher rate of restricted rotation of PI as compared to PC. In contrast to the order parameters, the correlation times were almost identical for both probes and showed little difference between vesicles and the Torpedo membranes. In contrast to Torpedo membranes, the time-dependent fluorescence anisotropy of the two lipid probes in the sarcolemmal membranes showed, after an initial fast decay, a subsequent gradual increase. This phenomenon was satisfactorily analyzed by assuming two populations of probe lipids with distinct lifetimes, rotational correlation times and molecular order. The order parameter of the population with a short lifetime compared with that of phospholipid vesicles, whereas the population with a long lifetime agreed with that of the Torpedo membranes.Abbreviations PI phosphatidylinositol - PC phosphatidylcholine - PA phosphatidic acid - PE phosphatidylethanolamine - PS phosphatidylserine, PnA, cis-parinaric acid: cis,trans,trans,cis-9,11,13,15-octadecatetraenoic acid - 2-PnA-PC 1-acyl, 2-parinaroyl-PC - 2-PnA-PI 1-acyl,2-parinaroyl-PI - DPH diphenylhexatriene - POPOP 1,4-di[2-(5-phenyloxazolyl)]-benzene - NMR nuclear magnetic resonance - ESR electron spin resonance - I parallel fluorescence intensity component - I perpendicular fluorescence intensity component - SET-buffer 0.25 M Sucrose, 1 mM EDTA, 10mM Tris-HCl, pH 7.4     

α-TOXIN BINDING PROTEINS IN THE ELECTRIC ORGAN OF TORPEDO MARMORATA STUDIED BY IMMUNOCHEMICAL METHODS

—Crossed immunoelectrophoretic techniques were developed to study the efficiency of the various purification steps in the isolation of nicotinic acetylcholine receptor (nAChR) from Torpedo mormorata electric organ. A new α-neurotoxin binding assay based on immunoelectrophoresis is also presented. In crude extracts of Torpedo electric organ membranes one type of receptor molecule (M ñ; 300 , 000) was found; an earlier described higher molecular form was shown to be an artifact of affinity chromatography. Polyvalent antibodies against Torpedo electroplaque membranes, antibodies against purified membrane proteins and against Naja naja siamensisα-neurotoxin revealed four α-neurotoxin binding antigens (including nAChR). Two of these, nAChR and T₂, were specific for electroplaque membrane and showed partial immunoidentity but different biochemical and physical properties.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.     

Purification and Partial Characterization of a Novel Neurotoxic Protein from Eggs of Black Widow Spiders (Latrodectus tredecimguttatus)

Up to now, there have been a few reports on the toxic components purified from black widow spider (Latrodectus tredecimguttatus) eggs. In the present study, a novel neurotoxic protein was purified from the eggs by gel filtration combined with ion‐exchange chromatography. Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry. The protein could block the neuromuscular transmission in mouse‐isolated phrenic nerve‐hemidiaphragm preparations completely in a reversible manner and activate tetrodotoxin‐sensitive sodium current in rat dorsal root ganglion cells. The N‐terminal sequence of the protein was identified by the Edman degradation to be N‐S‐I‐A‐D‐D‐R‐Y‐R‐W‐P‐G‐Y‐P‐G‐A‐G‐L‐I‐P‐Y‐I‐I‐D‐S—. When the sequence was used to search against protein database with a sequence query in Mascot engine there was no matched sequence or protein whereas the Basic Local Alignment Search Tool (BLAST) analysis indicated that no significant similarity was found. These results demonstrated that the protein (named Latroeggtoxin‐I) is a novel neurotoxic protein purified from the eggs of black widow spiders. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:337‐342, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21493     

Cholinergic-specific glycoconjugates

Cholinergic nerve terminals utilize glycoconjugates in several ways, as surface markers and as structural components of the synaptic vesicles present within them. The surface markers have been discovered immunochemically: antibodies raised against them are able specifically to sensitize the cholinergic subpopulation of mammalian brain synaptosomes to complement-mediated lysis. One such group of antigens (Chol-1) have been identified as a novel series of minor gangliosides having in common a sialylatedN-acetylgalactosamine residue. These gangliosides may constitute the major gangliosides at cholinergic terminals. A second surface antigen (Chol-2) is thought to be a protein with an epitope in common with aTorpedo electric organ ganglioside. Cholinergic synaptic vesicles are rich in a proteoglycan which appears to assist in the sequestration of acetylcholine within the vesicle and to stabilize the vesicle membrane during cycles of exocytosis and recovery. It may be the cholinergic equivalent of the chromogranins.Abbreviations AP affinity purified - ATPase adenosine 5-triphosphate phosphohydrolase - cer ceramido - ChAT choline acetyltransferase - Chol-1, –2 cholinergic-specific antigens - DA dopamine - DOG deoxyglucose - ELISA enzyme-linked immunosorption assay - EOD electric organ discharge - FAB fast atomic bombardment - GABA -aminobutyrate - GAG glycosaminoglycan - gal galactosyl - gaINAc N-acetylgalactosaminyl - glc glucosyl - Glu glutamate - 5-HT 5-hydroxytryptamine - LDH lactate dehydrogenase - NA noradrenaline - NGF nerve growth factor - S, S-S mono-, disialyl - SPM synaptosomal plasma membrane - TH tyrosine hydroxylase - TLC thin-layer chromatography - TSM Torpedo electromotor synaptosomal membrane - VIP vasoactive intestinal polypeptide - VPG vesicle proteoglycan Special issue dedicated to Dr. Leon Wolfe.     

The subcellular fractionation of the electric organ of Torpedo

Summary The acetylcholine-rich electric organ of Torpedo has been submitted to subcellular fractionation in an attempt to isolate nerve endings and synaptic vesicles derived from cholinergic neurones. Fractions containing small vesicles and granules as their only morphologically identifiable components also contained appreciable amounts of bound acetylcholine; however, it was not possible to demonstrate a specific enrichment of any one fraction with respect to bound acetylcholine as has been possible in brain. The tissue proved difficult to homogenize and few detached nerve endings (synaptosomes) were formed. A low-speed fraction rich in Na, K- activated adenosine triphosphatase contained numerous membrane fragments with tubular appendages derived from the non-innervated surface of electroplaques. Homogenization in media isotonic with elasmobranch plasma (e.g. 0.5 M sucrose + 0.33 M urea) was essential to preserve the structure of osmotically sensitive organelles (e.g. mitochondria).We wish to express our gratitude to Dr. R. D. Keynes who arranged the supply of Torpedos and to Mr G. H. C. Dowe and Miss L. Swales for skilled technical assistance. The electron microscopic facilities were provided by a grant from the Wellcome Trust and the work was supported by a grant no. NB-03928-02 (to V.P.W.) from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. During the period of this work Dr. Sheridan held a Postdoctoral Fellowship of the U.S. Public Health Service and Dr. Israël was an Exchange Scholar of the Medical Research Council.We are also most grateful to Professor Sir Bryan Matthews, C.B.E., Sc. D., F.R.S., for providing aquarium facilities in the Physiological Laboratory of Cambridge University.     

MOLECULAR PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM DIFFERENT SPECIES INVESTIGATED BY ISOELECTRIC FOCUSING AND ION EXCHANGE ADSORPTION

—The isoelectric point, surface charge and K_m for choline of choline acetyltransferase from different species were determined. Choline acetyltransferase from mouse and monkey brain was resolved into three molecular forms with isoelectric points at 7·1, 7·5, 8·4 and 7·0, 7·35, 8·35 respectively, whereas choline acetyltransferase from the electric organ of Torpedo and from rabbit brain showed a molecular form with isoelectric point 6·6 and 6·9, respectively. With the exception of rabbit brain enzyme, there was a good correlation between the isoelectric points and surface charges of the different choline acetyltransferases. The K_m's for choline were 0·66, 0·88, 0·92 and 3·5 mM for monkey, mouse, rabbit and Torpedo choline acetyltransferase respectively. The separated molecular forms of mouse and monkey enzymes did not show any significant difference in their affinity for choline.