Mamushi Research Articles

Chromosome evolution and speciation of reptiles. I. Karyotype of the Japanese mamushi, Agkistrodon blomhoffii blomhoffii (Viperidae, Crotalinae) with special regard to the sex chromosomes.

The karyotype of the Japanese mamushi, Agkistrodon blomhoffii blomhoffii collected in Gifu-ken is investigated directly from the born marrow preparations. The chromosome number of males and females of this species is 2n=36, consisting of 8 macrochromosome pairs and 10 microchromosome pairs. The pair no. 4 is the sex chromosomes. In the male it is a metacentric homomorphic pair (ZZ), while in the female a metacentric and subtelocentric heteromorphic pair (ZW). The length of the W-chromosome is about two third of the Z-chromosome. Among 10 microchromosomes, the largest pair no. 9 is confirmed to be metacentric.

Rabbit platelet calcium ATPase differs from the human erythrocyte (Ca 2+ + Mg 2+)-ATPase in its response to three purified phospholipases A 2, exogenous phospholipids and calmodulin

Human erythrocyte ( Ca 2+ + Mg 2+ )-ATPase and calcium ATPase of rabbit platelets were compared by their responses to a variety of treatments. These included three purified phospholipases A 2 (acidic, neutral and basic) from Agkistrodon halys blomhoffii, as well as several phospholipids and lysophospholipids. The erythrocyte enzyme was stimulated 2–3-fold by all three phospholipases with maximal stimulation occurring at different concentrations of the three enzymes. The basic phospholipase was the most potent, followed by the neutral and acidic enzymes in that order. The calcium ATPase activity of the platelet was also stimulated by phospholipase treatment, but only by 10–20%. The stimulatory activity was attributable to hydrolysis of a very small portion of the total membrane phospholipid. Inactivation of the phospholipases by heating or chemical modification with p- bromophenacyl bromide abolished their ability to stimulate. Addition of polyphosphoinositides stimulated both ATPases. However, another acidic phospholipid, lysophosphatidic acid, stimulated only the erythrocyte enzyme and failed to affect the platelet calcium ATPase. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) had no effect on either enzyme, while the platelet-activating factor ( 1-O- alkyl-2-acetyl-sn- glycero-3-phosphocholine ), its lyso compound and lysoPC inhibited both ATPases. Calmodulin stimulated the erythrocyte enzyme, but did not affect the platelet calcium ATPase. These results demonstrate that the protein-lipid interactions operative in the erythrocyte and platelet calcium ATPases are quite different.

PH dependence of the binding constant of a phospholipase A2 from Agkistrodon halys blomhoffii venom to micelles of n-hexadecylphosphorylcholine.

The phospholipase A2 from the venom of A. halys blomhoffii was titrated with micellar n-hexadecylphosphorylcholine (an analog of lysolecithin) by following the tryptophyl fluorescence change at 25 degrees C and ionic strength 0.1. The data were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 10.0 and 6.7 for the apoenzyme and its Ca2+ complex, respectively. The binding constant of the enzyme to the substrate micelle was found to be enhanced by Ca2+ binding to the enzyme. The pH dependence of the binding constant of the apoenzyme to the micelle was well interpreted in terms of pK shifts of two ionizable groups from 5.16 to 5.67 and from 6.45 to 6.6. The pH-dependence curve for the enzyme-Ca2+ complex, which lacked the former transition, was interpreted in terms of the pK shift of a single ionizable group from 5.55 to 5.76. The former ionizable group was assigned as Asp 49, to which Ca2+ ion can coordinate, and the latter as His 48 in the active site. No participation of the alpha-amino group with a pK value of 7.30 was observed. The binding constant of the enzyme to the substrate micelle, Kmic = 0.45-2.3 X 10(6) M-1, was found to be far greater than that to the monomeric substrate, Kmon = 0.2-1.0 X 10(4) M-1. This was interpreted in terms of the presence of an additional weak substrate-binding site in the enzyme molecule.

The role of phospholipase A2 lysines in phospholipolysis of Escherichia coli killed by a membrane-active neutrophil protein.

Purified rabbit bactericidal/permeability-increasing protein at bactericidal concentrations is a membrane-perturbing agent that triggers hydrolysis of envelope phospholipids of a phospholipase A-less Escherichia coli (S17) mutant by a highly basic (pI greater than 10) phospholipase A2, purified from Agkistrodon halys blomhoffii snake venom. Most other purified phospholipases A2 do not degrade the phospholipids of E. coli killed by the bactericidal protein. To study the role of enzyme charge in bactericidal protein-dependent phospholipid hydrolysis, lysines of the Agkistrodon phospholipase A2 were modified, either by carbamylation (decreases net charge), or by reductive methylation (no delta charge). Incorporation of [14C]cyanate or [14C]formaldehyde and amino acid analysis served to monitor modification. Modification appears to be limited to epsilon-NH2 groups. Incorporation of up to 5 mol of cyanate or formaldehyde/mol of enzyme did not affect catalytic activity. In contrast, incorporation of, on average, 1 mol of either reagent/mol of protein reduced by 80% the activity of the enzyme toward E. coli S17 killed by the bactericidal protein. Since this loss is similar with carbamylation and reductive methylation, the role of the epsilon-NH2 group in the bactericidal protein-dependent hydrolysis seems independent of charge. Thus, the lysines in this phospholipase A2 are not essential for catalysis and substrate binding, but are essential for the action of this enzyme on E. coli killed by the bactericidal protein.

Participation of an ionizable group with pK 8.55 in the reaction of p-bromophenacyl bromide with His 48 of cobra venom phospholipases A2.

The chemical reaction of p-bromophenacyl bromide (BPB) with His 48 of cobra venom phospholipases A2 (N. naja siamensis, N. naja kaouthia, and N. naja atra) was studied at 25 degrees C and ionic strength 0.1 by following the decreases in the fluorescence intensity of 8-anilino-1-naphthalene sulfonate (ANS) and/or in the enzymatic activity. The effect of the BPB concentration on the pseudo-first-order rate constant, chi obs, was studied for the N. naja atra enzyme and the dissociation constants of a noncovalent intermediate were determined to be 5.6 x 10(-4) and 1.8 x 10(-4) M at pH 8.4 and 9.3, respectively. The ph-dependence curve of chi obs, obtained at fixed concentration(s) of BPB was found to be biphasic for all three enzymes. The analysis showed that two ionizable groups with pK values of 7.3 and 8.55 participated in this reaction. The reaction of BPB with the modified enzyme of N. naja atra lacking the N-terminal octapeptide, which had been prepared by the CNBr-cleavage method, was followed by measuring the decrease in the tryptophyl fluorescence. Since this modified enzyme showed a monophasic pH-dependence curve lacking the latter transition, the former group was assigned to His 48 and the latter to the alpha-amino group located nearby in the active site. The pK value of His 48 determined at present was in good agreement with that estimated from the pH dependence of the binding constant of Ca2+ (Teshima et al. (1981) J. Biochem. 89, 13-20). The pK value of the alpha-amino group of this enzyme, 8.55, was found to be somewhat higher than that of the A. halys blomhoffii enzyme, 7.3 (Ikeda & Samejima (1981) J. Biochem. 90, 799-804 and Ikeda et al (1981) J. Biochem. 90, 1125-1130), but quite similar to that for the porcine pancreatic enzyme, 8.4 (van Dam-Mieras et al. (1976) Nobel Symp. 34, 177-197 and Slotboom et al. (1978) Biochemistry 17, 4593-4600).

Identification of domains of phosphatidylcholine in human erythrocyte plasma membranes. Differential action of acidic and basic phospholipases A2 from Agkistrodon halys blomhoffii.

Highly purified acidic (pI 4.9) and basic (pI 8.7) phospholipases A2 from snake (Agkistrodon halys blomhoffii) venom hydrolyzed approximately 20% and 60%, respectively, of the phosphatidylcholine (PC) of intact human erythrocytes prior to hemolysis. Sequential use of the acidic enzyme followed by the basic phospholipase A2 or vice versa manifested a characteristic PC hydrolysis pattern. For example, when acidic enzyme had hydrolyzed nearly 20% of this substrate, a subsequent treatment with the basic enzyme hydrolyzed only an additional 40% of the PC before hemolysis. On the other hand, in experiments where hydrolysis of about 20% of PC of erythrocytes was achieved by a short term incubation with the basic enzyme, then a further treatment of the same cells with the acidic enzyme caused only 10% additional PC hydrolysis before hemolysis. This demonstrated that the acidic enzyme hydrolyzed one domain of PC in the intact erythrocytes, whereas the basic enzyme hydrolyzed not only the same but also another domain of PC in membranes. Analysis of fatty acids released by the action of these two phospholipases A2 on erythrocytes indicated further characteristic differences. In particular, the ratio of released saturated to unsaturated fatty acids was significantly higher with the acidic enzyme as compared with the basic phospholipase A2. These results provide firm support to the conclusion that there are different domains of PC in human erythrocyte membranes and that the acidic and basic phospholipase A2 of A. halys blomhoffii can be used to identify them.

PH dependence of the binding constant of Ca2+ to the phospholipase A2 of A. halys blomhoffii. Participation of ionizable groups with pK values of 5.16, 6.45, and 7.30.

The pH dependence of the binding constant of Ca2+ to the phospholipase A2 of A. halys blomhoffii was studied at 25 degrees C and an ionic strength of 0.1 by the tryptophyl fluorescence method and aromatic circular dichroism (Ikeda and Samejima (1981) J. Biochem. 89, 1175-1184), and was compared with those for cobra venom phospholipases A2 (Teshima et al. (1981) J. Biochem. 89, 13-20) and for porcine pancreatic enzyme (Pieterson et al. (1974) Biochemistry 13, 1439-1445). The shape of the pH-dependence curve was closer to that for the porcine enzyme than those for the cobra enzymes. The data were analyzed on the basis of our previous findings (Ikeda and Samejima (1981) J. Biochem., 90, 799-804) that the pK value of the ionizable group (alpha-amino group) is perturbed from 7.30 to 6.30 on the Ca2+ binding, and that the protonation of another group corresponding to Asp 49 of the porcine enzyme with a pK value of 5.16 competes with the Ca2+ binding. An additional ionizable group with a pK value of 6.45 was found to participate in the Ca2+ ion binding, and this was assigned to the His residue corresponding to His 48 in the active site of the porcine enzyme.

Potentiating effects of bradykinin-related substances on bradykinin-induced contraction of guinea pig ileum and hypotension in rats.

Bradykinin-related substances were studied in vitro and in vivo for bradykinin-potentiating activity. Of the ten peptides which augmented the contractile response of isolated guinea pig ileum to bradykinin, des-Pro2-bradykinin was the most potent. Its potency was 1.9 times that of potentiator B, a bradykinin potentiating peptide obtained from the venom of Agkistrodon halys blomhoffii. This peptide also potentiated the vasode-pressing activity of bradykinin in rats and inhibited angiotensin converting enzyme. These findings indicate that des-Pro2-bradykinin, like venom peptides, potentiates the activity of bradykinin, possibly by inhibiting angiotensin converting enzyme (kininase II).

The isolation and characterization of a third or neutral phospholipase A 2 in the venom of Agkistrodonhalys blomhoffii: An improved fractionation procedure for all three enzymes

The isolation of a new, third phospholipase A 2 from Agkistrodon halys blomhoffii is described. On the basis of a p I value of 6.9, it is termed a neutral phospholipase A 2. It was characterized as to its amino acid content, activity towards phosphatidylcholine, heat stability, and hemolytic behavior on human erythrocytes. A comparison of these characteristics with those of the acidic and basic phosphplipases A 2 established the uniqueness of the neutral enzyme. Two particularly important observations were concerned with the complete stability of the three phospholipases on heating at 100°C at pH 6.0 in the presence of 10 mM Ca 2+, but variable stability in the absence of Ca 2+, and the significant lack of hemolytic activity by the acidic (p I 4.9) phospholipase A 2 as compared to the neutral (p I 6.9) and basic (p I 8.7) enzyme which produced extensive hemolysis of human erythrocytes. Other facets of the characteristics of these phospholipases are discussed.

Effects of Poisonous Metals on the Biosynthesis of Biological Membranes : Inhibitory Effect of Cadmium on the Phospholipase A<SUB>2</SUB> Activity of Snake Venom of Agkistrodon halys blomhoffii

Kinetic studies on the inhibition of phospholipase A2 (from Agkistrodon halys blomhoffii) activity with cadmium ion demonstrated that cadmium ion inhibits the enzymic activity competitively with respect to calcium ion and phosphatidylcholine. The Km value was 6.3×10-3M for phosphatidylcholine and the Ki value of cadmium ion was 1.4×10-4M, while, for the calcium ion, the Km value was 2.8×10-4M and K1 value of cadmium ion was 1.4×10-4M. Cadmium-metallothionein isolated from the liver of cadmium-injected mouse did not show any effect on the phospholipase A2 activity and this fact suggests the protective effect of metallothionein against toxic effect of cadmium on lipid metabolism in living cells.

Snake venom NAD nucleosidase: Its occurrence in the venoms from the genus Agkistrodon and purification and properties of the enzyme from the venom of A. halys blomhoffii

Nicotinamide adenine dinucleotide nucleosidase (NAD glycohydrolase, EC 3.2.2.5) was demonstrated in venoms of various snakes. Among the venoms from 37 species of Viperidae, Crotalidae and Elapidae, venom of snakes in the genera Bungarus and Agkistrodon showed the highest activities. No NAD nucleosidase activity was detected in venoms of the Naja genus. A NAD nucleosidase found in the venom of Agkistrodon halys blomhoffii was purified by successive column chromatographies on DEAE-cellulose and DEAE-Sephadex A-50 and gel-filtration on Sepharose 6B. Final purification was over 25-fold with 20 per cent yield. The purified enzyme showed maximal activity at pH 7.5 and hydrolysed β-NAD +, NADP + and 3-acetylpyridine adenine dinucleotide. No hydrolysis could be observed on α-NAD +, NADH, NADPH and NMN +. The Km values for β-NAD +, NADP + and 3-acetylpyridine adenine dinucleotide were 8.3 × 10 −4 M and 7.4 × 10 −4 M, respectively. Over 60 per cent inhibition of β-NAD + hydrolysis was observed in the presence of 10 −3 M of HgCl 2 and cysteine.

Crotalus adamanteus phospholipase A 2-α: Subunit structure, NH 2-terminal sequence, and homology with other phospholipases

Automated Edman degradation of reduced and carboxymethylated phospholipase A 2-α from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase A 2-α with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH 2-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH 2-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.

A Simple Method for Preparation of Snake Venom Phosphodiesterase almost Free from 5'-Nucleotidase1

A simple method, involving NAD+-Sepharose chromatography, was developed for the preparation of snake venom phosphodiesterase (EC 3.1.4.1) almost free from 5'-Nucleotidase (EC 3.1.3.5). Using an NAD+-Sepharose 4B column, phosphodiesterase was eluted in the unadsorbed fraction, whereas 5'nucleotidase was strongly adsorbed. The latter enzyme was desorbed when 0.2 M sodium bicarbonate buffer containing 1mM beta-NADH was used as a solvent. The affinity column could be used at least four times without any decrease of potency, and the method was applicable for the preparation of phosphodiesterase from the venoms of rattlesnake (Crotalus adamanteus) and Japanese mamushi (Agkistrodan halys blomhoffi).

Synthesis and properties of new bradykinin potentiating peptides.

In a study of the relationship between structure and activity of bradykinin potentiating peptides (BPP), six analogs and homologs of peptides occurring in the venoms of Bothrops jararaca and Agkistrodon halys blomhoffii were synthesized and assayed in the isolated guinea pig ileum and rat uterus. None of the peptides had bradykinin-like activity and their bradykinin potentiating activity was much greater in the guinea pig ileum than in the uterus. The following observations were made with the guinea pig ileum. The introduction of Gln as the eigth residue in potentiator B (pGlu-Gly-Leu-Pro-Pro-Arg-Pro-Lys-Ile-Pro-Pro) and potentiator C (pGlu-Gly-Leu-Pro-Pro-Gly-Pro-Pro-Ile-Pro-Pro) produced a small increase in their bradykinin potentiating activity. Removal of the two N-terminal residues of [Gln8]-potentiator B and [Gln8]-potentiator C led to alterations in activity that paralleled those described earlier for the parent compounds (potentiators B and C). The peptide with the sequence pGlu-Trp-Pro-Ary-Pro-Lys-Trp-Ala-Pro was seven times as active as BPP5a, while the most potent natural BPP, a nonapeptide from B. jararaca venom, is reported to be only four times as active as BPP5a. An analog of the above-mentioned nonapeptide containing Pro8 instead of Ala8 was only as active as BPPa. For all of the peptides, as well as for potentiatrs B and C and BPP5a, the concentration vs. potentiating activity curves had similar shapes, with a plateau at twofold potentiation and a maximum potentiation of 10- to 11-fold. A direct action on the bradykinin receptors may be responsible for the effects observed at lower BPP concentration while the effects at higher concentrations may be due to kininase inhibition. The potentiating activities of potentiator B and its Gln8 analog persisted after the peptides were removed from the medium. This "sensitizing activity" was not observed with any of the other peptides.

The bradykininase activities of extracts of dog lung.

1. Homogenates of dog lungs freed from blood inactivate bradykinin. The bradykininase activity is present in soluble and particulate subcellular fractions.2. The fraction with the highest relative specific activity is that sedimenting between 8,700 g and 78,000 g. This fraction has been studied in more detail.3. There is evidence for two bradykinin inactivating enzymes in this fraction with apparent K(m) values of 7.5 muM and 120 muM.4. The bradykininase activity is not dependent on the concentration of chloride ion.5. The bradykininase activity is inhibited by EDTA, 2:3-dimercaptopropanol, nickel ions and some of the peptides from the venom of Bothrops jararaca and of Agkistrodon halys blomhoffii but not by 2-mercaptoethanol or N-ethylamaleimide.6. Angiotensin II in either 15 or 170 mM chloride ion is not an inhibitor of bradykininase activity but angiotensin I at either chloride concentration is an inhibitor. It is proposed that chloride is not necessary for binding of angiotensin I to converting enzyme but is necessary to ensure the correct orientation of substrate for hydrolysis to proceed.

EVALUATION OF EMERGENCY TREATMENT OF ADDER BITE WITH 5% TANNIC ACID SOLUTION

There are thirteen species of vipers distributing in Japan. Among them, adder (Mamushi in Japanese : Agkistrodon halys blomhoffii) is most common except Amami Island, Kagoshima Prefecture and Okinawa Prefecture.Although the injection of anti-Mamushi serum to the Mamushi bite cases is very effective, we used to hesitate the use of the serum because of possible occur of the serum disease. Under the background mentioned above, we have successfully treated nine patients of Mamushi bite in a farm village of Kyoto Prefecture by washing the wounds with 5% tannic acid solution which was first demonstrated by Okonogi et al. in 1970.This method is very easy and useful, and considered to be most appropriate in a case of the following conditions : (1) patient within 15 minutes after bite, (2) patient who has ever treated by anti-Mamushi serum, (3) patient who has allergic constitution, and (4) species of snake bited is doubtful whether it was Mamushi or not.

Application of mass spectrometry to sequence analysis of pyroglutamyl peptides from snake venoms: contribution to the confirmation of the amino acid sequence of bradykinin-potentiating peptides B, C and E isolated from the venom of Agkistrodon halys blomhoffii.

The amino acid sequences of the three bradykinin-potentiating peptides from the venom of Agkistrodon halys blomhoffii (potentiators B, C, and E), which had been previously presented by Kato and Suzuki using conventional stepwise techniques, were smoothly confirmed by means of mass spectrometry coupled with enzymatic cleavage techniques. In the course of the sequencing, it was suggested that the preparation of potentiator B previously purified is contaminated by a small amount of the peptides with analogous amino acid sequences. The results showed the usefulness of mass spectrometric method in the sequence determination of naturally occurring peptides having N-terminal pyroglutamic acid and abundant proline residues. Mass spectrometric method was also effective for the detection of a small amount of contaminating peptide in the sample.

Bradykinin-potentiating peptides from the venom of Agkistrodon halys blomhoffii. Isolation of five bradykinin potentiators and the amino acid sequences of two of them, potentiators B and C

Bradykinin-potentiating peptides from the venom of Agkistrodon halys blomhoffii. Isolation of five bradykinin potentiators and the amino acid sequences of two of them, potentiators B and C

Some properties of proteinase b in the venom of Agkistrodon halys blomhoffii

The properties of proteinase b (endopeptidase) isolated from the venom of snake, Agkistrodon halys blomhoffii, were studied. Proteinase b contained approx, 2 g-atoms of calcium per molecule as estimated by atomic absorption spectrophotometry and spectrophotometric titration with EDTA. After the removal of the calcium by electrodialysis or by treatment with EDTA (10 −2 M), the resulting enzyme molecule underwent some conformational change, followed by loss of the proteolytic activity. This conformational change and the concomitant loss of enzyme activity were found to be irreversible. The activity of venom proteinase b was slightly inhibited by -SH reagents, including p-chloromercuribenzoate (PCMB), monoiodoacetic acid, N-ethylmaleimide and 5,5′-dithio-bis-(2-nitrobenzoic acid). On prolonged incubation with the enzyme, PCMB caused significant inhibition. In the presence of 8 M urea and 10 −2 M EDTA, one mole of -SH group was found per enzyme molecule, while in the absence of urea and EDTA, only 20% of the -SH group was measurable. Thus, it may be suggested that the metal is probably involved in the stabilization of the enzyme molecule, and that the -SH groups are closely related to the enzyme activity of proteinase b.

Proteolytic, esterase and kinin-releasing activities of some Soviet snake venoms

The casein-, p-toluene-sulfonyl- l-arginine methyl ester- and benzoyl- l-arginine amide-hydrolyzing as well as the kinin-releasing activities of the viperids Vipera lebetina, Vipera ursini and Echis carinatus, the crotalids Agkistrodon halys halys and Agkistrodon halys blomhoffii and the elapid Naja naja oxiana venoms were studied. Viperid venoms were shown to contain kinin-releasing enzyme, as previously reported for crotalid venoms. The effect of inhibitors of proteolytic enzymes, such as aminocaproic acid, soy bean trypsin inhibitor and Trasylol, was studied on the kinin-releasing activity of the venoms mentioned above. Only the kinin-releasing activity of V. lebetina venom was inhibited by soy bean inhibitor.