Nobuo Tamiya

Sarafotoxins S6: several isotoxins from Atractaspis engaddensis (burrowing asp) venom that affect the heart.

Three isotoxins, named sarafotoxins S6a1, S6b and S6c, with strong cardiotoxic activity were isolated from the venom of a snake, Atractaspis engaddensis. All three sarafotoxins are homologous peptides (four or less than four residue replacements) consisting of 21 amino acid residues. Their structure and activity are novel among snake venom components.

Purification and properties of hydrogenases of different origins.

Abstract Hydrogenase of Desulfovibrio desulfuricans was purified. It was found that highly purified hydrogenase could catalyze the evolution of hydrogen from Na 2 S 2 O 4 in the presence of cytochrome c 3 or methyl viologen and not in the presence of ferredoxin, and the reduction by H 2 of ferricytochrome c 3 or methyl viologen but not of ferredoxin, methylene blue or hexacyanoferrate (III). Hence, H 2 :ferricytochrome c 3 oxidoreductase is proposed for the systematic name of Desulfovibrio hydrogenase. It could catalyze the H− 2 H exchange in the absence of added electron carriers. Hydrogenase of Clostridium pasteurianum (H 2 :ferredoxin oxidoreductase, EC 1.12.1.1) was purified and found to be specific to ferredoxin and not to cytochrome c 3 or methyl viologen. The optimum pH of the Desulfovibrio enzyme was 5–6 (cytochrome c 3 -dependent H 2 evolution), 8–9 (cytochrome c 3 reduction by H 2 ) or 6–8 (H− 2 H exchange reaction). K m value was 7.5 · 10 −5 for ferrocytochrome c 3 in the H 2 evolution reaction. H 2 production from Na 2 S 2 O 4 in the presence of cytochrome c 3 was irreversibly inhibited by CO, but that in the presence of methyl viologen was reversibly inhibited by CO.

Some chemical properties of the venom of the rattlesnake, Crotalus viridis helleri☆

Abstract The venom of the Southern Pacific Rattlesnake, Crotalus viridis helleri, was separated into three lethal and several non-lethal peaks by gel filtration. Peak I was a protein having a mol. wt of ca. 100,000 and an intravenous ld 50 of 0.58 mg/kg. Peak II had a mol. wt of ca. 30,000 and a ld 50 of 1·7 mg/kg. Peak III, the peptide, had a mol. wt of ca. 6000 and a ld 50 of 1·96 mg/kg and moved as a cation on strip and polyacrylamide gel electrophoresis. On ion exchange chromatography the peptide was resolved into three lethal fractions. The major fraction, C, was a basic polypeptide containing 43 amino acid residues with six half cystine residues. Its mol. wt was 4990, as calculated from its sequence, 7600 as estimated from Sephadex G-50 gel filtration and 5200 by SDS- disc gel electrophoresis. The differences are being studied. Analysis showed the peptide contained almost 20% lysine. On sequencing, the most basic amino acid residues were distributed in the N-terminal and C-terminal parts. The middle part was rather hydrophobic.

Structure of snake toxins and their affinity to the acetylcholine receptor of fish electric organ.

Abstract The affinity of sea snake and elapid snake venom components with neurotoxic activity, and their chemically modified derivatives, to the acetylcholine receptor of ray (Torpedo marmorata) electric organ was studied by following the competition in binding with labelled toxin α from Naja nigricollis venom. Some of the unusual toxins, which lack some of the ‘invariant residues’ among neurotoxins, have lower affinity. A parallel relationship between the affinity and ld 50 was observed.

Conformational changes in two neurotoxic proteins from snake venoms.

alpha-Neurotoxin from Naja nigricollis and erabutoxin b from Laticauda semifasciata, two homologous neurotoxic proteins, are studied by circular dichroism, ultraviolet spectroscopy and fluorescence in various water/trifluoroethanol mixtures. The data obtained show that the beta structure of alpha-neurotoxin is conserved in water as well as in the organic solvent. By contrast, erabutoxin b changes from the beta-structure in water to the helix type in trifluoroethanol. The latter induces similarly for both toxins a structural modification around tryptophan 29, a residue common to all neurotoxins known to date. The vicinity of tyrosine 25, another common amino acid, is also altered by the presence of the organic solvent as demonstrated by the sudden increase of reactivity of the phenolic ring towards iodine. The present work affords some evidence for the presence of a particular structure located around the two aromatic residues, which is common to all neurotoxins and able to rearrange independently from the rest of the molecule. Biological importance of this peculiar region is highly probable.

Purification and properties of several phospholipases A2 from the venom of Australian king brown snake (Pseudechis australis)

Thirteen isoenzymes of phospholipases A2 were purified from the venom of Australian king brown snake, Pseudechis australis. They (except phospholipase A2 Pa-9C) showed normal properties of snake venom phospholipases A2; the apparent mol. wts were about 13,000, the optimum pH values were around 8, calcium ion was indispensable for the enzymatic activity and the optimum calcium ion concentrations were more than 5 mM. Phospholipase A2 Pa-9C had a lag period at the initial stage of the enzymatic reaction. The enzymatic activities determined by the titration method using 1,2-dipalmitoylglycerophosphocholine as a substrate at 37 degrees C were 10,500 units/mg for Pa-1G and 75 units/mg for Pa-13. The lethal activities measured by i.v. injections in mice were 0.09 micrograms/g body wt for Pa-5 and 6.8 micrograms/g body wt for Pa-13. The lethal activity correlates with the enzymatic activity (correlation coefficient of 0.92), and both activities showed no relationship to the basicity of the enzyme. Pa-1G is the first acidic phospholipase A2 (pI = 6.4) with high neurotoxicity (0.13 micrograms/g body wt).

The properties and modification of tryptophan in a sea snake toxin, erabutoxin a

Abstract 1. 1.|The relative intensity of fluorescence by tryptophan 29 of erabutoxin a is dependent on the concentration of ethylene glycol in the solvent. This suggests that the tryptophan residue is not buried. 2. 2.|The tryptophan residue can be modified by N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide. The modified toxin has no lethal activity.

Neuromuscular effects of three phospholipases A2 from the venom of the Australian king brown snake Pseudechis australis

Three single chain phospholipases A2 (Pa-10A, Pa-11 and Pa-13) isolated from Australian king brown snake (Pseudechis australis) venom were tested for effects on neuromuscular transmission and muscle contractility on chick biventer cervicis and mouse diaphragm preparations. At 1 microgram/ml (about 85 nM) and higher, Pa-10A and Pa-11 reduced responses of both preparations to indirect stimulation in a concentration-dependent manner. Responses to direct muscle stimulation were generally reduced more slowly. Pa-11 also decreased membrane potentials of chick biventer muscle fibres and caused damage visible by light microscopy. Pa-13, which is about 50 times less active as a phospholipase A2, was also less potent in its pharmacological effects: 20 micrograms Pa-13 per ml were required to reduce responses of either preparation. The phospholipases A2 also caused a slow contracture. After block of responses to nerve stimulation, responses of the chick preparation to acetylcholine, carbachol and KCl could be obtained, although they were smaller than control and highly variable in different preparations. It is concluded that Pa-10A and Pa-11 produce muscle paralysis by reducing acetylcholine release and by a direct blockade of muscle fibre contractility. Pa-13 has similar, though less pronounced, activities.

Amino acid sequences of phospholipases A2 from the venom of an Australian elapid snake (king brown snake, Pseudechis australis).

Two basic phospholipases A2 (Pa-11 and Pa-13) have been isolated from the venom of an Australian elapid snake, Pseudechis australis (king brown snake). The reduced and S-carboxymethylated phospholipases A2 were digested with trypsin and the resulting peptides were purified by a combination of chromatography on a DEAE-cellulose DE-52 column and gel filtration procedures. Eleven main peptides from Pa-11 and 9 peptides from Pa-13 could account for the amino acid compositions of the respective enzyme molecules. The alignment of the tryptic peptides and unelucidated regions of the amino acid sequences of tryptic peptides were established by the analysis of the peptides obtained by chymotryptic and/or Staphylococcal protease digestions. Each phospholipase A2 consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. Although Pa-11 is enzymatically 30-times as active as Pa-13 and highly toxic as compared to Pa-13, they are highly homologous in their amino acid sequences. They are also homologous to the enzymes from mammalian pancreas and the other snake venom phospholipases A2, especially to those from snakes belonging to the subfamilies Acanthophiinae and Laticaudinae.

Isolation and properties of two phospholipases A2 from the venom of an Australian elapid snake (Pseudechis australis).

Two phospholipases A2 (Pa-11 and Pa-13) were purified from the venom of an Australian elapid snake (subfamily Acanthophiinae) Pseudechis australis (king brown snake) by chromatography on CM-cellulose CM-52 followed by gel filtration on a Sephadex G-75 column. The apparent molecular weights of the two phospholipases A2 (Pa-11 and Pa-13) were 14,000 and 13,500, respectively, by gel filtration analysis on a Sephadex G-75 column. Each enzyme molecule consists of a single polypeptide chain of 118 amino acid residues. The isoelectric points of Pa-11 and Pa-13 were 10.5 and 10.0, respectively. The optimum pH values of Pa-11 and Pa-13 for hydrolysis of egg-yolk phosphatidylcholine were 7.8 and 7.5, respectively. Pa-11 was lethal to mice (LD50 0.23 micrograms/g body weight), whereas Pa-13 showed no lethal activity at a dose level of 7.4 micrograms/g mouse. Each enzyme was inactivated by reaction with p-bromophenacylbromide on the sole histidine residue (Pa-11) or on one of the two histidine residues (Pa-13). Oxidation of the tryptophan residues in Pa-11 and Pa-13 with N-bromosuccinimide led to a decrease in the phospholipase A activity. A complete loss of both enzymic and lethal activities of Pa-11 was observed upon oxidation of one of the two tryptophan residues of the molecule.