Honeybee venom: a rich source of pharmacologically active peptides.
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Amines↗
Biomedical subjects
R C Hider
Publications and source records attributed to R C Hider.
Structure and pharmacology of elapid cytotoxins.
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Amino Acid Sequence↗
Thiol exchange catalysed refolding of small proteins utilizing solid-phase supports.
The study of isolated snake toxin refolding has been a valuable tool in the understanding of protein folding dynamics. We report here differences in the refolding characteristics of three toxin classes and introduce a novel method for overcoming disulphide mismatching and oligomer formation by utilizing solid-phase thiol exchange gels.
Chromatography, High Pressure Liquid↗
Origin of the positive 225-230 nm circular dichroism band in proteins. Its application to conformational analysis.
The 225-230 nm circular dichroism band found in many disulfide-containing proteins and peptides is sensitive to environmental changes. This band is assigned to the disulfide bond, the conformation of which influences both the intensity and lambda max of the band. This property can be used to monitor subtle conformation changes observed in many polypeptides. Examples using the alpha-neurotoxins of elapid venoms and neurohypophyseal hormones are discussed.
Amino Acid Sequence↗
Selection of hydroxypyridin-4-ones for the treatment of iron overload using in vitro and in vivo models.
The hydroxypyridin-4-one group of iron chelators show promise as potential compounds for the treatment of iron overload by the oral route. In the search for the compounds best suited for long term clinical use, a balance has to be struck between the desire to mobilise the maximum amount of iron and the wish to minimise the potential toxicity of such compounds. In this article we review the approach we have used to evaluate which of the hydroxypyridinones have the properties best suited for further development prior to clinical trials in man. The diversity of a number of closely related compounds substituted on the ring nitrogen have allowed us to study the properties of chelators responsible for cellular mobilisation of iron(III), as well as those which may contribute to their toxicity. The primary hepatocyte culture model has facilitated the investigation of the contribution of their iron binding constant, as well as the critical importance of their relative lipid solubility to both cellular iron mobilisation and toxicity. Similarly studies in mice have confirmed that the factors affecting cellular iron release also control iron excretion in whole animals. Further we have demonstrated that the acute toxicity of this group of compounds is closely linked to the size of the available iron pool.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant.
A series of bidentate hydroxypyridinone iron chelators that have therapeutic potential as oral iron chelators, have been studied systematically to determine which properties are the most critical for the mobilization of hepatocyte iron. The relationship between lipid solubility of the free and complexed forms of each chelator and hepatocyte iron release has been investigated as well as the contribution of the binding constant for iron (III). Hydroxypyridin-4-ones that were approximately equally soluble in lipid and aqueous phases were the most active compounds, the partition coefficient of the free chelator appearing to be more critical in determining iron release than that of the iron-complexed form. Highly hydrophilic chelators did not mobilize intracellular iron pools, whereas highly lipophilic compounds were toxic to hepatocytes. The contribution of the binding constant for iron (III) to cellular iron release was assessed by comparing hydroxypyridin-4-ones (log beta 3 = 36) and hydroxypyridin-2-ones (log beta 3 = 32), which possess similar partition coefficients. The results show that the binding for iron (III) is particularly important at low concentrations of chelator (less than 100 mumol/L) and that at higher concentrations (greater than 500 mumol/L) iron mobilization is limited by the available chelatable pool. Measurement of iron release with other chelators confirms the importance of both the lipid solubilities and iron (III)-binding constants to iron mobilization. The most active hydroxypyridin-4-ones released more hepatocyte iron than did deferoxamine when compared at equimolar concentrations. The results suggest that the ability of an iron chelator to enter the cell is crucial for effective iron mobilization and that once within the cell the binding constant of the chelator for iron (III) becomes a dominant factor.
Animals↗
Cobra cardiotoxins. Purification, effects on skeletal muscle and structure/activity relationships [published errtum appears in Eur J Biochem 1988 Feb 1;171(3):727].
A new preparative method for isolating homogeneous cardiotoxins from cobra venoms is described. The technique, based on reverse-phase high-pressure liquid chromatography, was used to isolate eight cardiotoxins of known sequence from four different venoms. In each case the method was found to be particularly efficient at removing trace quantities of contaminating phospholipase. Cardiotoxins isolated in this manner were found to retain their full biological activity. Without exception the purified cardiotoxins lacked powerful haemolytic activity at concentrations up to 0.01 mM (about 100 micrograms ml-1), although some lysis of human erythrocytes was induced at higher concentrations. The cardiotoxins displayed a wide range of depolarizing activity on cultured skeletal muscle, the lowest activity being associated with the highest LD50 value. Correlating variations in amino acid sequence and variations in depolarization potency revealed the importance of residues in the second and third loops, especially lysine-46, serine-48 and lysine-52, together with a number of hydrophobic residues. Further modifications of pharmacological activity were associated with the presence of additional basic residues in the first and second loops and to minor differences in secondary structure.
Amino Acid Sequence↗
Isolation and structure analysis of bee venom mast cell degranulating peptide.
The venom of Apis mellifera was processed by gel permeation chromatography on Sephadex G-50 and by reversed-phase HPLC. The initial gel permeation step was carried out in the presence of phosphate ions (0.5 M). Ion pair reagents were required to resolve the strongly basic peptides, secapin, mast cell degranulating (MCD-) peptide and apamin, by reversed-phase (RP) HPLC. Using this relatively simple procedure it is possible to isolate these peptides essentially free of melittin (less than 1 in 10(7)) and phospholipase (less than 1 in 10(5] in high yield. The CD spectrum and secondary structure analysis are reported for MCD-peptide and on this basis a solution structure is proposed for this toxin.
Animals↗
Malonate transport in human red blood cells.
Kinetic parameters of [2-14C]malonate uptake by the human erythrocyte membrane have been determined as Km, 24 mM and turnover number, 5 X 10(4) s-1. The translocation of this organic dianion is concentration, pH and temperature dependent. Competitive inhibition of malonate uptake by eosin and inorganic anions, strongly implies that a common route exists for both inorganic anions and organic dianions, namely the anion-exchange Band 3 protein. 14C-Malonate which is nonmetabolized in the erythrocyte, could be a useful probe for monitoring anion-exchange in reconstituted Band 3 systems.
Anion Exchange Protein 1, Erythrocyte↗
Effects of the pyrones, maltol and ethyl maltol, on iron absorption from the rat small intestine.
The pyrones, 3-hydroxy-2-methyl-4-pyrone (maltol) and 3-hydroxy-2-ethyl-4-pyrone (ethyl maltol) chelate iron with a high affinity and selectivity. The resulting 1:3 (metal-ligand) complexes, being neutral, are able to partition readily across cell membranes and thus may facilitate iron transport across the intestinal wall. Absorption of radioactive iron (59Fe) in the presence of these pyrones was investigated in male rats 1, 2, 4 and 6 h after intraduodenal administration of a 7 micrograms dose and compared with that of 59Fe given as the sulphate, gluconate, fumarate or complexed to EDTA. Total body absorption and distribution were calculated from the 59Fe content of various tissue samples. With all the iron preparations used, blood levels of 59Fe were highest 1 h after injection whilst the 59Fe content at the major site of deposition, i.e. the bone marrow, increased up to 6 h. No 59Fe was found in the urine. Total body absorption of 59Fe was significantly higher from the pyrones than from the other four preparations. Over the dose range 0.7-700 micrograms, the proportion of 59Fe absorbed from both iron maltol and iron sulphate decreased with increasing dose. Enhanced 59Fe uptake from maltol was evident at 0.7-70 micrograms but not at 700 micrograms suggesting that use of these pyrones will not result in iron overload. Absorption of 59Fe given into the stomach was slower in onset but was sustained longer presumably via a steady delivery of iron to the duodenum from the gastric reservoir.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
In vivo evaluation of hydroxypyridone iron chelators in a mouse model.
The 59Fe excretion caused by a range of bidentate N-substituted [R group = methyl (CP20), ethyl (CP21), propyl (CP22), isopropyl (CP23), butyl (CP24) or hexyl (CP25)] 3-hydroxypyrid-4-one chelators in iron-overloaded mice is presented. All the compounds cause significant iron excretion when given intraperitoneally, but that the most hydrophobic compounds, CP24 and CP25, were toxic except at low doses. The excretion caused by CP21, CP22 and CP23 were significantly greater than that caused by CP20 and slightly larger than that caused by an equivalent dose of desferrioxamine. These compounds (CP20 through CP24) also caused significant excretion of 59Fe when administered orally. Compounds CP21, CP22 and CP24 were significantly more active than compounds CP20 and CP23. It is concluded that the N-ethyl or N-propyl 3-hydroxypyrid-4-ones are the most promising compounds for clinical application. Preliminary experiments using a hexadentate pyrid-2-one, CP130, show that this causes significant 59Fe excretion both when given intraperitoneally or orally.
Administration, Oral↗
The conformation of apamin.
Energy minimization techniques are used as a tool to distinguish between different proposed models for the structure of the bee venom polypeptide apamin. The influence of electrostatic interactions on the resultant energies is noted. The model of Hider and Ragnarsson [(1980) FEBS Lett. 111, 189-193] is found to be of consistently low energy.
Apamin↗
The role of the phosphate group for the structure of phosphopeptide products of adenosine 3',5'-cyclic monophosphate-dependent protein kinase.
By c.d. studies it is shown that liver-pyruvate-kinase-related peptide substrates of cyclic AMP-dependent protein kinase have a high tendency towards non-random structures in non-aqueous media. When phosphorylated, the conformation tendencies decrease. This structural change is explained in terms of the formation of strong intrapeptide phosphate-guanidinium salt links. It is proposed that similar events occur at the catalytic site of protein kinase and that such an interaction could facilitate the removal of the phosphorylated products.
Binding Sites↗
Molecular conformation of alpha-bungarotoxin as studied by nuclear magnetic resonance and circular dichroism.
We have examined the circular dichroism and nuclear magnetic resonance spectra of a long neurotoxin, alpha-bungarotoxin, over a wide range of pH values and temperatures, and under high salt conditions. The observations are interpreted partly in terms of the known crystal structure of this polypeptide. We support earlier findings of a greater degree of beta-sheet structure in solution than has been reported by X-ray crystallography and, importantly, the invariant residue associated with neurotoxicity, Trp29, is shown to be in a similar environment to that found in alpha-cobratoxin and LS III from Laticauda semifasciata. The implications of this observation for structure/function relationships are outlined.
Amino Acid Sequence↗
A proposal for the structure of conotoxin--a potent antagonist of the nicotinic acetylcholine receptor.
Conotoxin, a thirteen residue neurotoxic peptide, is demonstrated, by circular dichroism measurements, to possess a high content of alpha-helix. The location of this helix in the sequence is severely limited by the structural preferences of individual amino acids and by the positioning of the two cystine links. Comparisons are made between the reactive site of elapidae snake venom postsynaptic neurotoxins and the surface of the conotoxin molecule.
Animals↗
The influence of harmaline on the movements of sodium ions in smooth muscle of the guinea pig ileum.
The effect of changing the extracellular concentration of both Na+ and K+ on the longitudinal muscle of the guinea-pig ileum was studied in the presence and absence of harmaline. A decrease in extracellular Na+ concentration was found to produce a dose-dependent contractile response, which may suggest the existence of a Na-Ca exchange mechanism in this muscle. Harmaline (2 X 10(-4) M) was found to reversibly inhibit this contraction and was also found to selectively block the tonic component of high-K induced contradictions. In view of the fact that harmaline is a non-competitive inhibitor of Ca-induced contractions (Hider et al., Europ. J. Pharmacol., 71, 87, 1981), the action of harmaline was interpreted as being a specific inhibitor of the Na-Ca exchange mechanism, binding specifically to Na+ coordination sites.
Alkaloids↗
The influence of melittin on the rotation of band 3 protein in the human erythrocyte membrane.
The rotational mobility of band 3, a protein constituent of the human erythrocyte membrane, was measured by observing the flash-induced transient dichroism of the triplet probe eosin maleimide. In the presence of melittin, a pharmacologically active polypeptide from honey bee (Apis mellifera) venom, a dose-dependent loss of rotational mobility was detected. With acetylated melittin, the ability to immobilize is reduced. Succinylated melittin, however, is devoid of immobilizing activity. The possible relevance of these findings to the normal mode of action of melittin was examined by comparing the relative abilities of the native, acetylated and succinylated melittins to lyse erythrocytes and synergize with phospholipase A2, another constituent of bee venom. For both these properties, the order of effectiveness is native melittin greater than acetyl melittin greater than succinyl melittin = 0, the same as their order of effectiveness in immobilizing band 3. A mechanism is proposed in which melittin is anchored in the membrane by its hydrophobic N-terminus, while its cationic C-terminal moiety binds to negatively charged residues on membrane proteins. This leads either directly or indirectly to protein aggregation and hence loss of mobility. From a detailed comparison of the different effects of the melittin derivatives, it is concluded that melittin may function in vivo by aggregating membrane proteins in order to allow phospholipase A2 to gain access to the membrane bilayer and commence catalysis.
Anion Exchange Protein 1, Erythrocyte↗
A comparison of eel electroplax and snake venom acetylcholinesterase.
The effects of some cholinergic ligands, harmala alkaloids and local anesthetics on the activity of eel electroplax and Naja naja siamensis venom acetylcholinesterase have been studied. In most cases, eel electroplax was found to be more susceptible towards inhibition than the venom acetylcholinesterase. No major difference was observed with respect to the type of inhibition in both enzymes. The activation of the two enzyme preparations by inorganic cations (Ca2+, Mg2+ and Na+) showed a similar pattern. In both preparations, the onset of activation was detectable at much lower concentration with the divalent metal ions than with the monovalent Na+. Antagonism between Ca2+ and decamethonium, tubocurarine and tetracaine in both enzymes approached competitive kinetics. The onset of substrate inhibition is delayed by Ca2+ (30 mM) in both enzymes. It is suggested that the Ca2+ binding site overlaps with the substrate inhibitory site. It is concluded that cobra venom acetylcholinesterase has similar allosteric binding sites to those of eel electroplax.
Acetylcholinesterase↗