Search PubMed⌕ Search

Biomedical subjects

W R Gray

Publications and source records attributed to W R Gray.

At least 37 records · Page 2Linked to original sources
PubMed1995

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.

Action Potentials↗
PubMed1995

Rat corticostatin R4: synthesis, disulfide bridge assignment, and in vivo activity.

We have synthesized significant amounts of the most potent member of the rat corticostatins that inhibits ACTH-induced corticosteroid and compared its structure to that of the natural hormone. The cystine bridging arrangement that corresponds to that reported for a human defensin (3-31, 5-20, 10-30) was determined. The in vitro corticostatic activity of the synthetic rat corticostatin R4 paralleled that of the natural R4. Biological studies in vivo showed that doses of 8 or 12 mg corticostatin/kg effectively interfered with corticosterone release in stressed rats. We conclude that in the assays that were used, the biological activity of the synthetic and natural molecules was identical. The availability of significant amounts of synthetic material will make possible studies investigating the physiological role played by corticostatins in modulating the activity of the hypothalamic-pituitary-adrenal axis.

Adrenal Glands↗
PubMed1994

Delta-conotoxin GmVIA, a novel peptide from the venom of Conus gloriamaris.

A novel peptide toxin, delta-conotoxin GmVIA, was purified from the venom of Conus gloriamaris, a mollusc-hunting snail. It consists of 29 amino acids, including six Cys residues: [sequence: see text] The pattern of disulfide connectivity (4-19, 12-24, and 18-29) is the same as for the omega-conotoxins, which are Ca2+ channel ligands. However, the peptide does not compete with omega-conotoxin for binding to membrane preparations from frog, rat, and chick brain. Instead, initial electrophysiological results suggest that the peptide induces action potential broadening in molluscan neurons by slowing down Na+ current inactivation. Synthetic delta-conotoxin GmVIA was prepared by solid-phase methods and appeared identical in all respects to the natural material. The chromatographic behavior of native and reduced delta-conotoxins is quite remarkable, suggesting that the disulfides form a core which forces hydrophobic residues to point out toward the solvent.

Amino Acid Sequence↗
PubMed1994

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.

Animals↗
PubMed1994

A nicotinic acetylcholine receptor ligand of unique specificity, alpha-conotoxin ImI.

We report the isolation, characterization, and total synthesis of a small peptide ligand for nicotinic acetylcholine receptors (nAChRs). It is highly active against the neuromuscular receptor in frog but not in mice. In contrast, it induces seizures when injected centrally in mice and rats, suggesting that it may target neuronal nAChRs in mammals. Although such receptors may be important in both normal cognition and the pathophysiology of several neuropsychiatric disorders, there are few ligands to discriminate between the multiple receptor subtypes. The new peptide is a highly divergent alpha-conotoxin from the snail Conus imperialis, which preys on polychaete worms. In this article, the purification, structural analysis, synthesis, and preliminary physiological characterization of alpha-conotoxin ImI (alpha-CTx-ImI) are reported. The sequence of the peptide is: Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2. The peptide shows striking sequence differences from all alpha-conotoxins of fish-hunting Conus, but its disulfide-bridging is similar: [2-8; 3-12]. We suggest that cone venoms may provide an array of ligands with selectivity for various neuronal nAChR subtypes.

Amino Acid Sequence↗
PubMed1993

Disulfide structures of highly bridged peptides: a new strategy for analysis.

A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide chain be split between close or adjacent Cys residues. The water-soluble tris-(2-carboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra- and interchain disulfides; -Cys.Cys- pair), endothelin and apamin (two disulfides; -Cys.x.Cys- pair), conotoxin GI and isomers (two disulfides; -Cys.Cys- pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys- pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.

Alkylation↗
PubMed1993

Echistatin disulfide bridges: selective reduction and linkage assignment.

Echistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with tris-(2-carboxyethyl)-phosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 degrees C a single bridge linking Cys 2-Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 degrees C the bridges Cys 7-Cys 32 and Cys 8-Cys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 degrees C to a peptide that retains a single bridge linking Cys 20-Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 degrees C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2-11; 7-32; 8-37; 20-39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.

Alkylation↗
PubMed1993

A new Conus peptide ligand for Ca channel subtypes.

A cDNA clone encoding a new omega-conotoxin was identified from Conus magus. The predicted peptide was chemically synthesized using a novel strategy that efficiently yielded the biologically active disulfide-bonded isomer. This peptide, omega-conotoxin MVIID, targets other voltage-gated calcium channels besides the N-subtype and exhibits greater discrimination against the N-channel subtype than any other omega-conotoxin variant to date. Consequently, omega-conotoxin MVIID may be a particularly useful ligand for calcium channel subtypes that are not of the L- or N-subclasses. Of the eight major sequence variants of omega-conotoxins that have been elucidated, four come from Conus magus venom. We suggest that sequence variants from the same venom may be designed to optimally interact with different molecular variants of calcium channels; such omega-conotoxin sets from a single venom may therefore be useful for helping to identify novel calcium channel subtypes.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structure of an Fv from a human IgM immunoglobulin.

An IgM(kappa) immunoglobulin from a patient (Pot) with Waldenstrom's macroglobulinemia was hydrolyzed with pepsin to release a fragment consisting of the 'variable' (V) domains of the light and heavy chains plus eight residue 'tails' from the 'constant' (C) domains. The crystal structure of this fragment was determined at 2.3 A resolution by molecular replacement and crystallographic refinement methods. When examined separately, the light chain component closely resembles another human kappa chain (Rei) in both the beta-pleated sheet regions and the 'hypervariable' loops. The conserved pleated sheets in the heavy chain are similar to those in the human Kol IgG1 protein, but the third hypervariable loop in particular is different from that in any immunoglobulin structure described to date. As in the Kol protein, this loop blocks the access to any internal active site along the light-heavy chain interface. Unlike the Kol protein, however, the loop does not protrude beyond the boundaries of a conventional antigen combining site. Instead, it forms a very compact structure, which fills almost all residual space between the domains. This is an example of one dominant complementarity-determining region (CDR) essentially negating the diversity possible with five other CDRs in the two chains. Ordered water molecules are associated with light chain constituents along the interface, but not with CDR3 of the heavy chain. In screening exercises the Pot IgM failed to bind a wide variety of peptides. Together, the results suggest that ligand binding can only occur on external surfaces of the protein. These surfaces carry a limited number of side chains usually assigned to CDRs in more typical antibodies.

Amino Acid Sequence↗
PubMed1992

Novel alpha- and omega-conotoxins from Conus striatus venom.

Three neurotoxic peptides from the venom of Conus striatus have been purified, biochemically characterized, and chemically synthesized. One of these, an acetylcholine receptor blocker designated alpha-conotoxin SII, has the sequence GCCCNPACGPNYGCGTSCS. In contrast to all other alpha-conotoxins, SII has three disulfide bonds (instead of two), has no net positive charge, and has a free C-terminus. The other two paralytic peptides are Ca channel-targeted omega-conotoxins, SVIA and SVIB. omega-SVIA is the smallest natural omega-conotoxin so far characterized and has the sequence CRSSGSPCGVTSICCGRCYRGKCT-NH2. Although omega-conotoxin SVIA is a potent paralytic toxic in lower vertebrate species, it was much less effective in mammals. The third toxin, omega-conotoxin SVIB, has the sequence CKLKGQSCRKTSYDCCSGSCGRSGKC-NH2. This peptide has a different pharmacological specificity from other omega-conotoxins previously purified from Conus venoms; only omega-conotoxin SVIB has proven to be lethal to mice upon ic injection. Binding competition experiments with rat brain synaptosomal membranes indicate that the high-affinity binding site for omega-conotoxin SVIB is distinct from the high-affinity omega-conotoxin GVIA or MVIIA site.

Amino Acid Sequence↗
PubMed1992

Conopressin G, a molluscan vasopressin-like peptide, alters gill behaviors in Aplysia.

Superfusion of an invertebrate vasopressin structural analogue, conopressin G, over the abdominal ganglion of an in vitro preparation of Aplysia californica has significant neurophysiological and behavioral effects. Both the amplitude of the siphon-evoked gill withdrawal reflux and concomitant activity in gill motor neurons are reduced in the presence of conopressin G. Moreover, the frequency of spontaneous gill movements and their neural correlate, interneuron II activity, are increased. These behavioral modifications strongly resemble those that occur during the food-aroused behavioral state in intact Aplysia. In addition, conopressin G superfusion reduces both the excitability of gill motor neurons and the strength of gill contractions in response to gill motor neuron discharges elicited by direct depolarizing current. A role for conopressin G or a similar peptide in the modulation of gill behaviors associated with the food-aroused state is suggested.

Amino Acid Sequence↗
PubMed1991

alpha-Conotoxins, small peptide probes of nicotinic acetylcholine receptors.

alpha-Conotoxins, a family of small peptides from the venoms of the Conus marine moluscs, are selective, snake alpha-neurotoxin-competitive antagonists of the nicotinic acetylcholine receptor. A new alpha-conotoxin, SIA, has been purified, sequenced, and synthesized. Cross-linking with bivalent reagents and photoaffinity labeling of the acetylcholine receptor with alpha-conotoxin yield covalent adducts. Surprisingly, cross-linking to other subunits is considerably more efficient than to the alpha subunit. The relative efficiency of photoactivatable cross-linking to different subunits of the receptor is a function of placement of the photoactivatable group on the toxin. Since the structures of alpha-conotoxins can be solved by 2D NMR [see Pardi et al. (1989) Biochemistry 28, 5494-5508; Kobayashi et al. (1989) Biochemistry 28, 4853-4860], this family of toxins should provide a set of new ligands for probing the acetylcholine receptor with considerable precision.

Affinity Labels↗
PubMed1991

Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase. Structural and functional properties.

Proteinase 3 (PR-3) is a human polymorphonuclear leukocyte (PMNL) serine proteinase that degrades elastin in vitro and causes emphysema when administered by tracheal insufflation to hamsters (Kao, R. C., Wehner, N. G., Skubitz, K. M., Gray, B. H., and Hoidal, J. R. (1988) J. Clin. Invest. 82, 1963-1973). We have determined the primary structure of several PR-3 peptides and have analyzed catalytic properties of the enzyme. The enzyme has considerable amino acid sequence homology with two other well characterized PMNL neutral serine proteinases, elastase and cathepsin G. Furthermore, the NH2-terminal amino acid sequence of PR-3 is identical to that of the target antigen of the anti-neutrophil cytoplasmic autoantibodies associated with Wegener's granulomatosis. PR-3 degrades a variety of matrix proteins including fibronectin, laminin, vitronectin, and collagen type IV. It shows no or minimal activity against interstitial collagens types I and III, respectively. The analysis of peptides generated by PR-3 digestion of insulin chains and the activity profile against a panel of chromogenic synthetic peptide substrates show that PR-3 prefers small aliphatic amino acids (alanine, serine, and valine) at the P1 site. The elastase-like specificity of PR-3 is consistent with its striking sequence homology to elastase at substrate binding sites. PR-3 is inhibited by alpha 1-proteinase inhibitor (ka = 8.1 x 10(6) M-1 S-1; delay time = 25 ms) and alpha 2-macroglobulin (ka = 1.1 x 10(7) M-1 S-1; delay time = 114 ms) but not by alpha 1-anti-chymotrypsin. In contrast to elastase and cathepsin G, PR-3 is not inhibited by secretory leukoprotease inhibitor and is weakly inhibited by eglin c. Thus, PR-3 is distinct from the other PMNL proteinases.

Amino Acid Sequence↗
PubMed1989

Candida glabrata metallothioneins. Cloning and sequence of the genes and characterization of proteins.

Southern blot analysis has identified several metallothionein gene sequences in a human pathogenic yeast Candida glabrata. Two of these genes encoding proteins designated MT-I and MT-II have been cloned and sequenced. No introns were found in either of the genes. The complete primary structure of MT-II was also determined by protein sequencing methods. As isolated, MT-I and MT-II consist of 62 and 51 amino acids, respectively. The only residues predicted from the nucleotide sequence but not present in the isolated protein are the amino-terminal methionines in each sequence. MT-I contains 18 cysteines, 14 of which are present as Cys-X-Cys motifs and two additional cysteines in a Cys-X-X-Cys sequence. The sequence of MT-II contains 16 cysteinyl residues, 14 of which are in Cys-X-Cys sequences. Fluorescence spectroscopy indicates the presence of Cu(I)-thiolate bonds in both proteins. The binding stoichiometries are 11-12 for MT-I and 10 for MT-II. Under certain nutritional conditions, a truncated form of MT-II was also produced. Northern analysis of the total cellular RNA from copper-treated cells showed that both MT-I and MT-II genes are regulated by this metal ion in a concentration-dependent fashion. The concentrations of MT-II mRNA appeared to be higher than that of MT-I mRNA at all concentrations of copper sulfate tested. Both genes are inducible by silver but not by cadmium salts. Cadmium ions, however, are effective in reducing the control levels of both MT-I and MT-II mRNAs.

Amino Acid Sequence↗
PubMed1989

mu-conotoxin GIIIA, a peptide ligand for muscle sodium channels: chemical synthesis, radiolabeling, and receptor characterization.

The peptide conotoxin GIIIA from Conus geographus L. venom, which specifically blocks sodium channels in muscle, has been synthesized by a solid-phase method. The three disulfide bridges were formed by air oxidation. After HPLC purification, the synthetic product was shown to be identical with the native conotoxin GIIIA from Conus geographus. A high specific activity, 125I derivative of mu-conotoxin was prepared and used for binding assays to the Na channel from Electrophorus electric organ. Specific binding could be abolished by competition with tetrodotoxin. The radiolabeled toxin was specifically cross-linked to the Na channel. These studies demonstrate that mu-conotoxin GIIIA can be used to define the guanidinium toxin binding site and will be a useful ligand for understanding functionally important differences between Na channel subtypes.

Animals↗