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W R Kem

Publications and source records attributed to W R Kem.

54 records · Page 3Linked to original sources
PubMed1989

Interfacial activity of an ion channel-generating protein cytolysin from the sea anemone Stichodactyla helianthus.

The ability of a purified sea anemone (Stichodactyla helianthus) protein cytolysin to interact with a variety of interfaces was investigated by means of the Wilhemy plate method. At the air:water and lipid:water interfaces, the toxin lowered the surface pressure most readily as the aqueous phase pH increased towards the isoelectric point (9.8) of the toxin. The affinity of the toxin for both the phospholipid:water and the oil:water interfaces was much greater than for the air:water interface. Although the toxin had previously been found to avidly bind to sphingomyelin-containing phospholipid dispersions and bilayers, it failed to display any preferential interaction with a sphingomyelin monolayer relative to one of dipalmitoylphosphatidyl-choline under identical conditions, even when the monolayers were maintained at 40 dynes/cm, a pressure considered to produce phospholipid packing densities similar to those observed in cell membranes. Unlike many other membrane-active protein cytolysins, the ability of Stichodactyla cytolysin to penetrate these phospholipid monolayers was not affected by the initial surface pressure over the range 0-32 dynes/cm. However, at 40 dynes/cm initial packing pressure, the surface pressure generated by the cytolysin was similarly reduced in both sphingomyelin and dipalmitoylphosphatidylcholine monolayers. Our results suggest that the protein cytolysin initially binds reversibly to cell and artificial bilayer membranes in a non-specific manner; sphingomyelin domains in the bilayer then provide optimal conditions for insertion into the membrane and subsequent assembly of a stable multimeric complex which functions as an ion channel.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1988

Separation and characterization of four different amino acid sequence variants of a sea anemone (Stichodactyla helianthus) protein cytolysin.

A basic protein cytolysin previously isolated from the Caribbean sea anemone Stichodactyla helianthus was shown by CM cellulose chromatography to consist of four isotoxins possessing different N-terminal amino acid sequences. These are designated as toxins I-IV in order of increasing isoelectric point. The estimated molecular sizes (17,400-18,200) of toxins I-III were very similar; toxins I and II posses one additional amino acid at their amino terminus relative to toxin III. Under denaturing conditions, toxin IV behaved as a significantly larger (19,600) polypeptide; Edman sequencing established that it possesses a seven residue extension at the N-terminal end relative to toxin III. None of the variants contained half-cystines or reducing sugars. Toxin III contributed 83% of the total purified cytolytic (hemolytic) activity, toxin II 14%, and the relatively insoluble toxins I and IV together only contributed about 3% of the total cytolytic activity. Cytolysin III lysed Ehrlich ascitic tumour cells, but when administered intraperitoneally in nonlethal doses to mice already inoculated with this tumour, it failed to protect the mice against the tumour. Comparison of the partial amino acid sequence of equinatoxin, another sea anemone protein cytolysin, with that of Stichodactyla cytolysin III indicates they are highly homologous. Many other cytolytic proteins isolated from sea anemones share these properties with Stichodactyla cytolysins: (1) selective inhibition of hemolytic activity by preincubation with sphingomyelin, (2) a molecular size of 10,000-20,000, and (3) an isoelectric point of 9 or above.

Amino Acid Sequence↗
PubMed1987

Effects of nucleus basalis lesions on the muscarinic and nicotinic modulation of [3H]acetylcholine release in the rat cerebral cortex.

Presynaptic muscarinic and nicotinic receptors in the cerebral cortex reportedly inhibit and increase acetylcholine (ACh) release, respectively. In this study, we investigated whether these receptors reside on cholinergic nerve terminals projecting to the cerebral cortex from the nucleus basalis magnocellularis (nbm). Adult male rats received unilateral infusions of ibotenic acid (5 micrograms/1 microliter) in the nbm. Two weeks later, cerebral cortical cholinergic markers (choline acetyltransferase activity, high-affinity choline uptake, and coupled ACh synthesis) were significantly reduced in synaptosomes prepared from the lesioned hemispheres compared to contralateral controls. The depolarization-induced release of [3H]ACh from these synaptosomes was also reduced in the lesioned hemispheres, reflecting the reduced synthesis of transmitter. However, the nbm lesions had no effect on the inhibition of release induced by 100 microM oxotremorine. Synaptosomal [3H]ACh release was not altered by nicotine or the nicotinic agonists anabaseine and 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine. Nicotine (10-100 microM) did increase [3H]ACh release in control and lesioned hemispheres in cortical minces, but to a similar extent. These results suggest that neither muscarinic nor nicotinic receptors modulating ACh release reside on nbm-cholinergic terminals.

Acetylcholine↗
PubMed1983

Primary structure of Stoichactis helianthus cytolysin III.

The primary structure of Stoichactis helianthus cytolysin III has been determined by automated Edman degradation of the intact protein and of peptides derived therefrom by hydrolysis with trypsin and staphylococcal protease and by chemical cleavage with cyanogen bromide and o-iodosobenzoic acid. As a result of these studies, the positions of all 153 amino acid residues of toxin III have been unambiguously determined. Most regions of sequence were determined two times in different types of digests of the protein. A number of highly hydrophobic regions of sequence, which may be functionally significant, have been identified, including a region rich in tyrosine and tryptophan (residues 86-98). The secondary structure of toxin III has been predicted by Chou-Fasman analysis (Chou, P.Y., and Fasman, G.D. (1978) Annu. Rev. Biochem. 47, 251-276) of the primary structure. The predicted secondary structure contains 16% alpha-helix and 31% beta-structure.

Amino Acid Sequence↗
PubMed1983

Cobra polypeptide cytotoxin I and marine worm polypeptide cytotoxin A-IV are potent and selective inhibitors of phospholipid-sensitive Ca2+-dependent protein kinase.

The effects of a number of polypeptide cytotoxins and neurotoxins on various protein kinases were examined. It was found that cobra cytotoxin I and marine worm cytotoxin A-IV effectively and specifically inhibited phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) relative to myosin light chain kinase and cyclic nucleotide-dependent protein kinases. Inhibition of PL-Ca-PK by these cytotoxins could be overcome by phosphatidylserine. Neurotoxins, in comparison, were much less effective inhibitors. The present findings indicated that these polypeptide cytotoxins, unlike other agents reported to date, were selective inhibitors of PL-Ca-PK and could be used to differentiate Ca2+-dependent events regulated by phospholipid or calmodulin.

Bee Venoms↗
PubMed1981

Structure and action of heteronemertine polypeptide toxins. Amino acid sequence of Cerebratulus lacteus toxin B-II and revised structure of toxin B-IV.

The primary structure of Cerebratulus lacteus toxin B-II has been investigated by automated Edman degradation of the reduced, carboxymethylated protein and of tryptic peptides derived from the maleylated protein. As a result of these studies, the identity of all 55 amino acid residues in the polypeptide chain has been unambiguously determined. Hydroxyproline, an amino acid not normally found in nonfibrous proteins, occupies position 10 of toxin B-II. The sequence analysis of B-II led us to re-examine the sequence of Cerebratulus toxin B-IV. The revised structure of toxin B-IV is presented herein. Like B-II, toxin B-IV contains hydroxyproline at position 10 of its sequence. Comparison of the sequences of toxins B-II and B-IV reveals a high degree of homology between these two polypeptides, particularly within the NH2-terminal two-thirds of each chain.

Amino Acid Sequence↗
PubMed1980

Structure and action of heteronemertine polypeptide toxins. Primary structure of Cerebratulus lacteus toxin A-III.

The primary structure of Cerebratulus lacteus cytotoxin A-III has been determined by automated Edman degradation of the reduced, carboxymethylated protein and of peptides derived therefrom by hydrolysis with trypsin and staphylococcal protease. As a result of these studies, the positions of all 95 amino acid residues have been determined unambiguously. The COOH-terminal half of the protein is more hydrophobic than is the NH2-terminal half. The predicted secondary structure of toxin A-III contains 38% alpha helix and 14% beta sheet, in excellent agreement with the values obtained experimentally by circular dichroism spectropolarimetry. Virtually all of the predicted helix lies in a single long stretch starting at position 63, just beyond the last residue of half-cystine in the sequence. This region of helix is also the most hydrophobic portion of the protein and may be involved in the mechanism of its cytotoxic action.

Amino Acid Sequence↗
PubMed1978

Purification and characterization of the cytotoxic Cerebratulus A toxins.

Mucus secreted from the skin of a marine worm, Cerebratulus lacteus, contains a family of polypeptide cytotoxins (A toxins) in addition to the previously reported polypeptide neurotoxins (B toxins). The A toxins were purified by Sephadex G-50 chromatography and then CM-cellulose gradient chromatography at pH 7.5 and pH 3.5. The three most abundant A toxins (designated according to their order of CM-cellulose elution) were homogeneous by gel electrophoreses, amino acid composition, and by NH2-terminal and COOH-terminal partial sequence analyses. Each of the three A toxins consists of a single basic polypeptide chain of 93 to 99 residues, cross-linked by three or four disulfide bonds, lacking reducing sugar and cysteinyl residues. The three A toxins rapidly lysed human red cells and Ehrlich ascites tumor cells at 1 to 10 microgram/ml concentrations. On a molar basis toxin A-III is about 4 times more active than melittin (bee venom lysin) and over 10 times more active than cardiotoxin (elapid snake lysin) upon human red cells. Purified A toxins lacked phospholipase A activity. The cytoxins as well as the neurotoxins were concentrated within the body wall integument.

Amino Acids↗
PubMed1977

Structure and action of heteronemertine polypeptide toxins: disulfide bonds of Cerebratulus lacteus toxin B-IV.

The positions of the disulfide bonds in Cerebratulus lacteus toxin B-IV were investigated by hydrolysis of the unreduced protein with a variety of proteases. The resulting peptides were purified by gel filtration, ion exchange chromatography, and preparative paper electrophoresis and chromatography. Determination of the amino acid compositions of the cystine-containing peptides purified demonstrated the existence of disulfide bonds linking half-cystine residues 12 and 24, 21 and 51, and 35 and 38. The fourth bond, involving half-cystines 10 and 47, was assigned by difference.

Amino Acid Sequence↗
PubMed1976

Structure and action of heteronemertine polypeptide toxins. Primary structure of Cerebratulus lacteus toxin B-IV.

The amino acid sequence of Cerebratulus toxin B-IV, a crustacean-selective axonal toxin occurring in the marine worm C. lacteus, was determined by Edman degradation of the tryptic and staphylococcal protease peptides obtained from the reduced, carboxymethylated toxin. All four of the anticipated maleylated tryptic peptides, ranging in size from 8 to 23 residues, and three staphylococcal protease peptides, ranging from 9 to 35 residues, were isolated in pure form by gel filtration followed by either ion exchange chromatography or preparative paper electrophoresis. The order of the maleylated tryptic peptides was based upon the sequences of the staphylococcal protease peptides. As might be expected, toxin B-IV displays no homology with the elapid nicotinic receptor toxins. In addition, toxin B-IV is structurally unrelated to a group of scorpion neurotoxins which, like B-IV, affect action potential generating mechanisms.

Amino Acid Sequence↗
PubMed1976

Purification and characterization of a new family of polypeptide neurotoxins from the heteronemertine Cerebratulus lacteus (Leidy).

Mucus secreted from the integument of the marine heteronemertine Cerebratulus lacteus (Leidy) contains two major types of polypeptide neurotoxin: the A toxins (11,000 daltons) which are lethal to a variety of animal species including mammals, and the B toxins (6,000 daltons) which appear to be selectively toxic for crustaceans. Both types of toxin were readily obtained from live worms by stimulation and collection of mucus with 1% acetic acid followed by batch adsorption of the basic polypeptide fraction upon CM-cellulose. Separate A and B toxin fractions were obtained by gel chromatography. Four toxins were purified from the B toxin fraction by CM-cellulose gradient elution chromatography. Each consists of a single polypeptide chain with NH2-terminal alanine and three (B-I) or four (B-II, B-III, B-IV) intrachain disulfide bonds. All four B toxins contain large proportions of lysine and exhibit identical gel electrophoretic mobilities at neutral pH. The B toxins lack cysteine and reducing sugar moieties. Toxin B-I differs considerably from the others in its amino acid composition and its shorter chain length (49 instead of 53 or 54 residues). The other three toxins have similar compositions, but lack proline, methionine, and phenylalanine. All four polypeptides show crustacean-selective toxicities which vary over a 30-fold range; B-II possesses the highest toxicity. As a working hypothesis it is suggested that the Cerebratulus B toxins are isotoxins which act via a common receptor present in crustacean axon membranes.

Amino Acids↗
PubMed1976

Hoplonemertine worms -- a new source of pyridine neurotoxins.

Two pyridine bases were isolated from the marine hoplonemertine Amphiporus angulatus (Fabricius) and identified by mass and PMR-spectroscopy as 2, 3'-bipyridyl and 3, 2'; 3', 2"; 4", 3"'-tetrapyridyl (Nemertelline). Nemertelline, the first tetrapyridyl natural product to be reported, shows a structural resemblance to the tobacco constituent nicotelline. The crustacean toxicity of 2, 3' -bipyridyl is very similar to that of nicotine, but its mammalian toxicity is negligible.

Animals↗
PubMedDate not supplied

Alzheimer's drug design based upon an invertebrate toxin (anabaseine) which is a potent nicotinic receptor agonist.

Naturally occurring toxins can often serve as useful chemical tools for investigating signalling processes in nervous and other systems. Tetrodotoxin and alpha-bungarotoxin are prime examples of toxins which are widely used in neurobiological research. Some toxins may also become molecular models for designing new drugs. Usually drugs are small, non-peptide molecules, as these display better bioavailability, longer durations of action and are less likely to generate immune responses. The relatively large size and conformational flexibility of peptides and protein toxins makes them more challenging molecular models for rational drug design. This article considers a marine invertebrate toxin, anabaseine, and describes how manipulation of the structure of this alkaloid has provided a drug candidate which selectively stimulates mammalian brain alpha7 nicotinic receptors. Numerous anabaseine analogs were synthesized and subjected to a variety of pharmacological, behavioral and toxcicological tests. This led to the choice of GTS-21 (also known as 3-(2,4-dimethoxybenzylidene)-anabaseine or DMXBA), as a drug candidate for the treatment of Alzheimer's dementia. The chemical and pharmacological properties of GTS-21 are compared with those of the initial lead compound, anabaseine.

Alzheimer Disease↗
PubMedDate not supplied

Synthesis and biological activity of six monosubstituted analogs of a sea anemone polypeptide neurotoxin.

Monosubstituted analogs of Stichodactyla helianthus neurotoxin I (ShI), a sea anemone polypeptide, were synthesized by solid-phase methods. The analogs were selected to probe the importance of charged residues in the amino-terminal region; ionizable side chains were replaced with a corresponding neutral isosteric amino acid side chain. Following oxidation of the three disulfide bonds and refolding, the analogs were purified by gel, ion-exchange and reversed-phase chromatography. Circular dichroism and fluorescence spectral analyses indicate that the analogs have folded structures very similar to the native toxin. Crustacean paralytic bioassays and neuronal receptor binding assays of ShI analogs show that the anionic triad Asp 6-Asp 7-Glu 8 is essential for activity, as replacement of any of these side chains with the corresponding amide reduces toxicity and binding at least 1000-fold. Substitution at Lys 4 or Asp 11 reduces biological activity at least 10-fold; these residues are apparently not as critical as the polyanionic region. Replacing the N-terminal Ala residue with a Tyr residue has only a slight effect upon crab toxicity and axolemma binding. Our results suggest that several amino acid side chains near the N-terminus of this sea anemone neurotoxin are important for binding to crab neuronal sodium channels.

Amino Acid Sequence↗