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PLTX II, a presynaptic calcium channel blocker in Drosophila isolated from the plectreurys spider venom, is a 44-residue peptide containing ten Cys residues and an O-palmitoylated threonine amide at the carboxy-terminus. In this study, the palmitoylated peptide was synthesized in solution by applying our maximum protection strategy using the HF method at the final deprotecting step. Before designing the synthesis, we examined the stability of the palmitoyl moiety under the conditions for the synthesis of the peptide using several model peptides. The O-palmitoyl group was confirmed to be stable during elongation of the peptide bonds, but was partially removable during the deprotection reaction in HF. The depalmitoylation reaction in HF was temperature- and time-dependent. Therefore, the decision was made to protect the Asp residues with benzyl ester, since it is more susceptible to HF than cyclohexyl ester, which is now commonly used in the Boc-based, solid-phase synthesis. Thus, the HF reaction was carried out at -10 degrees or -15 degrees C for 1 h in order to reduce the extent of the depalmitoylation reaction. The resulting palmitoylated and depalmitoylated products were separated, the remaining Acm groups were removed using Hg(OAc)2, and then the completely deprotected peptides were folded to their native forms. The final palmitoylated peptide was proven to be identical with the natural one using various HPLC systems and by bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

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Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes calcium from intracellular stores in many cells. The synthesis of cADPR from NAD+ and its subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclase and a cADPR hydrolase, respectively. The ADP-ribosyl cyclase cloned from the ovotestis of the marine invertebrate Aplysia californica has amino acid sequence homology to the human lymphocyte surface antigen CD38. CD38 has been shown to catalyze both the formation and the hydrolysis of cADPR. In this study, we produced soluble, enzymatically active CD38 using recombinant expression techniques in bacteria and yeast. We engineered a gene coding for a soluble form of CD38 by excision of the region of the gene coding for the N-terminal amino acids representing the putative membrane spanning sequence and short putative intracellular sequence. For expression in bacteria (Escherichia coli), this construct was cloned into the pFlag-1 plasmid which allows induced, periplasmic expression and relatively simple purification of the soluble CD38. For expression in yeast (Pichia pastoris) the CD38 sequence was further modified to eliminate four putative N-linked glycosylation sites and the resulting construct was expressed as a secreted protein. Both systems produce soluble enzymes of approximately 30 kDa and both recombinant enzymes display similar cyclase and hydrolase activities.

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Cyclic nucleotides such as cAMP and cGMP are second messengers subserving various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD+ and is a mediator of Ca2+ mobilization in various cellular systems. The synthesis and degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyclase and cADPR hydrolase. CD38, a differentiation antigen of B lymphocytes, has recently been shown to be a bifunctional enzyme catalyzing both the formation and hydrolysis of cADPR. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD+, identical to that catalyzed by NADase. The difficulties in detecting the formation of cADPR have led to frequent identification of CD38 as a classical NADase. In this study, we show that both ADP-ribosyl cyclase and CD38, but not NADase, can cyclize nicotinamide guanine dinucleotide (NGD+) producing a new nucleotide. Analyses by high performance liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except with guanine replacing adenine. Compared to cADPR, cGDPR is a more stable compound showing 2.8 times more resistance to heat-induced hydrolysis. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Spectroscopic analyses show that cGDPR is fluorescent and has an absorption spectrum different from both NGD+ and GDPR, providing a very convenient way for monitoring its enzymatic formation. The use of NGD+ as substrate for assaying the cyclization reaction was found to be applicable to pure enzymes as well as crude tissue extracts making it a useful diagnostic tool for distinguishing CD38-like enzymes from degradative NADases.

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Toxins from spider venom, originally purified for their ability to block synaptic transmission in Drosophila, are potent and specific blockers of Ca2+ currents measured in cultured embryonic Drosophila neurons using the whole-cell, patch-clamp technique. Differential actions of toxins from two species of spiders indicate that different types of Drosophila neuronal Ca2+ currents can be pharmacologically distinguished. Hololena toxin preferentially blocks a non-inactivating component of the current, whereas Plectreurys toxin blocks both inactivating and non-inactivating components. These results suggest that block of a non-inactivating Ca2+ current is sufficient to block neurotransmitter release at Drosophila neuromuscular junction.

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Studies of presynaptic events in synaptic transmission may be facilitated through the use of specific ligands for functional components of the transmitter release mechanism and through the use of genetics. For this purpose, neurotoxins that affect neuromuscular transmission in Drosophila have been identified and purified from Plectreurys spider venom (PLTX). One class of toxins causes irreversible presynaptic block, probably by blocking calcium entry or by acting on other closely associated processes. These toxins have been highly purified and are peptides of about 7 kDa in molecular weight. They specifically block transmitter release at nanomolar concentrations and may be useful in further biochemical studies.

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The bag cell neurons of the marine mollusk Aplysia are a putative multitransmitter system which utilizes two or more neuropeptides that are enzymatically cleaved from a common precursor protein. It has been proposed that one of the neuropeptides, egg-laying hormone (ELH), acts nonsynaptically as a neurotransmitter in the abdominal ganglion by diffusing long distances to target neurons compared to conventional transmitters acting at synapses. To test this idea further, we investigated the physiological properties of neurotransmission mediated by ELH. We found that ELH acts directly to duplicate two types of responses produced by a burst discharge of the bag cells: prolonged excitation of LB and LC cells, and the previously described effect of ELH, burst augmentation of cell R15. Analysis of perfusate collected after electrical stimulation of the bag cells showed that the peptide is released in sufficient quantity to diffuse long distances within the ganglion without being completely inactivated. To mimic the way the peptide is thought to be released physiologically, ELH was arterially perfused into the ganglion. The response normally produced by bag cell activity was duplicated by 0.5 to 1.0 microM concentrations of ELH and showed no rapid desensitization. ELH had no effect on cells that are unaffected by bag cell activity and no effect on cells that are inhibited (LUQ cells) or transiently excited (cells L1 and R1) by bag cell activity. Acidic peptide, another peptide encoded on the ELH precursor protein, was found to be synthesized and released by the bag cells, but it had no effect on the cells we tested. We conclude that the combined properties of ELH neurotransmission resemble the properties of transmission at autonomic nerve endings on cardiac and smooth muscle rather than those of conventional synaptic transmission. ELH released from bag cells is dispersed throughout the interstitial and vascular spaces of the ganglion to produce responses in the cells that have receptors for the peptide. The results also suggest that ELH mediates only a subset of the responses induced by bag cell activity; they are consistent with data indicating that the other responses are mediated by other bag cell peptides derived from the same precursor protein as ELH.

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1. The bag cells are a group of neuroendocrine cells located in the abdominal ganglion of Aplysia. Accumulated evidence suggests they synthesize and release egg-laying hormone (ELH), a peptide that induces egg laying. In this and the following paper (37) we describe five types of prolonged neural responses in cells of the isolated abdominal ganglion that are produced by stimulated bag cell activity. 2. Prolonged, 5- to 40-min bursts of spike activity were triggered in the normally silent bag cells by local stimulation of one of the bag cell clusters with brief, 0.6- to 2-strains of pulses. This local stimulation minimized the possible effects of the stimulus on other ganglion cells and initiated bag cell activity similar to what has been recorded in intact animals at the initiation of egg laying. 3. Following onset of triggered bag cell activity there is an increase in the amplitude of the bursting pacemaker potential in cell R15 that results in augmented bursting activity in this autoactive cell for up to 3 h. The increase begins in less than 1 min and reaches a maximim after 8--20 min. In two other bursting pacemaker cells, L3 and L6, there is a second type of response, slow inhibition, consisting of a smoothly graded hyperpolarization that begins in 5--14 s, reaches a peak value of 10--20 mV after 30 s, and results in a decrease in the spontaneous spike activity of these cells for 3 h or longer. Both types of responses are contingent on the occurrence of bag cell activity, they depend on prolonged bag cell activity for their normal expression, and they occur in the absence of the fast interactions characteristic of conventional synapses. 4. The results reveal at the level of intracellular recordings prolonged actions of peptide-secreting neuroendocrine cells on the central nervous system. The role of ELH as a putative mediator of one or more of these actions is discussed.

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1. A survey of identified cells of the abdominal ganglion of Aplysia was undertaken to determine the extent of bag cell influence in the ganglion. Bursts of bag cell spike activity lasting 5--40 min were elicited by brief, 0.6- to 2 s local stimulation while recording simultaneously from bag cells and other ganglion cells with intracellular electrodes. 2. Slow inhibition occurs in giant cell R2, neurosecretory cells R3-R14, and ink-gland motoneurons, L14A, B, C. The cells remain hyperpolarized for from 15 to 60 min. 3. Transient excitation occurs in mechanoreceptor cells L1 and R1. The cells are strongly depolarized by a slow excitatory potential that lasts for about 10 min and produces spike activity for 3--7 min. 4. Prolonged excitation occurs in some cells of the LB and LC identified cell clusters. The cells are depolarized and spike activity is increased for 3 h or more. 5. A biphasic response occasionally occurs in the command interneuron L10. Inhibition of this cell lasts 10--15 min and is followed by excitation for several hours. Excitation is accompanied by facilitation of synaptic potentials for 40--60 min in cells innervated by L10; the facilitation apparently results from the increase in L10 firing rate. 6. The results indicate that the bag cells have multiple types of actions and affect large numbers of ganglion neurons. All effects have the slowly graded onsets and prolonged durations to be expected of hormonally mediated interactions. 7. Previous studies have indicated that in intact animals the bag cell burst discharge initates a stereotyped egg-laying behavioral pattern that persists for several hours (3, 27). The present data support the hypothesis that certain elements of egg-laying behavior and homeostasis are regulated by a direct action of the bag cells on the central nervous system.

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Egg laying hormone, a peptide neurohormone with an approximate molecular weight of 6000, was isolated from the region of the abdominal ganglion of Aplysia that contains the neuroendocrine bag cells and purified by gel filtration chromatography, isoelectric focusing, and dialysis. A 1-min local application of egg laying hormone to the identified neuron R15 produced prolonged (greater than 1 hr) augmentation of impulse activity in this neuron. The distinctive quality and prolonged duration of the response are apparently identical to the previously described response to electrically elicited bag cell activity. The results provide evidence that egg laying hormone is the mediator of this prolonged neuronal interaction.

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