Analogies and differences between omega-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells.

The characteristics of the binding sites for the Conus magus toxins omega-conotoxin MVIIC and omega-conotoxin MVIID, as well as their effects on K+-evoked 45Ca2+ entry and whole-cell Ba2+ currents (IBa), and K+-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [125I] omega-conotoxin GVIA to bovine adrenal medullary membranes was displaced by omega-conotoxins GVIA, MVIIC and MVIID with IC50 values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [125I] omega-conotoxin MVIIC, which was displaced by omega-conotoxins MVIIC, MVIID and GVIA with IC50 values of around 30, 80 and 1.200 nM, respectively. The sites recognized by omega-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC50 values of around 1 nM). Both omega-conotoxin MVIIC and MVIID blocked IBa by 70-80%; the higher the [Ba2+]o of the extracellular solution the lower the blockade induced by omega-conotoxin MVIIC. This was not the case for omega-conotoxin MVIID; high Ba2+ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba2+. A further difference between the two toxins concerns their reversibility; washout of omega-conotoxin MVIIC did not reverse the blockade of IBa while in the case of omega-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by omega-conotoxin GVIA, suggesting that it is associated with N-type Ca2+ channels. Blockade of K+-evoked 45Ca2+ entry produced results which paralleled those obtained by measuring IBa. Thus, 1 microM of each of omega-conotoxin GVIA and MVIIA inhibited Ca2+ uptake by 25%, while 1 microM of each of omega-conotoxin MVIIC and MVIID caused a 70% blockade. K+-evoked catecholamine secretory responses were not reduced by omega-conotoxin GVIA (1 microM). In contrast, at 1 microM both omega-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca2+ channels that contribute to the regulation of secretion and are sensitive to omega-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for omega-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca2+ channels or an entirely new subtype of voltage-dependent high-threshold Ca2+ channel.

Purification, characterization, synthesis, and cloning of the lockjaw peptide from Conus purpurascens venom.

The major groups of Conus peptides previously characterized from fish-hunting cone snail venoms (the alpha-, mu-, and omega-conotoxins) all blocked neuromuscular transmission. A novel activity, the "lockjaw peptide", from the fish-hunting Conus purpurascens, caused a rigid (instead of flaccid) paralysis in fish and increased excitability at the neuromuscular junction (instead of a block). We report the purification, biological activity, biochemical and preliminary physiological characterization, and chemical synthesis of the lockjaw peptide and the sequence of a cDNA clone encoding its precursor. Taken together, the data lead us to conclude that the lockjaw peptide is a vertebrate-specific delta-conotoxin, which targets voltage-sensitive sodium channels. The sequence of the peptide, which we designate delta-conotoxin PVIA, is (O = 4-trans-hydroxyproline) EACYAOGTFCGIKOGLCCSEFCLPGVCFG-NH2. This is the first of a diverse spectrum of Conus peptides which are excitotoxins in vertebrate systems.

The distribution of omega-conotoxin MVIICnle-binding sites in rat brain measured by autoradiography.

An analogue of omega-conotoxin MVIIC, [125I]omega-MVIICnle, has been employed in an autoradiographic assay to define the distribution of binding sites in rat brain of this neuronal calcium channel antagonist. In comparison with N-type channels (labeled by [125I]omega conotoxin GVIA), omega-MVIICnle sites are much denser in cerebellum (molecular layer) than in forebrain. Binding in thalamus is also comparatively high for omega-MVIICnle. Under these conditions, [125I]omega-MVIICnle binding to rat brain sections is not displaceable by the N-channel antagonist, omega-conotoxin GVIA. The calcium channel blocker [125I]omega-conotoxin MVIICnle labels a unique set of binding sites in mammalian brain.

Toxityping rat brain calcium channels with omega-toxins from spider and cone snail venoms.

Different types of voltage-sensitive Ca2+ channels in the brain can be defined by specific ligands: L-type Ca2+ channels are uniquely sensitive to dihydropyridines, and N-type Ca2+ channels are selectively blocked by the Conus peptide omega-CTX-GVIA. Cloning data have revealed additional calcium channel types in mammalian brain for which selective ligands would be desirable. We describe binding experiments involving three newer ligands that block dihydropyridine- and omega-CTX-GVIA-resistant Ca channels: omega-Aga-IIIA and omega-Aga-IVA from venom of the spider Agelenopsis aperta and omega-CTX-MVIIC from Conus magus. [125I]omega-Aga-IVA binds with high affinity (IC50 = approximately 50 nM) to receptors in rat brain which may correspond to P-like calcium channels. A second high-affinity site (IC50 = approximately 1 nM) is defined by [125I]omega-CTX-MVIIC, proposed here to be on an "O"-type calcium channel. [125I]omega-Aga-IIIA targets homologous binding sites present on multiple Ca channel types. The IIIA sites overlap with the binding sites for [125I]omega-CTX-GVIA and [125I]omega-CTX-MVIIC. The IIIA sites do not overlap with the site defined by omega-Aga-IVA. Thus toxin ligands may be highly specific for a particular Ca channel (i.e., GVIA for the N-type channel) or exhibit broader specificity (i.e., omega-Aga-IIIA, which appears to bind L-, N-, P-, and O-type Ca2+ channels).

Heterodimeric structure of the spider toxin omega-agatoxin IA revealed by precursor analysis and mass spectrometry.

We report the first molecular characterization of a precursor sequence for a small, Ca2+ channel blocking, peptide spider toxin, omega-agatoxin IA. By integrating information generated from a molecular genetic approach using agatoxin cDNAs with data provided from mass spectrometry of the mature toxin, we were able to deduce the likely mechanisms by which the toxin precursor peptide is processed to its mature heterodimeric form. A particularly interesting feature of the prepropeptide is the occurrence of two glutamate-rich sequences interposed between the signal sequences, the major peptide toxin, and the minor toxin peptide. Excision of the more distal glutamate-rich region appears to be signaled by flanking arginine residues but likely occurs only after a disulfide linkage has formed between the major and minor chains of the mature toxin. Our molecular genetic approach toward characterizing this toxin will allow us to quickly generate a series of spider sequences from which mature toxin structures can be deduced and eventually expressed. Additionally, this approach will provide insights into the evolutionary divergence observed among spider peptide toxins.

Precursor structure of omega-conotoxin GVIA determined from a cDNA clone.

The Ca2+ channel blocking neurotoxin, omega-conotoxin GVIA, is a 27-amino acid peptide with three disulfide bonds. We have determined the precursor structure of this peptide by analyzing a cDNA clone obtained from a Conus geographus venom duct library. The omega-conotoxin GVIA prepropeptide is 73 amino acids in length comprising a 22 amino acid signal sequence, an intervening region of 23 amino acids, and finally, a 27 amino acid toxin region. A C-terminal glycine residue is later processed to a C-terminal amide moiety. The omega-conotoxin GVIA precursor exhibits regions of strong homology to the previously characterized King-Kong peptide precursor, but shows surprising divergence as well. The possible significance of the precursor organization is discussed.

Cadang-cadang disease of coconut palm.

Cadang-cadang disease has caused the death of over 30 million coconut palms, and no control measures are known. The causal viroid is the smallest known pathogen. Field diagnostic tests will soon be developed to allow mapping of the distribution and spread of the viroid and this should lead to the identification of appropriate control measures.

The pyridine nucleotide cycle. Studies in Escherichia coli and the human cell line D98/AH2.

Different metabolic steps comprise the pyridine nucleotide cycles in Escherichia coli and in the human cell line HeLa D98/AH2. An analysis of the 32P-labeling patterns in vivo reveals that in E. coli, pyrophosphate bond cleavage of intracellular NAD predominates, while in the human cell line, cleavage of the nicotinamide ribose bond predominates. In E. coli, intracellular NAD is processed differently from extracellular NAD. Conversion of intracellular NAD to nicotinic acid mononucleotide (NaMN) can be demonstrated in intact cells. We have also assayed and purified an enzyme, NMN deamidase, which converts NMN to NaMN. These data suggest that in E. coli, the predominant intracellular pyridine nucleotide cycle operative under our experimental conditions is: NAD leads to NMN leads to NaMN leads to NaAD leads to NAD Thus, a metabolic event requiring pyrophosphate bond cleavage of NAD, such as DNA ligation, initiates most NAD turnover. In the human cell line, the data are consistent with the following NAD turnover cycle: (formula, see text) Whereas in E. coli, ADP-ribosylation does not make a quantitatively important contribution, we suggest that in HeLa cells, ADP-ribosylation events initiate NAD turnover.