Amino acid sequence of Trimeresurus flavoviridis phospholipase A2

The amino acid sequence of phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was determined. The enzyme subunit has a molecular weight of 13,764 and consists of a single polypeptide chain of 122 amino acids and seven disulfide bonds. The fragmentation was conducted by digesting the reduced and S-carboxymethylated derivative of the protein with Achromobacter protease I, chymotrypsin, and trypsin, respectively. Achromobacter protease I peptides were used for alignment and to establish overlaps over chymotryptic and tryptic peptides. The automated Edman degradation of the S-carboxymethylated protein, which was extended to the N-terminal 30 amino acid residues, supplemented the deletions found with the enzymatic peptides alone. T. flavoviridis phospholipase A2 was found to be highly (65-67%) homologous in sequence to the enzymes from T. okinavensis, Crotalus adamanteus, and Crotalus atrox (viperid family) and less (35-44%) homologous to those from elapid snakes and mammalian pancreas. The T. flavoviridis enzyme appears to be similar in secondary structure composition to the C. atrox enzyme.     

Acquisition of native structure from reduced Trimeresurus flavoviridis phospholipase A2

Oxidation of reduced T. flavoviridis phospholipase A2 under suitable conditions resulted in recovery of its active, native structure. The oxidized product was eluted at the same positions as native phospholipase A2 on reserved phase column and DEAE-Toyopearl 650M column chromatographies. The native and regenerated proteins were also identical in mobility on polyacrylamide gel electrophoresis and in circular dichroism spectra. Des-octapeptide(1-8)-phospholipase A2 (L-fragment), which shows only greatly reduced activity, was reduced and oxidized but no effective reformation of the native structure was found, indicating that the entire sequence is required for efficient reorganization.     

Interactions of monodispersed and micellar substrates with a phospholipase A2 from Trimeresurus flavoviridis

Bindings of the phospholipase A2 from Trimeresurus flavoviridis to the monodispersed and micellar n-alkylphosphorylcholines (n-CnPC) were studied at 25 degrees C and ionic strength 0.2 by the aromatic CD and tryptophyl fluorescence methods, respectively. The bindings to micelles of the substrate analog 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 = 9-13. The binding constant to the micelle was about 40 times greater than it was to the monodispersed substrate. The binding constant to the micellar substrate analog increased on the binding of Ca2+ to the enzyme and decreased on modification of the N-terminal alpha-NH2 group, whereas the binding to the monodispersed substrate analog was independent of pH, of the Ca2+ binding, and of the chemical modification of the alpha-NH2 group. The kinetics of the hydrolyses of monodispersed and micellar dihexanoylphosphatidylcholines (diC6PC) were studied at 25 degrees C and ionic strength 0.2 by the pH-stat method in the presence of saturating amounts of Ca2+. The catalytic center activity, kappa cat, as well as the binding constant, 1/Km, for the micellar substrate, were found to be much greater than those for the monodispersed substrate. The binding constant, 1/Km, of the monodispersed substrate was independent of pH; this was in good agreement with that of the substrate analog described above. The pH-dependence curve of kappa cat for the monodispersed substrate exhibited two transitions, one below pH 6.5 and the other above pH 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the tryptophan residue located in the calcium binding site of Trimeresurus flavoviridis phospholipase A2

When phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased linearly with increase in the extent of oxidation of tryptophan residues. Oxidation of two of the four tryptophan residues caused an apparent loss of activity. The accessibilities of the tryptophan residues were analyzed with differently oxidized phospholipase A2 preparations and were determined to be in the following order: Trp-3 approximately Trp-30 greater than Trp-68 greater than Trp-108. The magnitude of the difference spectrum with a negative peak at 292 nm which is produced upon the binding of Ca2+ in the vicinity of tryptophan residue(s) decreased in a concave manner with increase in the extent of oxidation of tryptophan residues and was greatly diminished when 2 mol of tryptophan residues were oxidized. The activity and Ca2+-induced difference spectrum are thus related to either Trp-3 or Trp-30 or both. Des-octapeptide(1-8)-phospholipase A2 (L-fragment) is 14% as active as phospholipase A2 and is able to give a Ca2+-induced difference spectrum which is smaller than, but similar to, that of phospholipase A2. Its activity and the magnitude of the Ca2+-induced difference spectrum decreased along similar paths with increase in the amount of tryptophan residues oxidized, but in a manner indicating that two tryptophan residues are apparently responsible for the activity and the Ca2+-induced difference spectrum. The order of accessibility of the tryptophan residues of L-fragment was Trp-30 approximately Trp-108 greater than Trp-68. Trp-108, however, could be excluded from the residues located in the active site by reference to the tertiary structure of homologous Crotalus atrox phospholipase A2. Thus, Trp-30 is located in the Ca2+ binding site and is responsible for the activity of L-fragment. It is thus concluded that in phospholipase A2 Trp-30 is located in the Ca2+ binding site. From the concave decrease of relative magnitude of the Ca2+-induced difference spectrum and the linear decrease of relative activity upon oxidation of phospholipase A2, it may be assumed that both Trp-3 and Trp-30 are required to produce the Ca2+-induced difference spectrum, while only Trp-30 need be intact for activity. Anomalous binding of Ca2+ was observed for oxidized phospholipase A2.     

Interactions of Trimeresurus flavoviridis phospholipase A2 and its N-terminal octapeptide-removed and p-bromophenacylated derivatives with acridine and anilinonaphthalene dyes

Interactions of dimeric Trimeresurus flavoviridis (the Habu snake) phospholipase A2 (PLA2), des-octapeptide(1-8)-PLA2 (L-fragment) (14% of PLA2 activity), and p-bromophenacyl bromide (BPB)-inactivated PLA2 (BP-PLA2) with dyes, namely, proflavine, 1-anilinonaphthalene-8-sulfonate (Ans), and 2-toluidinylnaphthalene-6-sulfonate (Tns), were investigated. All dyes were bound in a 1:1 molar ratio to the subunit of the proteins. Proflavine was bound most strongly to PLA2 and Ans and Tns were bound to the three proteins with comparable affinities. Capabilities of the dyes for inhibiting alkylation of His-47 of PLA2 with BPB were in the following order: Ans greater than proflavine greater than Tns. Fluorescences of Ans and Tns that were increased in the presence of PLA2 were further greatly enhanced upon the addition of Ca2+, with concomitant formation of the ternary complexes. Ca2+, however, inhibited, competitively or noncompetitively, the bindings of the dyes to PLA2. All dyes were bound to the active site of PLA2 but with different orientations. Inactivation of L-fragment with BPB was inhibited by the dyes in the following order: Tns greater than proflavine approximately Ans. Addition of Ca2+ to the binary complexes formed from L-fragment and Ans or Tns caused no additional enhancement of fluorescence in spite of the formation of the ternary complexes. The active site structures are different between PLA2 and L-fragment, and the N-terminal octapeptide moiety of PLA2 possibly plays a role in maintaining the optimally arranged active site structure of the molecule. Comparison of the data suggests that the N-terminal moieties of PLA2S from snakes of an elapid family and from mammalian pancreas are essential for catalysis of a micellar substrate, whereas those of PLA2S from snakes of a viperid family, such as T. flavoviridis, are not. BP-PLA2 bound Ca2+ and was similar to L-fragment in terms of the fluorescence measurements. It appears that the active site of PLA2 has a space large enough to accommodate p-bromophenacyl, Ans or Tns, and Ca2+ together. Comparison of the emission maxima of Ans and Tns complexed with the three proteins indicated that Tns could be a useful fluorescent probe informing us of the state (disorder) of the active site of PLA2.     

Investigation of an active site histidine-aspartate couple in Trimeresurus flavoviridis phospholipase A2

When Trimeresurus flavoviridis phospholipase A2 was reacted with methyl p-nitrobenzenesulfonate, its activity decreased following first-order kinetics. The pH dependence of the rate constants of inactivation showed that His-48 with an apparent pKa of 6.5 controls the reaction. In the pH region below 6.5, N1-methylhistidine was predominantly formed. On the other hand, N1,N3-dimethylhistidine was almost exclusively produced in the pH region above 6.5. No N3-methylhistidine was detected at any pH tested. Such observations suggested that the first methylation occurred at the N1-position of the imidazole ring followed by a second methylation at the N3-position, and that His-48 couples the carboxylate of Asp-99 at the N3-position of the imidazole ring, in accord with the interaction observed in the crystal structure of homologous Crotalus atrox phospholipase A2. As it has been reported that, in the reaction of chymotrypsin with methyl p-nitrobenzenesulfonate at pH 7.8, only monomethylation occurred at the N1-position of the His-57 imidazole group (Nakagawa, Y. & Bender, M.L. (1970) Biochemistry 9, 259-267), the nature of the active site histidine-aspartate couple of T. flavoviridis phospholipase A2 seems not to be identical with that of chymotrypsin.     

Contractile activity of Trimeresurus flavoviridis phospholipase A2 on guinea pig ileum and artery.

Trimeresurus flavoviridis phospholipase A2 (PLA2) induced strong contractions of the smooth muscles of guinea pig ileum and artery in a concentration-dependent manner (10(-10)-10(-6) M). When the same dose of PLA2 was administered in repetition to the ileal preparation, the contraction diminished progressively and was no longer recovered even by consecutive washings. The enzymatically inactive derivative of PLA2, in which His-47 was p-bromophenacylated, was unable to elicit contraction. Also, no activity was observed when the Ca(2+)-free medium was used. The contraction induced by PLA2 was inhibited completely by 1.0 x 10(-6) M indomethacin, but not by nordihydroguaiaretic acid. These results imply that the PLA2-induced contraction is due essentially to the hydrolytic action of the enzyme against phospholipid membranes to liberate arachidonic acid that is then converted to pharmacologically active prostaglandins. In guinea pig artery, PLA2 caused both contraction and relaxation.     

Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake

Inhibitors (PLIs) against snake venom gland phospholipases A₂ (PLA₂s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA₂ isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.     

Isolation and fundamental properties of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis

Phospholipase A2 inhibitor was purified from the blood plasma of Habu, Trimeresurus flavoviridis, by Sephadex G-200 gel filtration, DEAE-cellulose chromatography, and Blue-Sepharose CL-6B column chromatography. The purified inhibitor was shown to be a glycoprotein with a molecular weight of about 100K. It was found to consist of four subunits whose molecular weights were around 20-24K. In order to examine the inhibition mechanism of the inhibitor, the interaction of the inhibitor with a phospholipase A2 from T. flavoviridis venom was examined by Sephadex G-100 gel filtration. One inhibitor molecule was found to bind directly to one phospholipase A2 molecule in both the presence and absence of Ca2+. The inhibitor inhibited the phospholipase A2 from T. flavoviridis venom with an apparent dissociation constant, Ki, of 1.7 X 10(-10) M, but not the porcine pancreas enzyme or the Agkistrodon halys blomhoffii enzyme belonging to the same family, Crotalidae, as T. flavoviridis, or the phospholipase C from Bacillus cereus.     

Preliminary crystallographic study of phospholipase A2 from the venom of Trimeresurus flavoviridis (habu snake)

Crystallization and a preliminary crystallographic study of Trimeresurus flavoviridis (habu snake) phospholipase A2 (PLA2) were carried out. Although crystals were obtained from various solutions, crystals suitable for X-ray analysis could be obtained from polyethylene glycol solutions only when a repeated seeding technique was applied starting from twinned crystals. The crystal is monoclinic with space group P21, with a = 44.1, b = 55.7, c = 48.8 A, and beta = 92.4 degrees. An asymmetric unit contains a dimer consisting of two identical subunits made of 122 amino acids. The crystal reflects X-rays beyond 2.5 A. A Pt derivative gave a good isomorphous crystal.     

Transition of environmental polarity of tyrosine-76 of Trimeresurus flavoviridis phospholipase A2 upon active site ligand binding

Modification of Trimeresurus flavoviridis phospholipase A2 with a 5-fold molar excess of tetranitromethane produced 40% active mononitrotyrosyl phospholipase A2 in which Tyr-76 was specifically nitrated. This is in contrast to the case of mammalian pancreatic phospholipases A2 where Tyr-70 but not Tyr-76 was nitrated. When Ca2+ was bound to T. flavoviridis mononitrotyrosyl phospholipase A2, nitrated tyrosine (Tyr(NO2))-76 moved from a less polar site to a polar site with the decrease of the pKa value of its hydroxyl group. Nitration of Tyr-76 did not influence the binding affinity to Ca2+. Addition of laurylphosphorylcholine to mononitrotyrosyl phospholipase A2 in the presence of Ca2+ caused the movement of Tyr(NO2)-76 from a polar environment to a less polar environment with the rise in the pKa value. Tyrosine-76 is located in the site whose environmental polarity is affected by the binding of the ligands to the active site. As Tyr-76 is located in the site not proximal to the active site, it could be assumed that the conformational change induced by the binding of the ligands extends to the region remote from the active site in T. flavoviridis phospholipase A2. This might provide evidence of long-range diffusional coupling between remote sites in the noncooperative globular protein.     

Proton NMR resonance assignments and surface accessibility of tryptophan residues of a dimeric phospholipase A2 from Trimeresurus flavoviridis

Proton NMR spectra of a dimeric phospholipase A₂ from Trimeresurus flavoviridis have been recorded. N-1 proton resonances of the tryptophan indole rings have been detected and assigned to specific positions, Trp-3/Trp-30, Trp-68 and Trp-108, by comparing the spectra of the enzyme derivatives with tryptophans oxidized to differing extents. Photo-CIDNP experiments have revealed that Trp-68 and Trp-108 are exposed while Trp-3 and Trp-30 are buried in the molecule. This is consistent with the X-ray crystal structure of a homologous phospholipase A₂ from Crotalus atrox where residues 3 and 30 are located at a dimer interface, but inconsistent with the results of stepwise oxidation of tryptophan residues.     

Polymorphisms of Trimeresurus flavoviridis Venom Gland Phospholipase A2 Isozyme Genes

Trimeresurus flavoviridis venom gland phospholipase A₂ (PLA₂) genes pgPLA 1a and pgPLA 2a encode Asp-49-PLA₂ and genes pgPLA lb and pgPLA 2b encode an isozyme of Asp-49-PLA₂. Polymorphisms were found in pairs of pgPLA la and pgPLA 2a and of pgPLA lb and pgPLA 2b for individuals of T. flavoviridis. The occurrence of both homozygotes and heterozygotes was demonstrated.     

Pharmacological action of the phospholipase A2 from the venom of Trimeresurus flavoviridis on the smooth muscle of the rat stomach

Phospholipase A2 (PLA2) from the venom of the snake Trimeresurus flavoviridis produced an increase in resting tension of isolated strips of rat stomach fundus. The contractions of the fundus strips induced by the PLA2 were significantly inhibited by treatment with 10(-6) M indomethacin and in Ca2+-free medium, while treatment of the fundus strips with nordihydroguaiaretic acid caused a marked potentiation of the PLA2-induced contraction. Atropine (10(-6) M), chlorpheniramine (10(-6) M) and methysergide (10(-6) M) had no effects on the contractions induced by PLA2, while tetrodotoxin (10(-6) M) significantly potentiated the contraction. From these results, it appears that exogenously applied PLA2 may cause contraction of the rat stomach fundus through the liberation of endogenous arachidonic acid which may then be transformed into prostaglandins.     

Amino acid sequences of the two subunits of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. Sequence homologies with pulmonary surfactant apoprotein and animal lectins

Phospholipase A2 inhibitor (PLI), purified from the blood plasma of the Habu snake (Trimeresurus flavoviridis), was separated into two distinct subunits, PLI-A and PLI-B. These subunits were shown to be glycoproteins with molecular weights of around 21,000-22,000. When they were deglycosylated chemically with trifluoromethanesulfonic acid, the molecular weights were found to be 17,000. Their amino acid sequences were determined by alignment of peptides obtained by lysyl endopeptidase digestion and Staphylococcus aureus V8 protease digestion. PLI-A and PLI-B were each composed of 147 amino acid residues with one residue, Asn103, being for N-linked glycosylation, and the molecular weights of their protein portions were calculated to be 16,368 and 16,408, respectively. Each subunit contained four cysteine residues, all of which exist in disulfide linkages (Cys64-Cys141 and Cys119-Cys133). The sequences of PLI-A and PLI-B showed 89.9% homology to each other. When the sequences were compared with those of lipocortins, no significant homologies were detected. But the sequences were significantly homologous to those of COOH-terminal carbohydrate recognition portions of pulmonary surfactant apoprotein and animal lectins.     

Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.