Search PubMed⌕ Search

Biomedical subjects

L J Cruz

Publications and source records attributed to L J Cruz.

At least 55 records · Page 3Linked to original sources
PubMed1988

Phylogenetic specificity of cholinergic ligands: alpha-conotoxin SI.

The alpha-conotoxins are small peptide neurotoxins from the venom of fish-hunting cone snails which block nicotinic acetylcholine receptors (nAChRs). We describe the purification, characterization, and chemical synthesis of a new alpha-conotoxin from Conus striatus, alpha-conotoxin SI. In contrast to other AChR ligands, alpha-SI discriminates between different vertebrate nAChRs. The sequence of alpha-conotoxin SI is Ile-Cys-Cys-Asn-Pro5-Ala-Cys-Gly-Pro-Lys10-Tyr-Ser-Cys-NH2. This sequence was confirmed by chemical synthesis. A des-Ile-alpha-SI derivative was also synthesized and is biologically active. Although alpha-conotoxin SI is highly homologous to previously described alpha-conotoxins, it has one noteworthy sequence feature which may account for its novel biological specificity. In all other alpha-conotoxins, there is a positively charged amino acid at residue 9; in alpha-conotoxin SI, this is replaced by proline. The discovery that different alpha-conotoxins can vary by orders of magnitude in their apparent affinity for different vertebrate receptors demonstrates that alpha-conotoxins will be useful probes for investigating phylogenetic differences between vertebrate nAChRs.

Amino Acid Sequence↗
PubMed1987

Total synthesis and further characterization of the gamma-carboxyglutamate-containing "sleeper" peptide from Conus geographus venom.

The total synthesis of the Gla-containing "sleeper" peptide (Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 ) from Conus geographus is described. A new strategy for the synthesis of acid-sensitive peptide amides was developed, which allowed complete deprotection and cleavage of the L-gamma-carboxyglutamate-containing peptide from the 2,4-dimethoxybenzhydrylamine resin. Synthetic sleeper peptide, after preparative high-performance liquid chromatography (HPLC) purification, was shown to be identical with the native peptide by all criteria (coelution experiments of HPLC, sequence analysis, and biological activity). In addition, a developmental switch in the behavioral symptoms induced by the peptide after intracerebral administration in mice was documented. At low doses of the peptide (4-30 pmol/g), a sleeplike state was induced in mice under 2 weeks old; in contrast, older mice became markedly hyperactive. It is proposed that, in the presence of Ca2+, the sleeper peptide assumes an alpha-helical configuration in which all the gamma-carboxyglutamate residues are located on the same side of the alpha-helix.

1-Carboxyglutamic Acid↗
PubMed1987

Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms.

The vasopressin-oxytocin family of peptides is of very ancient lineage, found in organisms as diverse as hydra and man. Although these peptides have been intensively studied in vertebrates, the presumably more extensive invertebrate series was defined primarily by immunological methods. In this report, we describe the purification and structures of two peptides of the vasopressin-oxytocin family from molluscs ("Conopressins"), which were found in the venom of fish-hunting marine snails of the genus Conus. The biological activity observed when the two snail peptides are injected intracerebrally into mice is very similar to that elicited by the vertebrate neurohypophyseal hormones and presumably reflects their actions upon a common receptor in the brain. The sequences of the purified peptides reveal unique features not found in the vertebrate peptide series, most notably an additional positive charge. These are the first members of the invertebrate series of the vasopressin-oxytocin family to be characterized biochemically. The sequences of these peptides are: from Conus geographus venom, Lys-conopressin-G, Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2; and from Conus striatus venom, Arg-conopressin-S, Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH2.

Amino Acid Sequence↗
PubMed1987

Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom.

The omega-conotoxins from the venom of fish-hunting cone snails are probably the most useful of presently available ligands for neuronal Ca channels from vertebrates. Two of these peptide toxins, omega-conotoxins MVIIA and MVIIB from the venom of Conus magus, were purified. The amino acid sequences show significant differences from omega-conotoxins from Conus geographus. Total synthesis of omega-conotoxin MVIIA was achieved, and biologically active radiolabeled toxin was produced by iodination. Although omega-conotoxins from C. geographus (GVIA) and C. magus (MVIIA) appear to compete for the same sites in mammalian brain, in amphibian brain the high-affinity binding of omega-conotoxin MVIIA has narrower specificity. In this system, it is demonstrated that a combination of two omega-conotoxins can be used for biochemically defining receptor subtypes and suggested that these correspond to subtypes of neuronal Ca2+ channels.

Amino Acid Sequence↗
PubMed1987

Characterization of the omega-conotoxin target. Evidence for tissue-specific heterogeneity in calcium channel types.

omega-Conotoxin GVIA (omega-CgTx-VIA) is a 27 amino acid peptide from the venom of the fish-hunting snail, Conus geographus, that blocks voltage-activated Ca channels. The characterization of a biologically active, homogeneous 125I-labeled monoiodinated Tyr22 derivative of omega-conotoxin GVIA and its use in binding and cross-linking studies are described. The 125I-labeled toxin is specifically cross-linked to a receptor protein with an apparent Mr of 135,000. The stoichiometry between omega-conotoxin and nitrendipine binding sites in different chick tissues was determined. Skeletal muscle has a high concentration of [3H]nitrendipine binding sites (greater than 1000 fmol/mg) but no detectable omega-conotoxin sites (less than 7 fmol/mg). Brain microsomes have both binding sites, but omega-conotoxin targets are in excess. These results, combined with recent electrophysiological studies (E. W. McCleskey, A. P. Fox, D. Feldman, L. J. Cruz, B. M. Olivera, R. W. Tsien, and D. Yoshikami, unpublished results), define four types of Ca channels in chick tissues, N, T, Ln (omega sensitive), and Lm (omega insensitive), and are consistent with the hypothesis that the alpha-subunits of certain neuronal Ca2+ channels (Ln, N) are the molecular targets of omega-conotoxin GVIA.

Amino Acid Sequence↗
PubMed1987

Neuronal calcium channel inhibitors. Synthesis of omega-conotoxin GVIA and effects on 45Ca uptake by synaptosomes.

We previously described a 27-amino acid peptide neurotoxin from the venom of Conus geographus, omega-conotoxin GVIA, which inhibits neuronal voltage-activated calcium channels. In this paper we describe the total synthesis of omega-conotoxin GVIA and demonstrate that it efficiently blocks voltage-activated uptake of 45Ca by standard synaptosomal preparations from chick brain. Dihydropyridines do not block 45Ca uptake under these conditions. Thus, the omega-conotoxin-sensitive, but dihydropyridine-insensitive uptake of 45Ca2+ by chick brain synaptosomes serves as a functional assay for a Ca channel target of omega-conotoxin. The use of synthetic GVIA should rapidly accelerate our understanding of the molecular biology of Ca2+ channels and their role in neuronal function.

Animals↗
PubMed1987

Omega-conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle.

Blockade of Ca2+ channels by omega-conotoxin GVIA, a 27 amino acid peptide from the venom of the marine snail Conus geographus, was investigated with patch-clamp recordings of whole-cell and unitary currents in a variety of cell types. In dorsal root ganglion neurons, the toxin produces persistent block of L- and N-type Ca2+ channels but only transiently inhibits T-type channels. Its actions appear to be neuron-specific, since it blocks high-threshold Ca2+ channels in sensory, sympathetic, and hippocampal neurons of vertebrates but not in cardiac, skeletal, or smooth muscle cells. Block occurs through direct interaction of the toxin with an external site closely associated with the Ca2+ channel, without apparent involvement of a second messenger or dependence on channel gating. The tissue and channel-type specificity and the directness and slow reversibility of the block are features that favor use of omega-conotoxin as a tool for purifying particular neuronal Ca2+ channels and defining their physiological function.

Animals↗
PubMed1987

Transmitter release from presynaptic terminals of electric organ: inhibition by the calcium channel antagonist omega Conus toxin.

Cholinergic synaptosomes from electroplax of the ray Ommata discopyge release both ATP and ACh when depolarized with high K+ concentration in the presence of Ca2+. Others have shown that the ATP and ACh are released in the molar ratio found in isolated synaptic vesicles. Thus, it is assumed that the release of ATP reflects exocytosis of synaptic vesicles, and that transmitter release can be indirectly monitored by assaying ATP release. We present further evidence for this assumption and examine the effects of presynaptic neurotoxins on this ATP release. As expected for transmitter release, we find that depolarization-evoked ATP release is supported by Sr2+ and Ba2+ and is inhibited by the Ca channel antagonists Co2+ and Mn2+. Likewise, the presynaptic toxins omega-CmTX and omega-CgTX, omega peptides from the venom of the marine snails Conus magus and Conus geographus, respectively, inhibit 80% of the depolarization-evoked ATP release. Half-maximal inhibition of ATP release occurs with approximately 0.5 microM of either toxin. The toxins' effects are reversible, and when toxin is washed away, the time dependence of recovery of release is approximately first order and half complete within 40 min with omega-CmTX and 15 min with omega-CgTX. The Ca2+ ionophore A23187 induces Ca2+-dependent ATP release from resting synaptosomes. As would be expected of a Ca channel antagonist, omega-CmTX does not affect this ionophore-induced release. Leptinotarsin-d (LPTd), a putative Ca channel agonist from the Colorado potato beetle, evokes Ca2+-dependent ATP release from resting synaptosomes. omega-CmTX does not block LPTd-evoked release of ATP, which suggests that omega-CmTX and LPTd act at different sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1986

Calcium channel antagonists. Omega-conotoxin defines a new high affinity site.

The omega-conotoxins, a class of Ca2+ channel antagonists from fish-hunting marine snails, have recently been described (Olivera, B. M., McIntosh, J. M., Zeikus, R., Gray, W. R., Varga, J., Rivier, J., de Santos, V., and Cruz, L. J. (1985) Science, 230, 1338-1343). One of these peptide neurotoxins, omega-conotoxin GVIA, was radiolabeled with iodine, and the 125I-labeled toxin was shown to bind specifically to high affinity sites on chick brain synaptosomes. The toxin-receptor complex was extremely stable; addition of an excess of unlabeled toxin did not cause significant displacement of the labeled toxin after 2 h. Binding competition data suggest that omega-conotoxin defines a new high affinity receptor site affecting voltage-activated Ca2+ channels, distinct from both the verapamil and dihydropyridine target sites.

Animals↗
PubMed1985

Peptide neurotoxins from fish-hunting cone snails.

To paralyze their more agile prey, the venomous fish-hunting cone snails (Conus) have developed a potent biochemical strategy. They produce several classes of toxic peptides (conotoxins) that attack a series of successive physiological targets in the neuromuscular system of the fish. The peptides include presynaptic omega-conotoxins that prevent the voltage-activated entry of calcium into the nerve terminal and release of acetylcholine, postsynaptic alpha-conotoxins that inhibit the acetylcholine receptor, and muscle sodium channel inhibitors, the mu-conotoxins, which directly abolish muscle action potentials. These distinct peptide toxins share several common features: they are relatively small (13 to 29 amino acids), are highly cross-linked by disulfide bonds, and strongly basic. The fact that they inhibit sequential steps in neuromuscular transmission suggests that their action is synergistic rather than additive. Five new omega-conotoxins that block presynaptic calcium channels are described. They vary in their activity against different vertebrate classes, and also in their actions against different synapses from the same animal. There are susceptible forms of the target molecule in peripheral synapses of fish and amphibians, but those of mice are resistant. However, the mammalian central nervous system is clearly affected, and these toxins are thus of potential significance for investigating the presynaptic calcium channels.

Amino Acid Sequence↗
PubMed1985

Conus geographus toxins that discriminate between neuronal and muscle sodium channels.

We describe the properties of a family of 22-amino acid peptides, the mu-conotoxins, which are useful probes for investigating voltage-dependent sodium channels of excitable tissues. The mu-conotoxins are present in the venom of the piscivorous marine snail, Conus geographus L. We have purified seven homologs of the mu-conotoxin set and determined their amino acid sequences, as follows, where Hyp = trans-4-hydroxyproline. GIIIA R.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro6]GIIIA R.D.C.C. T.P.Hyp.K.K.C.K.D.R.Q.C.K.Hyp.Q.R.C.C.A-NH2 [Pro7]GIIIA R.D.C.C.T.Hyp.P.K.K.C.K.D.R.Q.C.R.Hyp.Q.R.C.C.A-NH2 GIIIB R.D.C.C.T.Hyp.Hyp.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2 [Pro6]GIIIB R.D.C.C.T.P.Hyp.R.K.C.K.D.R.R. C.K.Hyp.M.K.C.C.A-NH2 [Pro7]GIIIB R.D.C.C.T.Hyp.P.R.K.C.K.D.R.R.C.K.Hyp.M.K.C.C.A-NH2 GIIIC R.D.C.C.T.Hyp.Hyp.K.K.C.K.D.R.R.C.K.Hyp.L.K.C.C.A-NH2. Using the major peptide (GIIIA) in electrophysiological studies on nerve-muscle preparations and in single channel studies using planar lipid bilayers, we have established that the toxin blocks muscle sodium channels, while having no discernible effect on nerve or brain sodium channels. In bilayers the blocking kinetics of GIIIA were derived by statistical analysis of discrete transitions between blocked and unblocked states of batrachotoxin-activated sodium channels from rat muscle. The kinetics conform to a single-site, reversible binding equilibrium with a voltage-dependent binding constant. The measured value of the equilibrium KD for GIIIA is 100 nM at OmV, decreasing e-fold/34 mV of hyperpolarization. This voltage dependence of blocking is similar to that of tetrodotoxin and saxitoxin as measured by the same technique. The tissue specificity and kinetic characteristics suggest that the mu-conotoxins may serve as useful ligands to distinguish sodium channel subtypes in different tissues.

Amino Acid Sequence↗
PubMed1985

A sleep-inducing peptide from Conus geographus venom.

A novel peptide toxin, which causes a sleep-like state upon intracerebral injection in mice, has been purified to homogeneity from the venom of the piscivorous marine snail Conus geographus L. It elicits no obvious effects when injected i.p. into either mice or fish. The purified toxin is a highly acidic heptadecapeptide with no cystine residues (Lys1, Arg1, Asx2, Ser1, Glx7-8, Gly1, Ile1, Leu2). This composition is in marked contrast to those of other conotoxins, which are basic and disulphide-bridged. The N-terminal residue is Gly and the COOH-terminal sequence is Ser-Asn-NH2.

Amino Acids↗
PubMed1984

Gamma-carboxyglutamate in a neuroactive toxin.

The venom of a fish-hunting cone snail (Conus geographus) contains a novel toxin, the "sleeper" peptide, which induces a sleep-like state in mice when injected intracerebrally. We demonstrate that this peptide contains 5 mol of gamma-carboxyglutamate (Gla) in 17 amino acids. The amino acid sequence of the sleeper peptide is Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2.

1-Carboxyglutamic Acid↗
PubMed1984

Purification and sequence of a presynaptic peptide toxin from Conus geographus venom.

A novel toxin, omega-conotoxin (omega-CgTX), from the venom of the fish-eating marine mollusc Conus geographus has been purified and biochemically characterized. Recently, this omega-conotoxin has been shown to inhibit the voltage-activated entry of Ca2+, thus providing a potentially powerful probe for exploring the vertebrate presynaptic terminal [Kerr, L. M., & Yoshikami, D. (1984) Nature (London) 308, 282-284]. The toxin is a basic 27 amino acid peptide amide with three disulfide bridges. An unusual feature is a remarkable preponderance of hydroxylated amino acids. The sequence of omega-CgTx GVIA is Cys-Lys-Ser- Hyp-Gly5-Ser-Ser-Cys-Ser-Hyp10-Thr-Ser-Tyr-Asn-Cys15-C ys-Arg-Ser- Cys-Asn20-Hyp-Tyr-Thr-Lys-Arg25-Cys-Tyr-NH2.

Amino Acid Sequence↗
PubMed1983

Conotoxin MI. Disulfide bonding and conformational states.

The toxic peptide from Conus magus venom (conotoxin MI) is a 14-amino acid peptide (McIntosh, M., Cruz, L. J., Hunkapiller, M. W., Gray, W. R., and Olivera, B. M. (1982) Arch. Biochem. Biophys. 218, 329-334) which inhibits the acetylcholine receptor. In this work we have confirmed the primary structure and established the disulfide bonding configuration (Cys 3-Cys 8; Cys 4-Cys 14) by direct chemical synthesis of the toxin with specific disulfide bridges. Natural and synthetic toxins were compared by several methods. Fast atom bombardment mass spectroscopy confirmed that the synthetic product had the expected molecular mass and number of exchangeable hydrogens. Ultraviolet CD spectra were closely comparable in shape and magnitude for the two materials, which were also identical in biological activity and chromatographic behavior. We have also established that, although the peptide is highly cross-linked with two disulfide bridges, it can slowly equilibrate between two conformations. A simulation analysis suggests that the conformers have half-lives of approximately 12 and approximately 72 min at 0 degrees C, decreasing approximately 2-fold for every 10 degrees C increase in temperature.

Amino Acid Sequence↗
PubMed1981

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.

Escherichia coli↗