Publications (17)25.49 Total impact
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Article: Subunit structure and inhibition specificity of alpha-type phospholipase A2 inhibitor from Protobothrops flavoviridis.
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ABSTRACT: The alpha-type phospholipase A2 inhibitor (PLIalpha) in the plasma of the Habu snake, Protobothrop flavoviridis, was shown to be a trimer of two homologous subunits, PLIalpha-A and PLIalpha-B, each of which contains one C-type lectin-like domain (CTLD). Since one molecule of trimeric PLIalpha binds stoichiometrically to one molecule of P. flavoviridis acidic phospholipase A2 (PLA2), the trimeric structure is critical for its inhibitory activity. Hydrophobic chromatography separated the purified P. flavoviridis PLIalpha into four different trimeric subspecies, A3-PLIalpha, A2B-PLIalpha, AB2-PLIalpha, and B3-PLIalpha, with different combinations of the two subunits. The trimeric PLIalpha could be reconstituted from the purified subunits, and the four different trimeric subspecies were formed through random association of the two subunits. The inhibitory activity of the PLIalpha-A homotrimer (A3-PLIalpha) was more specific than that of the PLIalpha-B homotrimer (B3-PLIalpha). This difference in inhibitory properties between the two homotrimers was probably caused by the amino acid differences at residues 10-37.Toxicon 05/2008; 51(5):787-96. · 2.51 Impact Factor -
Article: Structural analysis of the interaction between Shiga toxin B subunits and linear polymers bearing clustered globotriose residues.
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ABSTRACT: We previously developed linear polymers bearing clustered trisaccharides of globotriaosylceramide (Gb3) as orally applicable Shiga toxin (Stx) neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. These results provide the molecular basis for the interaction between Stx B subunits and Gb3 polymers.Infection and Immunity 04/2006; 74(3):1984-8. · 4.16 Impact Factor -
Article: Molecular cloning of the major lethal toxins from two kraits (Bungarus flaviceps and Bungarus candidus).
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ABSTRACT: The major lethal toxins present in the venoms of the red-headed krait, Bungarus flaviceps, and the Malayan krait, Bungarus candidus, have both been purified. Each consists of two polypeptide chains, A and B, joined by a disulfide bond. In the present study, primary structures of these toxins were determined by Edman degradation and by nucleotide sequencing of the cDNA clones. Amino acid sequencing of the N-terminus and enzymatically digested peptides revealed that the A and B chains were highly homologous to those of beta-bungarotoxins (beta-Bgts) from Bungarus multicinctus, respectively. We isolated cDNA clones encoding the A and B chains from both B. flaviceps and B. candidus venom gland cDNA libraries using probes designed based on the cDNA sequence of beta-Bgt from B. multicinctus. Two isoforms of the A chain and one isoform of the B chain were obtained from B. flaviceps, and one isoform of the A chain and two isoforms of the B chain were obtained from B. candidus. Both of the two A chains from B. flaviceps are made up of 119 amino acids and comprise 15 cysteine residues, while the A chains of beta-Bgt from other Bungarus species including B. candidus comprise 13 cysteine residues. The B chains from both species are composed of 59 amino acid residues and comprise seven cysteines. In conclusion, the lethal toxin from B. flaviceps is considered to be a novel isoform of beta-Bgt, which has a different pattern of cysteine residues from known beta-Bgts.Toxicon 04/2006; 47(4):416-24. · 2.51 Impact Factor -
Article: Complete amino acid sequence and phylogenetic analysis of a long-chain neurotoxin from the venom of the African banded water cobra, Boulengerina annulata.
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ABSTRACT: The sequence of a long-chain neurotoxin (71 amino acid residues, 10 half-cystines) from the venom of the African banded water cobra (Boulengerina annulata) was determined by Edman degradation. It exhibits high sequence similarity with long-chain neurotoxins from the venoms of four species of African cobras (genus Naja), which are collectively more similar to the Boulengerina toxin than to those of Asian Naja species. These results are discussed in the light of phylogenetic hypotheses on the relationships of African cobras.Toxicon 07/2004; 43(7):855-8. · 2.51 Impact Factor -
Article: Oral therapeutic agents with highly clustered globotriose for treatment of Shiga toxigenic Escherichia coli infections.
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ABSTRACT: Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.The Journal of Infectious Diseases 03/2004; 189(3):360-8. · 6.41 Impact Factor -
Article: Isolation, toxicity and amino terminal sequences of three major neurotoxins in the venom of Malayan krait (Bungarus candidus) from Thailand.
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ABSTRACT: We isolated the most lethal toxins in the venom of the Malayan krait (Bungarus candidus), one of the medically most important snake species in southeast Asia. Three beta-BTx like basic neurotoxins, T1-1, T1-2, and T2, with PLA2 activity were isolated from pooled venom of eight B. candidus from southern Thailand by cation-exchange chromatography, followed by adsorption chromatography on hydroxylapatite and RP-HPLC, with 14-, 16-, and 4-fold increases in toxicity compared to crude venom. The LDs50 determined in mice weighing 18-20 g were 0.26, 0.22, and 0.84 micro g per mouse with i.v. injection. T1-1 and T1-2 possessed comparable lethal toxicities to those of beta1-BTx, the most toxic neurotoxin in B. multicinctus venom, and the major neurotoxin in B. flaviceps venom. The apparent molecular weights of the native toxins were approximately 25-25.5 kDa. They consist of two polypeptide chains with apparent molecular weights of 15.5-16.5 and 8-8.5 kDa, respectively. The amino terminal sequences of the two chains of each of the toxins determined by Edman degradation exhibited considerable similarity with those of the A-chains and B-chains of beta-BTxs in the venom of Bungarus multicinctus.Journal of Biochemistry 01/2004; 134(6):799-804. · 2.37 Impact Factor -
Article: Identification of alpha-bungarotoxin (A31) as the major postsynaptic neurotoxin, and complete nucleotide identity of a genomic DNA of Bungarus candidus from Java with exons of the Bungarus multicinctus alpha-bungarotoxin (A31) gene.
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ABSTRACT: The Malayan krait (Bungarus candidus) is one of the most medically significant snake species in Southeast Asia. No specific antivenom exists to treat envenoming by this species. Death within 30 min after its bite has been reported from Java, suggesting the presence of highly lethal postsynaptic neurotoxins in the venom of these snakes. We purified and identified the major postsynaptic toxin in the venom of B. candidus from Java. The toxin was indistinguishable from alpha-bungarotoxin (A31), a toxin originally isolated from Bungarus multicinctus, in its mass (7983.75 Da), LD50 (0.23 microg/g in mice i.p.), affinity to nicotinic acetylcholine receptors, and by its 40 N-terminal amino acid residues as determined by Edman degradation. Identity with alpha-bungarotoxin was confirmed by cloning and sequencing a genomic DNA from B. candidus which encodes the 74 amino acid sequence of alpha-bungarotoxin (A31) and part of its signal peptide, revealing complete identity to the alpha-bungarotoxin (A31) gene in exon and 98.9% identity in intron sequences. The entire mitochondrial cytochrome b gene of the krait species B. candidus from Java and B. multicinctus from Taiwan was sequenced for comparison, suggesting that these snakes are phylogenetically closely related. alpha-Bungarotoxin appears to be widely present and conserved in Southeast and East Asian black-and-white kraits across populations and taxa.Toxicon 10/2003; 42(4):381-90. · 2.51 Impact Factor -
Article: Isolation and characterization of two phospholipase as from the venom of Agkistrodon halys blomhoffii
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ABSTRACT: Two phospholipase A's (phosphatide acyl-hydrolase, EC 3.1.1.4) found in the venom of Agkistrodon halys blomhoffii were purified by successive chromatographies on ion exchangers and gel filtration on Sephadex G-100.The purified enzymes, A-I and A-II, each gave a single band on disk electrophoresis on polyacrylamide gel and a symmetrical schlieren sedimentation pattern. On isoelectric focusing analysis each protein also gave a single protein peak with phospholipase activity. Enzyme A-I had a sedimentation constant of 1.8 S and an average molecular weight, determined by two different methods, of 13 800 (± 500). The protein was strongly basic, as indicated by its isoelectric point of pH 10.0. Enzyme A-II had a sedimentation constant of 1.9 S, a diffusion constant of 13.35·10−7 cm2·sec−1, and an average molecular weight, determined by three different methods, of 13 700 (± 500). In contrast to enzyme A-I, enzyme A-II was acidic, as indicated by its isoelectric point of pH 4.0. Thus, the charge distributions of the protein molecules of the two enzymes differed significantly. Moreover, studies of circular dichroism in the spectral region between 210 and 310 nm revealed conformational differences between the side-chain chromophores of the two proteins, probably due to the tyrosyl and tryptophyl residues in these phospholipase molecules.Chemical analysis of the proteins of A-I and A-II gave, respectively, 14.44 and 15.45% total nitrogen and 2.98 and 3.18% total sulfur. Thus, both proteins had relatively high sulfur contents. However, no free sulfhydryl groups were detected on titration with p-chloromercuribenzoate (PCMB). Neither protein contained appreciable hexose, hexosamine or sialic acid.Biochimica et Biophysica Acta (BBA) - Protein Structure. -
Article: pH-Dependence of the Binding Constant of Monodispersed n-Dodecylphosphorylcholine to the Phospholipase A2 of A halys blomhoffii. Participation of Ionizable Groups with pK Values of 5.16 and 7.30
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ABSTRACT: The pH-dependence of the binding constant of monodispersed n -dodecylphosphoryl-choline to the phospholipase A2-II of A. halys blomhoffii and its Ca2+ complex was studied at 25�C and an ionic strength of 0.1 by the aromatic circular dichroism method (Ikeda & Samejima (1981) J. Biochem . 89, 1175–1184). The pH-dependence curve for the apoenzyme was found to be well interpreted in terms of pK shifts of two ionizable groups from 7.30 to 7.66 and from 5.16 to 4.80. The binding constant at infinitely lowered pH values for the Ca2+ complex was practically the same that for the apoenzyme. The pH-dependence curve for the Ca2+ complex had no transition corresponding to the ionization of a group with pK 5.16 and was interpreted in terms of the pK shift of only a single ionizable group from 6.30 to 6.65. This result indicates that the pK value of 7.30 of an ionizable group is perturbed to 6.30 on the Ca2+ binding and that the Ca2+ binding competes with the protonation of another ionizable group with pK value of 5.16. The latter group was assigned to an Asp residue corresponding to Asp 49 of pancreatic phospholipases A2, to which Ca2+ can coordinate (Verheij et al . (1980) Biochemistry 19 , 743–750 and Fleer et al . (1981) Eur. J. Biochem . 113 , 283–288). The pH-dependence of the ellipticity at 289 nm of the apophospholipase A 2 -II and its Ca2+ complex and their complexes with n -dodecylphosphorylcholine were studied in detail at 25�C and an ionic strength of 0.1. The effect of an ionizable group with a pK value of about 4.7 was observed and was most remarkable in the cases of the substrate complexes. -
Article: Kinetics of the Hydrolysis of Monodispersed Dihexanoyllecithin Catalyzed by the Phospholipase A2 from Agkistrodon halys blomhoffii Venom
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ABSTRACT: The hydrolysis of 1,2-dihexanoyl- sn -glycero-3-phosphorylcholine (diC 5 PC), catalyzed by the phospholipase A 1 from the venom of Agkistrodon halys blomhoffii , was studied at 25�C and the ionic strength of 0.1 in the presence of 3–33.3 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micelle concentration (cmc), were analyzed according to the Michaelis-Menten equation. The pH-dependence curve of the K m value exhibited only one transition below pH 8. The analytical results indicated that the p K value of 6.30 of an ionizable group changed to 6.54 on the binding of the monodispersed substrate. This ionizable group was assigned as the a-amino group on the basis of its p K value, which had been determined from the pH dependence of the binding constant of monodispersed n -dodecylphosphorylcholine ( n -C 12 PC) (Ikeda and Samejima (1981) J. Biochem . 90, 799–804, and Haruki et al . (1986) J. Biochem. 99, 99–109). The pH-dependence curve of the k cat value exhibited two transitions, below pH 6.5 and above pH 9.5. The analytical results indicated the participation of two ionizable groups with p K values of 5.55 and 10.50. Deprotonation of the former and protonation of the latter group were found to be essential for the catalysis. The former ionizable group was assigned as His 48 in the active site on the basis of its p K value, which had been determined from the pH dependence of the binding constant of Ca2+ (Ikeda et al. (1981) J. Biochem. 90, 1125–1130). The latter group was tentatively assigned as the invariant Tyr 52, which is located in close proximity to the irmdazole ring of His 48 (Dijkstra et al. (1983) J. Mol. Biol. 168, 163–179). These findings were compatible with the results of our previous study on the cobra (Naja naja atra) venom phospholipase A 2 , although in the case of the cobra enzyme the additional minor participation of the α-amino group was observed. -
Article: Kinetics of the Hydrolysis of Monodispersed Dihexanoyllecithin Catalyzed by a Cobra (Naja naja atra) Venom Phospholipase A2
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ABSTRACT: The hydrolysis of 1,2-dihexanoyl- sn -glycero-3-phosphorylcholine (diC 6 PC), catalyzed by a cobra ( Naja naja atra ) venom phospholipase A 2 was studied at 25°C and ionic strength 0.1 in the presence of 3–10 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical miceflar concentration (cmc), were analyzed accord ing to the Michaelis-Menten equation. The K m value was practically independent of pH (between pH 6.75 and 10.30). This finding was consistent with the result of a direct binding study on monodispersed n -alkylphosphorylcholines (Teshima et al . (1981) J. Biochem . 89, 1163–1174). The hydrolysis of the substrate was competitively inhibited by the presence of monodispersed n -dodecylphosphorylcholine ( n -C 12 PC). These results indicated that the substrate and n -C 12 PC compete for the same site on the enzyme molecule. The pH dependence curve of the kinetic parameter, k cat / K m , exhibited three transitions, below pH 8, between pH 8 and 9.5, and above pH 10. The analysis indicated the participation of three ionizable groups with pK values of 7.25, 8.50, and 10.4. The deprotonation of the first group and the protonation of the third group were found to be essential for the catalysis. The first group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Teshima et al . (1981) J. Biochem . 89, 13–20). The second group was assigned as the α-amino group on the basis of its pK value, which had been determined from the pH dependence of the rate constant for the reaction of p -bromophenacyl bromide (BPB) with His 48 (Teshima et al . (1984) J. Biochem . 96, 1903–1913). The third group was tentatively assigned as the invariant Tyr 52, located in close proximity to the imidazole ring of His 48. -
Article: Binding Mode of Phospholipase A2 with a New Type of Phospholipid Analog Having an Oxazolidinone Ring
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ABSTRACT: Inhibition of phospholipases A 2 (PLA 2 s) by a new type of monodispersed phospholipid analog, 3-dodecanoyl-4-phosphatidylcholinohydroxymethyl-2-oxazolidinone (oxazolidinone-PC), was investigated by the pH stat assay method using monodispersed 1,2-dihexanoyl- sn -glycero-3-phosphorylcholine (diC 6 PC) as the substrate. The PLA 2 s used were those from bovine pancreas and cobra ( Naja naja atra ) venom (Group I) and from Japanese mamushi ( Agkistrodon halys blomhoffii ) venom (Group II). This new-type substrate analog was shown to inhibit competitively both types of venom and bovine pancreatic enzymes by binding to the active site in a similar manner to the carboxamide-type analog 2-dodecanoyl-amino-1-hexanol-phosphocholine (amide-PC). The binding of a stereoiso-mer, ( R )-amide-PC, to N . naja atra (Group I) and A. halys blomhoffii (Group II) PLA 2 s was facilitated by the binding of Ca2+ to the enzymes. On the other hand, the binding of ( R )-oxazolidinone-PC to the N. naja atra (Group I) enzyme was found to be independent of Ca2+ binding, while its binding to the A. halys blomhoffii (Group II) enzyme was markedly facilitated by the binding of Ca2+ to the enzyme. The binding of ( R )-amide-PC to N. naja atra PLA 2 (Group I) was markedly influenced by the ionization state of the catalytic residue His 48, whereas the binding of ( R )-oxazolidinone-PC was found to be practically independent of the ionization state of this residue. The Ca2+ dependency and participation of the catalytic group His 48 in the binding of genuine substrate to both types of PLA 2 s were found to be very similar to those for the oxazolidinone-PC, but differed greatly from those for the amide-PC, indicating that the binding mode of oxazolidinone-PC is very similar to that of the genuine substrate, but very different from that of the amide-PC. -
Article: Subunit structure and inhibition specificity of α-type phospholipase A2 inhibitor from Protobothrops flavoviridis
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ABSTRACT: The α-type phospholipase A2 inhibitor (PLIα) in the plasma of the Habu snake, Protobothrop flavoviridis, was shown to be a trimer of two homologous subunits, PLIα-A and PLIα-B, each of which contains one C-type lectin-like domain (CTLD). Since one molecule of trimeric PLIα binds stoichiometrically to one molecule of P. flavoviridis acidic phospholipase A2 (PLA2), the trimeric structure is critical for its inhibitory activity. Hydrophobic chromatography separated the purified P. flavoviridis PLIα into four different trimeric subspecies, A3-PLIα, A2B-PLIα, AB2-PLIα, and B3-PLIα, with different combinations of the two subunits. The trimeric PLIα could be reconstituted from the purified subunits, and the four different trimeric subspecies were formed through random association of the two subunits. The inhibitory activity of the PLIα-A homotrimer (A3-PLIα) was more specific than that of the PLIα-B homotrimer (B3-PLIα). This difference in inhibitory properties between the two homotrimers was probably caused by the amino acid differences at residues 10–37.Toxicon 51(5):787-796. · 2.51 Impact Factor -
Article: pH Dependence of the Reaction Rate of His 48 with p-Bromophenacyl Bromide and of the Binding Constant to Ca2+ of the Monomeric Forms of Intact and {alpha}-NH2 Modified Phospholipases A2 from Trimeresurus flavoviridis
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ABSTRACT: The phospholipase A 2 of Trimeresurus flavoviridis was found to show monomer-dimer equilibria. Under conditions where the enzyme exists predominantly in the monomeric form, the chemical reaction rate of p -bromophenacyl bromide (BPB) with the catalytic group, His 48, was studied at 25°C and ionic strength 0.2 by measuring the residual enzymic activity using a fluorescent substrate, 1,2-bis[4-(1-pyreno)butanoyl]- sn -glycero-3-phosphorylcholine (diPBPC). The pH-dependence curve of the reaction rate for the intact enzyme was practically the same as that for the modified enzyme, in which the N-terminal α-NH 2 group had been selectively converted into an α-keto group. The pH-dependence curves were monophasic (sigmoidal) with a midpoint at pH 7.53, which corresponds to the p K a . value of His 48. The pH dependences of the binding constants of Ca2+ to the intact and the α-NH 2 modified enzymes were also studied at 25°C and ionic strength 0.2 by measuring the changes in the tryptophyl fluorescence and/or aromatic CD spectra. The pH-dependence data for the modified enzyme were interpreted in terms of participation of Asp 49 (p K a 8 5.40) and His 48 (p K a 7.53), assuming that the protonation of Asp 49 competes with the Ca2+ binding. The pH-dependence data for the intact enzyme were similarly interpreted in terms of participation of the α-NH 2 group (p K a 9.40) in addition to that of Asp 49 (p K a 5.40) and His 48 (p K a 7.53). The enzyme dimerization was found to reduce the binding constant of Ca2+ to the enzyme at neutral and acidic pH values, suggesting that the way in which one enzyme molecule is brought into contact with the other is unfavorable to the Ca2+ binding. This result seems compatible with the recent X-ray crystallographic data on the dimeric phospholipase A 2 of Crotalus atrox (Brunie et al . (1985) J. Biol. Chem . 260, 9742–9749), which belongs to the same family, Crotalidae, as T. flavoviridis . -
Article: pH Dependence of the Binding Constant of Ca2+ to the Phospholipase A2 of A. halys blomhoffii. Paritcipation of Ionizable Groups with pK Values of 5.16, 6.45, and 7.30
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ABSTRACT: The pH dependence of the binding constant of Ca2+ to the phospholipase A2 of A. halys blomhoffii was studied at 25�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 A 2 (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 an ionizable group (α-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 K value of 5.16 competes with the 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. -
Article: Kinetics of the Hydrolysis of Mixed Micelles of Dipalmitoyllecithin with Triton X-100 Catalyzed by a Phospholipase A2 from the Venom of Agkistrodon halys blomhoffii1
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ABSTRACT: The pH dependence of kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl- sn -glycero-3-phosphorylcholine (diC 16 PC) with Triton X-100, catalyzed by the intact and the N-terminal α-NH2-modified phospholipases A 2 (PLA 2 s) of Agkistrodon halys blomhoffii , was studied at 25°C and ionic strength 0.1 in the presence of saturating amounts of Ca2+. The pH dependence of the kinetic parameters for the hydrolysis of monodispersed diC 6 PC, catalyzed by the modified enzyme, was also studied under the same conditions, and the data were compared with the previous results for the intact enzyme [Teshima, K. et al . (1986) J . Biochem . 100, 1655–1662]. The pK values of the catalytic group, His 48, and Tyr 52 were found to shift from 5.55 to 7.00 and from 10.50 to 11.50, respectively, on binding of the micellar substrates to the enzyme. On the other hand, no participation of these ionizable groups was observed for the binding of the monodispersed substrate. On the basis of the present finding and the X-ray crystallographic studies on bovine pancreatic PLA 2 [Dijkstra, B.W. et al . (1981) J . Mol . Biol . 147,97–123] and on a PLA 2 of Crotalus atrox venom [Brunie, S. et al . (1985) J . Biol . Chem . 260, 9742–9749], the hydrogen-bonding of Tyr 73, which is involved in the lipid-water interface recognition site, to His 48 and Tyr 52 in the active center was strongly suggested to be important for the hydrolysis of micellar substrates. -
Article: Bindings of Ca2+ Ion and Monodispersed n-Alkylphosphorylcholines to Phospholipase A2-II of A. halys blomhoffii
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ABSTRACT: Bindings of monodispersed n -dodecyl-, n -decyl-, and n -octylphosphorylcholines ( n -C 12 PC, n -C 10 PC, and n -C 8 PC, respectively) to phospholipase A 2 II of A. halys blom-hoffii were studied by the aromatic circular dichroism (CD) or ultraviolet difference absorption method. The binding constants to the apoenzyme at around 25°C, pH 6.4–6.5, and ionic strength 0.1 were 1600–1800, 820, and 320 M−1 for n -C 12 PC, n -C 10 PC, and n -C, PC, respectively. The contribution of one methylene moiety in the alkyl chain of the substrate to the unitary free energy of binding was calculated to be 0.26 kcal/mol, which is smaller than that for porcine pancreatic enzyme, about 0.5 kcal/mol (van Dam-Mieras et al . (1975) Biochemistry 14, 5387–5394 and Verheij et al . (1980) Biochemistry 19, 743–750) and that for cobra enzymes, 0.39 kcal/mol (Teshima et al ., submitted to J. Biochem .). This suggests that the substrate binding site of phospholipase A 2 -II of A. halys blomhoffii is less hydrophobic than those of the enzymes of porcine pancreas and cobra venoms. Binding of Ca2+ to phospholipase A 2 -II was studied by the aromatic circular dichroism method. The binding constants at pH 6.53 and 8.32 were 1880 and 6300 M−1 respectively. If we assume that the pK value of His 48 in the active site is around 7, the result indicates a pK shift of His 48 by about 0.5 pH unit to the acidic side on binding of Ca2+. The binding constants of n -C 12 PC to the apoenzyme and Ca2+ complex at pH 8.2–8.4 were smaller than those at pH 6.4–6.5 by a factor of about two. This suggests a pK shift of His 48 by about 0.3 pH unit to the alkaline side on binding of the substrate. The binding constant of n -C 12 PC to the Ca2+-enzyme complex was smaller than that for the apoenzyme by a factor of about 2.5 to 2.7 at pH 8.2–8.4 and 6.4–6.5. This indicates that an electrostatic interaction between the bound Ca2+ ion and the negatively charged phosphate moiety of the substrate is not important in this case.
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Institutions
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2008
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Osaka University of Pharmaceutical Sciences
- Department of Biochemistry
Takatsuki, Osaka-fu, Japan
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