Separation of phospholipase A2 in Habu snake venom by glycyrrhizin (GL)-affinity column chromatography and identification of a GL-sensitive enzyme.

By means of glycyrrhizin (GL)-affinity and Mono S column chromatographies (HPLC), at least four GL-binding proteins (p25, p17, p15-1 and p15-2) in the two Superdex fractions (P-II and P-III fractions) from Habu snake venom were selectively purified. By determination of their N-terminal partial amino acid sequences, a metalloprotease (p25) and three GL-binding phospholipases A2 (gbPLA2s) [PA2Y (p17), PA21 (p15-1) and PA2B (p15-2)] were identified. PA2B (lysine-49 PLA2) was found to be the most sensitive to GL because (i) it strongly bound to a GL-affinity column; and (ii) its enzyme activity was selectively inhibited by low dose (ID50 = approx. 1.5 microM) of GL, but not by GA. Furthermore, these three gbPLA2s were phosphorylated by casein kinase II (CK-II) in vitro and GL inhibited the CK-II-mediated stimulation of their enzyme activities in vitro.

Two different endothelin B receptor subtypes mediate contraction of the rabbit saphenous vein.

To study endothelin receptor subtypes that mediate venous smooth muscle contraction, effects of some endothelin receptor agonists and antagonists on the rabbit lateral saphenous vein were examined and compared with those on the saphenous artery. In the artery, endothelin (ET)-1 elicited concentration-dependent contractions, while selective ETB-receptor agonists, IRL1620 (Suc-[Glu9,Ala11,15]ET-1(8-21)) and sarafotoxin 6c (S6c) had almost no effect. The ET-1-induced responses shifted in parallel to the right by BQ-123 (cyclo (-D-Trp-D-Asp-Pro-D-Val-Leu-)), an ETA-receptor antagonist, or PD142893 (Ac-D-Dip-Leu-Asp-Ile-Ile-Trp), an ETA/ETB-receptor antagonist, indicating the involvement of the ETA receptor in this response. In the saphenous vein, not only ET-1 and ET-3, but also ETB-receptor agonists, IRL1620, S6c and [Glu9]sarafotoxin 6b ([Glu9]S6b), produced concentration-dependent, BQ-123-insensitive contractions. PD142893 did not affect the ET-1-induced contraction, but it shifted greatly the IRL1620-induced concentration-response curve in parallel to the right. The major components of ET-3-, S6c- and [Glu9]S6b-induced contractions were resistant to PD142893. These results indicate that two different vasoconstrictive ETB-receptor subtypes, ETB1 (sensitive to IRL1620 and PD142893) and ETB2 (insensitive to IRL1620 and PD142893), are located on the smooth muscle of the saphenous vein.

Role of Ca2+ in the binding of phospholipase A2 with a monomeric substrate and with its amide-type analog.

Effects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by Group I phospholipases A2 (PLA2s) from Pseudechis australis, Naja naja atra, and bovine pancreas and by Group II enzymes from Vipera russelli russelli, Agkistrodon halys blomhoffii, and Trimeresurus flavoviridis, were studied by the pH-stat assay method at 25 degrees C, pH 7.5-8.2, and an ionic strength of 0.1 or 0.2 in the absence or presence of an amide-type substrate analog, 2-dodecanoyl-amino-1-hexanol-phosphoglycol. The binding of genuine substrate to the Group II enzymes and that of its analog to the Groups I and II enzymes were markedly facilitated by the binding of Ca2+ to the enzymes. On the other hand, the binding of genuine substrate to the Group I enzymes was found to be independent of the Ca2+ binding. The former result suggests that the structures of the Group II enzyme-genuine substrate complexes and both types of enzyme-analog complexes are generally stabilized by the Ca2+ binding, whereas the latter indicates that the structures of the Group I enzyme-genuine substrate complexes are already similar to those of their Ca2+ complexes and that, therefore, these enzyme-substrate interactions are independent of the Ca2+ binding.

Pharmacological properties of endothelin receptor subtypes mediating positive inotropic effects in rabbit heart.

The positive inotropic effect (PIE) of endothelin (ET) isoforms, ET-1 and ET-3, was similar in that 1) the PIE was associated with prolongation of isometric contractions, 2) the maximal response was approximately 60% of that to isoproterenol (Isomax), 3) the PIE was associated with acceleration of PI hydrolysis, and 4) it was selectively antagonized by phorbol 12,13-dibutyrate. Because the concentration-response curve for ET-1 was biphasic (whereas that for ET-3 was monophasic), ET-1 had a PIE greater than ET-3 up to 10(-8) M. ET-1 induced a PIE at 3 x 10(-14) M and higher, which reached a plateau of 10-20% of Isomax at 10(-12) M (first phase); the curve became steeper at 10(-9) M and higher (second phase), achieving the maximal response at 10(-7) M to 3 x 10(-7) M. An ETA-selective antagonist, BQ-123, did not affect the PIE of ET-1 up to 10(-7) M; it abolished the first phase at 10(-6) M but did not affect the second phase. BQ-123 at 10(-8) to 10(-6) M antagonized the PIE of ET-3, [Thr2]sarafotoxin S6b, and [Glu9]sarafotoxin S6b in a concentration-dependent manner. The PIE of ET-3 was abolished by 10(-6) M BQ-123. An ETB-selective partial agonist IRL-1620 neither elicited a PIE nor affected the PIE of ET-3. These findings indicate that the PIE of ET receptor agonists on rabbit ventricular myocardium cannot be totally explained by occupancy of the ETA or ETB receptor.

Cloning and sequence analysis of a snake, Atractaspis engaddensis gene encoding sarafotoxin S6c.

A 469 base pair genomic DNA, which encodes the mature region of a snake cardiotoxic peptide, sarafotoxin S6c, was isolated from the liver of the burrowing asp, Atractaspis engaddensis. The nucleotide sequence encoding the mature peptide region showed a high sequence homology with those of mammalian vasoconstrictor peptides, endothelin family as expected from the high homology of their amino acid sequences. In contrast, both of the upper and lower flanking sequences of sarafotoxin gene and the deduced amino acid sequence of the sarafotoxin precursor were quite different from those of endothelin family. These results suggest that the ancestral gene and biosynthetic pathway of sarafotoxins are different from those of endothelin.

Structure-activity relationships of sarafotoxins: chemical syntheses of chimera peptides of sarafotoxins S6b and S6c.

The three chimera peptides of sarafotoxins S6b (SRTb) and S6c (SRTc), [Thr2]SRTb, [Asn4]SRTb and [Glu9]SRTb, were synthesized chemically. From the comparisons of lethality, vasoconstrictor activity and receptor binding activity of SRTb, SRTa [( Asn13]SRTb), SRTc [( Thr2,Asn4,Glu9,Asn13]SRTb), [Thr2]SRTb, [Asn4]SRTb and [Glu9]SRTb, it appears that the Lys9 to Glu9 substitution greatly diminishes these activities while the Lys4 to Asn4 substitution does not affect them, and the Ser2 to Thr2 substitution or the Tyr13 to Asn13 substitution slightly diminishes these activities. These results suggest that the very low activities of SRTc are caused mainly by the Lys9 to Glu9 substitution, but not by the Ser2 to Thr2 substitution, which was suggested to be responsible for the weak bioactivities of SRTd [( Thr2,Ile19]SRTb).

Presence of non-selective type of endothelin receptor on vascular endothelium and its linkage to vasodilation.

We studied the role of non-selective type (ETB) of endothelin (ET) receptor in the vasculature, using a ligand specific to the ETB receptor, [Glu9]-sarafotoxin S6b ([Glu9]SRTb). Endothelium-containing rat thoracic aorta possessed specific binding sites for 125I-[Glu9]SRTb, which were almost eliminated by removal of the endothelium, while ET-3-specific binding sites were not detected in the endothelium-intact rat aorta. Only ETB receptor was detected in the membranes from the endothelium of porcine thoracic aorta. [Glu9]SRTb exerted only vasodilation in rat aortic ring. These findings indicate that ETB receptors are located on vascular endothelium and linked to vasodilation.

Studies on the venom components of the long-glanded coral snake, Maticora bivirgata.

The venom of the Asian long-glanded coral snake, Maticora bivirgata, was fractionated into five fractions, S1-S5, by passing through a Sephadex G-50 column. Fraction S2 contains two phospholipases A2, PLA2 I and PLA2 II, fraction S3 contains four cytotoxin homologues, maticotoxins A, C, D1 and D2, and fractions S4 and S5 contain a large amount (about 1 mg/specimen) of adenosine accompanied with smaller amounts of inosine and guanosine. The amino-terminal amino acid sequences of PLA2, I, PLA2 II and maticotoxin A suggest that Maticora bivirgata is closely related to Bungarinae, especially to genera Hemachatus and Naja.

Multiple subtypes of endothelin receptors in porcine tissues: characterization by ligand binding, affinity labeling and regional distribution.

To clarify the existence and the distribution of endothelin (ET) receptor subtypes, we have examined the pharmacological properties and the molecular weight (Mr) of 125I-ET-1 and 125I-ET-3 binding sites in various tissues of pigs. ET-1 and ET-2 showed almost identical potencies in displacing the bound 125I-ET-1 in all the tissues examined. ET-3, sarafotoxin S6b (SRT-b) and sarafotoxin S6c (SRT-c) displaced the 125I-ET-1 with the same sensitivity as ET-1 (IC50 = 0.1-1.4 nM) in brain, kidney, liver and adrenal, whereas the three peptides showed very weak competition (IC50 = 40-500 nM) against 125I-ET-1 binding in cardiac atria, aorta, lung, stomach and uterus. The computer analyses of the binding data suggested the presence of high (Kd1 = 0.04-0.29 nM) and low (Kd2 = 60-190 nM) affinity binding sites for ET-3 and SRT-b in lung and stomach. 125I-ET-3 bound to the high affinity sites in lung and stomach was displaced by ET/SRT isopeptides almost equipotently. Two proteins with Mr of 47,000 and 35,000 were affinity-labeled with 125I-ET-1 in cerebellum, while a protein with Mr of 123,000, in addition to the two proteins, was predominantly labeled in lung. The above findings indicated that two distinct subclasses of ET receptors, namely, ET-1-specific and ET/SRT family-common receptors were distributed in various proportions in mammalian tissues, and suggested that their molecular forms are also different.

Multiple subtypes of endothelin receptors in human and porcine tissues: characterization by ligand binding, affinity labeling, and regional distribution.

To characterize the properties and the distribution of endothelin (ET) receptor subtypes, we have examined the ligand selectivity and the molecular weight (Mr) of [125I]ET-1 and [125I]ET-3 binding sites in various tissues of human and pigs. ET-1 and ET-2 showed almost identical potencies in displacing the bound [125I]ET-1 in all of the tissues examined. ET-3, sarafotoxin S6b (SRTX-b), and sarafotoxin S6C (SRTX-c) displaced the [125I]ET-1 with nearly the same sensitivity as ET-1 (IC50 = 0.07-3.0 nM) in brain, kidney, liver, and adrenal, whereas the three peptides showed very weak competition (IC50 = 40-500 nM) against [125I]ET-1 binding in atria, aorta, lung, stomach, and uterus. The Bmax value for [125I]ET-3 was 83% of that for [125I]ET-1 in human liver membranes, whereas the Bmax for [125I]ET-3 was only 12% of that for [125I]ET-1 in human atrial membranes. [125I]ET-3 bound to liver and atrial membranes was displaced by ET/SRTX isopeptides almost equipotently. Two proteins with Mr of 110 and 50 kDa were specifically affinity-labeled with [125I]ET-1 in porcine lung membranes. The above findings indicated that two distinct subclasses of ET receptors, namely ET-1/ET-2-specific and ET/SRT family common receptors, were distributed in various proportions in mammalian tissues.

Vasodilator effects of sarafotoxins and endothelin-1 in spontaneously hypertensive rats and rat isolated perfused mesentery.

Sarafotoxins (SRTa, SRTb and SRTc) and ET-1 produced a potent vasodilator effect in spontaneously hypertensive rats in vivo and in rat isolated perfused mesenteries in vitro. Among these peptides SRTc demonstrated the most potent vasodilator activity, and was three times more active than SRTa in both preparations. These peptides induced endothelium-dependent vasodilatation in vitro and pretreatment with methylene blue inhibited this effect, while exposure to the antagonists of other vasodilators did not. In contrast, [nitrophenylsulfenylated Trp21]SRTc, SRTc(1-18) and reduced and S-carboxymethylated SRTc caused no vasodilatation in either animal model; the vasodilator effect of acetylated SRTc was less potent than that of SRTc. These results suggest that (i) the vasodilatations of these peptides may be exerted through the release of endothelium derived relaxing factor; (ii) the C-terminal Trp21 and disulfide bonds are essential; and (iii) the N-terminal amino group plays an important role in vasodilator activity.

Studies on the disulfide bridges of sarafotoxins. Chemical synthesis of sarafotoxin S6B and its homologue with different disulfide bridges.

Sarafotoxin S6b, a strong vasocontractile peptide with 21 amino acid residues containing two sets of disulfide bridges, was chemically synthesized. The retention time on a reversed-phase HPLC, lethal and vasocontractile activities of natural sarafotoxin S6b agree with those of the synthetic [Cys1-15, Cys3-11]-sarafotoxin S6b. The combination of the disulfide bridges of sarafotoxin S6b is the same as that of endothelin-1, a mammalian vasocontractile peptide, which shows a high degree of sequence homology and shares several common pharmacological properties with sarafotoxins.

Structure-receptor binding relationships of sarafotoxin and endothelin in porcine cardiovascular tissues.

The specific binding of 125I-sarafotoxin S6b was observed in the microsomal fractions from porcine thoracic aorta, and two vasoconstrictive peptides with strikingly homologous structures, sarafotoxin (SRT) and endothelin (ET), interact with a common receptor of the vasculature. The order of the potency of an each endothelin or sarafotoxin analogue as a competitor against 125I-sarafotoxin S6b binding was ET-1 greater than ET-2 greater than SRT S6b greater than ET-3 much greater than SRT S6c. The hydrophobic carboxyl-terminal tail and intramolecular disulfide bridges are essential for the binding activity. In addition, Ser4, Ser5 and Lys9 seem to be important for the activity while the 6th residue does not affect the activity.

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).

Amino acid sequences of eight phospholipases A2 from the venom of Australian king brown snake, Pseudechis australis.

The amino acid sequences of eight phospholipases A2 (Pa-1G, Pa-3, Pa-5, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-15) which had been isolated from the venom of Australian king brown snake (Pseudechis australis) were elucidated. Pa-1G, Pa-3 and Pa-15 showed micro-heterogeneity at the 103rd position and Pa-5 was separated into two components, Pa-5a ([Pro-18 and Tyr-61]Pa-5) and Pa-5b ([ Ser-18 and Phe-61]Pa-5). All the phospholipase A2 molecules except Pa-1Ga and Pa-1Gb which lack the 118th residue, consisted of a single chain of 118 amino acid residues including 14 half-cystine residues and all the common residues among phospholipases A2 from other sources. From comparison studies, Asp-50, Lys-58 and Asp-90 seem to be important for the toxicity, and we propose that the domain for the presynaptic toxicity consists of seven hydrophilic residues, i.e. Arg-43, Lys-46, Asp-50, Glu-54, Lys-58, Asp-90 and Glu-94.

Structure-activity relationship in vasoconstrictor effects of sarafotoxins and endothelin-1.

Sarafotoxins (SRTa, SRTb and SRTc) as well as endothelin-1 (ET-1) produced vasoconstrictions in rat thoracic aorta, rat isolated perfused mesentery and pithed rat in various of extents. The potency was ET-1 greater than SRTb greater than SRTa greater than SRTc at lower doses, but SRTb greater than ET-1 greater than SRTa greater than SRTc at higher doses. [Nitrophenylsulfenylated Trp21]SRTb and SRTb(1-19) caused no vasoconstriction. Either the reduction and carboxymethylation of Cys residues, the destruction of the intramolecular loop or the production of the non-natural disulfide bond, eliminated the constrictor activity. These results indicate that Trp21 and the intramolecular loop structure are essential, and Lys9 and Tyr13 may play some important roles for the vasoconstrictor activity of these peptides.

Effects of chemical modifications of Pa-11, a phospholipase A2 from the venom of Australian king brown snake (Pseudechis australis), on its biological activities.

Pa-11, a phospholipase A2 isolated from the venom of an Australian elapid snake Pseudechis australis, was chemically modified and its enzymic, neuromuscular and lethal activities were studied. Carboxymethylation of Met-8 gave a derivative with 2% of the enzymic activity and less than 3% of the lethal activity of native Pa-11; it had about 5% of the original ability to block directly and indirectly stimulated mouse phrenic nerve-hemidiaphragm preparations. Nitrophenylsulfenylation of tryptophanyl residues at positions 31 and 69 caused loss of all activities. Amidination of all 14 lysyl residues gave a derivative with 41% and 16% of the enzymic and lethal activities, respectively, but with less than 5% of the original neuromuscular blocking activity. Mono-carbamoylation of lysyl residues at positions 58, 63, 81 and 85 was achieved. The most abundant derivative, 58-carbamoyl-lysine Pa-11 was enzymically 130% and lethally 100% as active as native Pa-11, but it had only about 20% of the native's neuromuscular activity in vitro. 63-Carbamoyl-lysine Pa-11 had 10% of the enzymic and 20% of the lethal activities, respectively; however, it retained at least 50% of its ability to block neuromuscular transmission in vitro, while losing most of its activity to block directly stimulated muscle contractions. 81- and 85-Carbamoyl derivatives have the same enzymic and lethal activities as the original protein, but the 85 derivative had less than 10% of the native neuromuscular activity. Hence, modifications of lysine residues at positions 58, 63 and 85 seem to be particularly significant in altering the neuromuscular, but not enzymic, activity of Pa-11, perhaps by altering the ability of the toxin to bind to its target on nerve and muscle membranes. Modification at position 63 appeared to lead to a dissociation of effects on neuromuscular transmission and directly on muscle cells.

Amino acid sequence of a long-chain neurotoxin homologue, Pa ID, from the venom of an Australian elapid snake, Pseudechis australis.

Pa ID, a long-chain neurotoxin homologue, was isolated from the venom of an Australian elapid snake, Pseudechis australis, and its amino acid sequence was determined by conventional methods. Pa ID was an acidic protein (pI = 6.2) and consisted of 68 amino acid residues. It did not show binding activity to the acetylcholine receptor of an electric ray (Narke japonica) nor lethal effect on mice, though the amino acid sequence is homologous with those of long-chain neurotoxins isolated from other elapid snakes (homology, 39-51%). In the sequence of Pa ID, a structurally invariant residue (Tyr-22) and two functionally invariant residues (Val/Ala-49 and Lys/Arg-50) in snake venom neurotoxins are replaced by a cysteine, an arginine, and a methionine residue, respectively, and furthermore, four common residues in long-chain neurotoxins, Gly-17, Ala-43, Ser-59, and Phe/His-66 are replaced by a glutamic acid, a threonine, a threonine, and a valine residue, respectively. The conformational change of the protein molecule caused by these replacements and the removal of a positive charge at position 50 are probably the reasons why Pa ID has lost the lethality.

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.