Biomedical subjects

W R Kem

Publications and source records attributed to W R Kem.

At least 37 records · Page 2Linked to original sources

PubMed1994

Limited proteolysis study of structure-function relationships in Sh I, a polypeptide neurotoxin from a sea anemone.

The structure-function relationships of the neurotoxic polypeptide Sh I, from the sea anemone Stichodactyla helianthus, have been studied using limited proteolysis with trypsin and endoproteinase Lys-C. Major products from each of the proteolytic digests were characterised using N-terminal peptide sequencing and amino-acid analysis or mass spectrometry. Of the six possible tryptic cleavage sites in Sh I, the bonds adjacent to Arg-13 and Lys-47 were found to be the most susceptible, complete cleavage occurring within minutes. Cleavages adjacent to Lys-32 and Lys-46 proceeded more slowly and cleavage adjacent to Arg-45 was the slowest. The sixth potential site, adjacent to Lys-4, was not cleaved at all. All derivatives were inactive as crustacean neurotoxins. Cleavage with endoproteinase Lys-C generated two major products. Derivatives cleaved adjacent to Lys-32 and either Lys-46 or Lys-47 were isolated. Both were inactive, indicating that either cleavage adjacent to Lys-32 or the removal of the C-terminal lysine residue(s) was sufficient to abolish activity. Lys-4 again was refractory to cleavage. The sequence of cleavage events correlated well with the static accessibility of the lysyl and arginyl side chains and to a lesser extent with the accessibility of the carbonyl oxygen of susceptible peptide bonds, as measured from the solution structure of Sh I determined by 1H-NMR. In the case of Lys-4, the lack of cleavage by trypsin and endoproteinase Lys-C may reflect a lack of flexibility in this region. The effects of the various cleavages on biological activity emphasise that the surface of the protein near the reverse turn encompassing Asp-6, Asp-7 and Glu-8 is essential for activity.

Amino Acid Sequence↗

PubMed1994

A nicotinic agonist (GTS-21), eyeblink classical conditioning, and nicotinic receptor binding in rabbit brain.

The septo-hippocampal cholinergic system is of demonstrated involvement in eyeblink classical conditioning (EBCC). To determine if a nicotinic cholinergic agonist, GTS-21, would facilitate acquisition of EBCC in older rabbits, three doses (0.1, 0.5, 1.0 mg/kg) in sterile saline vehicle and vehicle alone were administered to older rabbits (n = 48; mean age = 29.8 months). A control group of vehicle-treated young rabbits (n = 12; mean age = 3.5 months) was included. Rabbits were conditioned for fifteen 90-trial sessions in the 750 ms delay paradigm with a tone conditioned stimulus and corneal airpuff unconditioned stimulus. Dependent measures of trials to learning criterion, percentage of conditioned responses (CRs) and CR amplitude consistently showed significant improvement in older rabbits treated with 0.5 and 1.0 mg/kg of GTS-21. Acquisition was similar in vehicle-treated young and GTS-treated older rabbits. Vehicle-treated older rabbits conditioned more poorly than vehicle-treated young rabbits. No non-associative learning effects were observed in GTS-21 treated animals. Nicotinic receptor binding was similar in all groups of older rabbits, indicating that GTS-21 administration over a 15-day period did not affect nicotinic receptors. Alzheimer's disease (AD) has been associated with significant loss of nicotinic cholinergic receptors, and patients diagnosed with probable AD are seriously impaired on EBCC. These results demonstrating that the nicotinic agonist, GTS-21, facilitated EBCC in older rabbits suggest that the compound should receive additional investigation for its potential to affect cognition in AD.

Aging↗

PubMed1994

Structure and membrane actions of a marine worm protein cytolysin, Cerebratulus toxin A-III.

Four homologous Cerebratulus lacteus A toxins are the first and as yet only protein cytolysins to be isolated from an ancient phylum of marine worms, the nemertines. The most abundant and toxic variant, toxin A-III, has been sequenced and its mechanisms of action studied in the most detail. It consists of a single basic polypeptide chain of 95 amino acid residues cross-linked by three disulfide bonds, and possesses a predominantly alpha-helical secondary structure. The C-terminal third of the toxin sequence is postulated to be a helical 'hairpin' structure involved in pore formation. Toxin A-III permeabilizes a variety of cells as well as liposomes made from a variety of phospholipids; apparently large pores are formed, as large proteins are released almost as rapidly as small organic molecules and inorganic ions. At sublytic concentrations, the toxin also inhibits protein kinase C and endogenous voltage-gated cation selective (sodium, calcium) channels occurring in the nervous and cardiovascular systems. A curious observation, also reported for colicins and some other protein cytolysins, was the conservation of toxin secondary structure upon insertion into phospholipid liposomes, despite the strong likelihood that significant changes in tertiary structure occur to provide a hydrophobic surface for interaction with membrane lipids. Because of its small size and presumed single helical hairpin secondary structure, Cl toxin A-III is an excellent molecular subject for investigating protein insertion into biological membranes and mechanisms of pore formation.

Amino Acid Sequence↗

PubMed1994

A novel nicotinic agonist facilitates induction of long-term potentiation in the rat hippocampus.

Long-term potentiation (LTP) can be modulated by a number of neurotransmitter receptors including muscarinic and GABAergic receptor types. We have found that a novel nicotinic agonist, 2,4-dimethoxybenzylidene anabaseine (DMXB), facilitated the induction of LTP in the hippocampus in a dose-dependent and mecamylamine-sensitive manner. DMXB displaced high affinity nicotinic [125I]alpha-bungarotoxin and [3H]acetylcholine binding in rat brain. Xenopus oocyte studies demonstrated that DMXB has agonist activity at alpha 7 but not alpha 4/beta 2 nicotinic receptor subtypes. These results indicated that DMXB is a novel nicotinic agonist with apparent specificity for the alpha 7/alpha-bungarotoxin nicotinic receptor subtype and indicate that nicotinic receptor activation is capable of modulating the induction of long-term potentiation.

Acetylcholine↗

PubMed1994

Nicotinic cholinergic system involvement in eyeblink classical conditioning in rabbits.

A nicotinic cholinergic antagonist, mecamylamine (MEC), was administered to rabbits tested on eyeblink classical conditioning (EBCC) in the 750-ms delay paradigm for 10 90-trial sessions. Nicotinic receptors were measured in 3 brain regions in 5 treatment groups: paired conditioned stimulus-unconditioned stimulus (CS-US) presentations with (a) vehicle, young; (b) vehicle, older; (c) 0.5 mg/kg MEC, young; unpaired CS-US with (d) 0.5 mg/kg MEC, young; and (e) vehicle, young. Daily MEC injections disrupted acquisition in young rabbits (769 trials to learning criterion vs. 323 trials for vehicle-treated young rabbits). MEC-treated young rabbits learned similarly to older rabbits. Brain nicotinic receptors were not affected by 10 daily MEC injections. To our knowledge, this experimental protocol, using a low MEC dose to selectively inhibit nicotinic cholinergic receptors, is the first to demonstrate a role for nicotinic cholinergic receptors in EBCC.

Animals↗

PubMed1994

Synthesis of the cardiac inotropic polypeptide anthopleurin-A.

The sea anemone polypeptide anthopleurin-A (AP-A) at nanomolar concentrations enhances myocardial contractility without affecting automaticity. It has a therapeutic index higher than that of the digitalis glycosides, and may serve as a molecular model for designing a new class of inotropic drugs acting on the myocardial Na channel at site 3. AP-A is a 49 residue peptide crosslinked by three disulfide bonds; its tertiary structure has been determined by NMR. Here we report the solid-phase synthesis of this polypeptide. Synthetic AP-A displayed CD and NMR spectra identical with those of the natural toxin; it possessed 94 +/- 15% of the inotropic activity of natural AP-A. Therefore, it is feasible to prepare various type 1 sea anemone toxin analogs by solid-phase chemical synthesis in order to identify side chains important for peptide folding and interaction with sodium channels.

Amino Acid Sequence↗

PubMed1992

1H-NMR study of neurotoxin B-IV from the marine worm Cerebratulus lacteus. Solution properties, sequence-specific resonance assignments, secondary structure and global fold.

Sequence-specific resonance assignments are reported for the 500-MHz 1H-NMR spectrum of the 55-residue neurotoxin B-IV, isolated from the heteronemertine worm Cerebratulus lacteus. A range of two-dimensional homonuclear correlated and NOE spectra was used in making these assignments, which include NH, C alpha H and C beta H resonances, as well as most resonances from longer-chain spin systems, with the exception of the ten Lys residues, where spectral overlap prevented complete, unambiguous assignments. The secondary structure of B-IV was identified from the pattern of sequential (i, i + 1) and medium range (i, i + 2/3/4) NOE connectivities and the location of slowly exchanging backbone amide protons. Two helices are present, incorporating residues 13-26 and 33-49, and the C-terminal five residues form a helix-like structure. A type-I reverse turn, involving residues 28-31 is present in a small loop linking the two major helices, and the N-terminus appears to be unordered at 27 degrees C, although it may adopt a more ordered conformation at lower temperatures. These elements of secondary structure, together with the four disulfide bonds in the protein, provide sufficient information to define the global fold of the molecule in solution. The pH and temperature dependence of the toxin have been investigated by 1H-NMR and the pKa values of several ionisable sidechains determined.

Amino Acid Sequence↗

PubMed1992

Synthesis and characterization of a disulfide bond isomer of omega-conotoxin GVIA.

Solid phase peptide synthesis and air oxidation of omega-conotoxin GVIA yielded, in addition to the desired product, an isomeric peptide which could be completely separated from the native toxin by repeated HPLC. A chymotrypsin-trypsin digest of this peptide, when subjected to HPLC peptide mapping, provided peptides identical with synthetic disulfide containing peptides predicted for the omega-conotoxin isomer containing C1-C2, C3-C5, C4-C6 cystinyl pairings. The 'shaking' potency (ED50 = 1500 pmoles/kg, i.c.v.) of the isomeric peptide upon cannulated rats was 1.3% of the potency of native conotoxin (ED50 = 20 pmoles/kg). Considering that all three disulfide pairings in the isomer are different from the native toxin, its retention of biological activity is of interest.

Amino Acid Sequence↗

PubMed1992

Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels.

The membrane actions of three recently isolated polypeptide neurotoxins from the sea anemones Stichodactyla helianthus (toxin ShI), Condylactis gigantea (toxin CgII) and Calliactis parasitica (toxin CpI) were investigated on action potentials and voltage-clamp membrane currents of the giant axon of the crayfish Procambarus clarkii. The first two toxins were also tested on the cockroach (Periplaneta americana) giant axon. All three toxins were particularly lethal to crustaceans, moderately toxic to an insect (cockroach), and essentially non-toxic to a mammal (mouse). ShI and CgII were 50- to 100-fold more potent on crayfish than on cockroach axons; this difference in activity was correlated with the relative reversibility of their effects on these arthropod axons. The crustacean selectivity of these toxins is therefore due largely to their greater affinity for crustacean sodium channels. All three toxins prolonged crayfish giant axon action potentials by selectively slowing Na channel inactivation without greatly affecting activation. Before toxin treatment, inactivation was nearly exponential, with a time constant less than 1 msec. After treatment, the inactivation time course could be described as the sum of two exponentially decaying components, plus a large steady-state component. The major component possessed the slower (10-20 msec) time constant. The steady-state component increased with depolarization, causing the sodium channel steady-state inactivation curve to reach a minimum between -60 and -20 mV and then increase at more positive potentials. All three toxins shifted the peak sodium current-voltage relation to the left. This voltage shift was greater at 20 degrees C than at 10 degrees C. Maintained membrane depolarization during toxin wash-in delayed the appearance of modified Na channels. Also, prolonged depolarization of toxin-treated axons converted modified sodium channels back to normal ones. The toxins did not affect potassium and leakage currents. Our results indicate that the three crustacean-active sea anemone toxins share a common electrophysiological action on arthropod sodium channels, at least at the macroscopic level.

Action Potentials↗

PubMed1990

Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study.

The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. A set of 470 nuclear Overhauser enhancement values was measured, of which 216 were used as distance restraints in the structure determination along with 15 dihedral angles derived from coupling constants. After restrained molecular dynamics refinement, the eight structures that best fit the input data form a closely related family. They describe a structure that consists of a core of twisted, four-stranded, antiparallel beta-sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution. Pairwise root mean square displacement values for the backbone heavy atoms of the eight best structures are 1.3 +/- 0.2A when the poorly defined loop is excluded and 3.6 +/- 1.0A for all backbone atoms. Refinement using restrained molecular dynamics improved the quality of the structures generated by distance geometry calculations with respect to the number of nuclear Overhauser enhancements violated, the size of the total distance violations and the total potential energies of the structures. The family of structures for S. heliathus neurotoxin I is compared with structures of related sea anemone proteins that also bind to the voltage-gated sodium channel.

Amino Acid Sequence↗

PubMed1990

Circular dichroism and laser Raman spectroscopic analysis of the secondary structure of Cerebratulus lacteus toxin B-IV.

The secondary structure of Cerebratulus lacteus toxin B-IV, a neurotoxic polypeptide containing 55 amino acid residues and four disulfide bonds, was experimentally estimated by computer analyses of toxin circular dichroism (CD) and laser Raman spectra. The CD spectrum of the toxin displayed typical alpha-helical peaks at 191, 208, and 222 nm. At neutral pH, the alpha-helix estimates from CD varied between 49 and 55%, when nonrepresentative spectrum analytical methods were used. Analysis of the laser Raman spectrum obtained at a much higher toxin concentration yielded a 78% alpha-helix estimate. Both CD and Raman spectroscopic methods failed to detect any beta-sheet structure. The spectroscopic analyses revealed significantly more alpha-helix and less beta-sheet for toxin B-IV than was predicted from its sequence. To account for the difference between the 49-55% helix estimate from CD spectra and the 78% helix estimate from the Raman spectrum, we postulate that some terminal residues are unfolded at the low toxin concentrations used for CD measurements but form helix at the high toxin concentration used for Raman measurements. Our CD observations showing that Cerebatulus toxin B-IV helix content increases about 15% in trifluoroethanol or at high pH are consistent with this interpretation.

Amino Acid Sequence↗

PubMed1990

Chemical synthesis of a neurotoxic polypeptide from the sea anemone Stichodactyla helianthus.

The sea anemone Stichodactyla helianthus neurotoxin I, a 48-residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (1H NMR) spectra of the HPLC-purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD50, 3.1 micrograms/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).

Amino Acid Sequence↗

PubMed1990

Quantitative structure-activity relationships for sea anemone polypeptide toxins.

Sea anemone polypeptides vary considerably in their affinities for sodium channels occurring in different excitable cells. The amino acid sequence variation in a set of six sea anemone type I polypeptide toxins (46-49 residues long), was parameterized using descriptor scales z1, z2, and z3, derived from a large number of amino acid physicochemical properties. The pharmacological properties of the toxins were represented by the results from four bioassays on crab, mouse, and rat brain and from rat heart. By means of the descriptor scales and the multivariate data analytical method PLS (partial least squares projections to latent structures), it was possible to develop quantitative structure-activity relationships (QSAR). Using the QSARs derived from the set of six polypeptide toxins the pharmacological properties of two homologous sea anemone polypeptide toxins were predicted. Thus it is shown that QSARs may be formulated for relatively long bioactive polypeptides. The QSARs indicate that 11 different amino acid positions may be of importance, but that positions no. 5, 21, 28, 34, 37, and 40 were of main importance in modeling the relative toxicities of the six polypeptides.

Amino Acid Sequence↗

PubMed1990

Reduction potentials of imine-substituted, biologically active pyridines: possible relation to activity.

Cyclic voltammetry data were obtained for a number of biologically active compounds which incorporate imine substitution on the pyridine nucleus. The reductions in acid (iminium ion formation) were for the most part reversible, and in the range of -0.5 to -0.7V. The toxic effect of these drugs is thought to be caused by the generation of reactive oxygen radicals that arise via charge transfer, or by disruption of electron transport chains.

Chemical Phenomena↗

PubMed1989

Binding of a radiolabeled sea anemone cytolysin to erythrocyte membranes.

Stichodactyla helianthus cytolysin III, a 17 kDa basic polypeptide isolated from a Caribbean sea anemone, is one of the most potent hemolysins yet found in a living organism. This toxin has been reported to form new ion channels in artificial lipid bilayer membranes. The ability of this toxin to attack cell membranes is greatly enhanced by the presence of sphingomyelin. In order to investigate the mechanism by which the cytolysin causes cell lysis, we have prepared a highly active [3H]cytolysin derivative by reductive methylation with sodium cyanoborohydride and [3H]formaldehyde. A dimethylated toxin derivative was used to investigate the basis for the differential lytic activity of this polypeptide upon erythrocytes from six mammalian species. Using both direct [3H]toxin binding and indirect (Thron method) binding techniques, we found that the interspecies differences are due to variable membrane susceptibilities toward the bound toxin, rather than to differences in membrane affinity for the toxin. Similarly, we showed the enhanced lytic activity of the toxin for rat erythrocytes at elevated pH to be caused by enhanced activity of the bound toxin.

Alkylation↗

PubMed1989

Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus.

An aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, contained a polypeptide neurotoxin (Sh I) selectively toxic to crustaceans. The polypeptide was purified by G-50 Sephadex, phosphocellulose, and sulfopropyl-Sephadex chromatography and shown to have a molecular size of 5200 daltons and a pI of 8.3. The amino acid sequence determined by automatic Edman degradations of whole RCM Sh I and of its clostripain, staphylococcal protease, and cyanogen bromide digest peptides is A1ACKC5DDEGP10DIRTA15PLTGT20VDLGS25CNAGW30EKCAS35YYTII40ADCCR45KKK . Only 33% of this sequence is identical with the sequence of Anemonia sulcata toxin II, a sea anemone toxin isolated from the taxonomic family Actiniidae. The six half-cystines are located in equivalent positions to those of the actiniid toxins and account for nearly half of the residues common to all of the toxins. However, 69% of the Sh I sequence is identical with that of toxin II from Heteractis paumotensis, another sea anemone belonging to the family Stichodactylidae. Stichodactylid toxins lack the initial N-terminal residue of actiniid toxins and possess three consecutive acidic residues at positions 6-8, a single tryptophan at position 30, and four consecutive basic residues at positions 45-48 (C-terminus). A rabbit IgG prepared by Sh I immunization bound Sh I with a K0.5 of 4.7 nM but failed to bind homologous actiniid (Anemonia sulcata II, Condylactis gigantea III) or bolocerid (Bolocera tuedae II) polypeptide neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

1H NMR study of the solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus.

The solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I) have been investigated by high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz. The pH dependence of the spectra has been examined over the range 1.1-12.2 at 27 degrees C. Individual pKa values have been obtained for the alpha-ammonium group of Ala-1 (8.6) and the side chains of Glu-8 (3.7), Tyr-36 (10.9), and Tyr-37 (10.8). For the remaining seven carboxyl groups in the molecule (from five Asp, Glu-31, and the C-terminus), four pKa values, viz., 2.8, 3.5, 4.1 and 6.4, can be clearly identified. The five Lys residues titrate in the range 10.5-11, but individual pKa values could not be obtained because of peak overlap. Conformational changes associated with the protonation of carboxylates occur below pH 4, while in the alkaline pH range major unfolding occurs above pH 10. The molecule also unfolds at elevated temperatures, having a transition temperature of ca. 55 degrees C at pH 5.25. Exchange of the backbone amide protons has been monitored at various values of pH and temperature in the ranges pH 4-5 and 12-27 degrees C. Up to 18 slowly exchanging amides are observed, consistent with the existence of a core of hydrogen-bonded secondary structure, most probably beta-sheet.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1989

Sequence-specific 1H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I.

Sequence-specific assignments are reported for the 500-MHz 1H nuclear magnetic resonance (NMR) spectrum of the 48-residue polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I). Spin systems were first identified by using two-dimensional relayed or multiple quantum filtered correlation spectroscopy, double quantum spectroscopy, and spin lock experiments. Specific resonance assignments were then obtained from nuclear Overhauser enhancement (NOE) connectivities between protons from residues adjacent in the amino acid sequence. Of a total of 265 potentially observable resonances, 248 (i.e., 94%) were assigned, arising from 39 completely and 9 partially assigned amino acid spin systems. The secondary structure of Sh I was defined on the basis of the pattern of sequential NOE connectivities, NOEs between protons on separate strands of the polypeptide backbone, and backbone amide exchange rates. Sh I contains a four-stranded antiparallel beta-sheet encompassing residues 1-5, 16-24, 30-33, and 40-46, with a beta-bulge at residues 17 and 18 and a reverse turn, probably a type II beta-turn, involving residues 27-30. No evidence of alpha-helical structure was found.

Amino Acid Sequence↗