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Biomedical subjects

C Takasaki

Publications and source records attributed to C Takasaki.

35 records · Page 2Linked to original sources
PubMed1989

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.

Acetylcholine↗
PubMed1989

Neuromuscular effects of a toxic phospholipase A2 and its nontoxic homologue from the venom of the sea snake, Laticauda colubrina.

A single chain phospholipase A2 (LcPLA-II) and a homologous protein lacking enzymatic activity (LcPLH-I) isolated from the venom of the Solomon Island sea snake (Laticauda colubrina) were tested for effects on neuromuscular transmission and muscle contractility on chick biventer cervicis and mouse hemidiaphragm preparations. LcPLA-II (7.5 nM-1.5 microM) blocked indirectly elicited muscle contractions of both preparations. Low concentrations of LcPLA-II caused little change in sensitivity to acetylcholine, carbachol and KCl. The homologue LcPLH-I (375 nM-1.5 microM) reduced the responses of the biventer cervicis preparation to indirect stimulation and abolished responses to acetylcholine and carbachol, but it did not block KCl responses. These effects were due to minor contamination by a post-junctional neurotoxin. LcPLH-I (375 nM-750 nM) had no effect on indirectly stimulated hemidiaphragm preparations. It is concluded that LcPLA-II blocks neuromuscular transmission by a prejunctional action, and that the homologue lacking phospholipase A2 activity also lacks neuromuscular activity.

Animals↗
PubMed1989

Phospholipases B from Japanese yellow hornet (Vespa xanthoptera) venom.

Two phospholipases B (Vx I and Vx II) were purified from the venom of the Japanese yellow hornet, Vespa xanthoptera, by sequential chromatography on Sephadex G-100, SP-Sephadex and Mono S columns. They are very similar to each other in molecular and enzymatic properties, though the specific activity of Vx I was one-fifth that of Vx II. They hydrolyze the acyl ester bonds at the 1-position of phosphatidylcholine and lysophosphatidylcholine, therefore, their enzymatic specificities were of the A1 and L1 types.

Amino Acids↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1988

Isolation, properties and amino acid sequences of a phospholipase A2 and its homologue without activity from the venom of a sea snake, Laticauda colubrina, from the Solomon Islands.

A phospholipase A2, Laticauda colubrina phospholipase A2 II (LcPLA-II), and a phospholipase A2 homologue, Laticauda colubrina phospholipase A2 homologue I (LcPLH-I), were isolated from the venom of the yellow-lipped sea snake, Laticauda colubrina, from the Solomon Islands. LcPLA-II showed phospholipase A2 activity towards egg-yolk phosphatidylcholine (24 mumol/min per mg at optimal conditions at 37 degrees C) and lethal potency (LD50 45 micrograms/kg body wt. intravenously in mice). Both of the activities were lost by treatment with p-bromophenacyl bromide. LcPLH-I showed neither phospholipase A2 activity nor lethal potency at a dose of 4.5 mg/kg body wt. in mice. It was not modified by the treatment with p-bromophenacyl bromide. LcPLA-II and LcPLH-I bound Ca2+ at a 1:1 molar ratio with KCa values of 105 microM and 44 microM at pH 8.0 respectively. Elucidation of the amino acid sequences of these two proteins showed that each protein consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. The two sequences are different from each other at 22 residues and highly homologous to those from other sources. The essential histidine residue for the phospholipase A2 activity at position 48 is replaced by an asparagine residue in the homologue LcPLH-I. Details of the separation of the peptides obtained by proteinase digestions of LcPLA-II and LcPLA-I and the determination of their amino acid sequences are given in Supplementary Publication SUP 50145 (14 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.

Acetophenones↗
PubMed1985

Isolation and amino acid sequence of a short-chain neurotoxin from an Australian elapid snake, Pseudechis australis.

A short-chain neurotoxin Pseudechis australis a (toxin Pa a) was isolated from the venom of an Australian elapid snake Pseudechis australis (king brown snake) by sequential chromatography on CM-cellulose, Sephadex G-50 and CM-cellulose columns. Toxin Pa a has an LD50 (intravenous) value of 76 micrograms/kg body wt. in mice and consists of 62 amino acid residues. The amino acid sequence of Pa a shows considerable homology with those of short-chain neurotoxins of elapid snakes, especially of true sea snakes.

Amino Acid Sequence↗
PubMed1985

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

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

Acetophenones↗
PubMed1983

[Simplified microdetermination of cerebral phospholipase A1, A2 and lysophopholipase].

The purpose of our study was to examine the ischemia induced enzymatic changes of decaylation-reacylation cycle of membrane phospholipids in dog brain. In this study, we developed new modified method for assay of phospholipase A1, A2 and lysophospholipase which is simpler and needs only a smaller amount of materials. For the first report, we introduced this new method and demonstrated some properties of phospholipase A1, A2 and lysophospholipase in dog brain. Crude enzyme solution for assays of phospholipase A1, A2 and lysophospholipase was gained from extraction of frozen brain with aceton, butanol and saline. The level of phosphorus in the enzyme extract was determined and only those extracts which had a level of phosphorus within a certain range were used. The substrates for assays were L-alpha-[beta-palmitoyl-1-14C] phosphatidylcholine, dipalmitoyl for phospholipase A1 and A2 and L-lysophosphatidylcholine-1-[1-14C] palmitoyl for lysophospolipase respectively. Each radioactive substrates was diluted with cold carrier lipid to give the proper specific activity. Reaction system including substrate, buffer [pH 7.0] and enzyme extract was incubated for 10 hours at 38 degrees C. But for the assay of phospholipase A1 and A2, enzyme solution was pre-incubated at 70 degrees C for 5 minutes. In our new method, reaction mixture was directly separated by TLC without extracting lipids. Enzyme activities were calculated from radio thin-layer chromatograms. Furthermore, we made a comparison between our method and the former one. The value of each enzyme activity was slightly higher in our method than in the former one. However, it was revealed that the results were reproducible in both methods.

Animals↗
PubMed1982

Isoelectric points of erabutoxins and monoacyl derivatives of erabutoxin b. Estimation of the pK values of amino groups in erabutoxins by using isoelectric-focusing data.

The isoelectric points of erabutoxins a, b and c, neurotoxic proteins of a sea snake, Laticauda semifasciata, were determined by density-gradient isoelectric focusing. The same measurement was also made with monoacyl derivatives of erabutoxin b, in which each one of all amino groups had been either acetylated or propionylated. Erabutoxins a and b showed the same isoelectric point at pH 9.68. The values for ]1-N alpha-acetyl-arginine]-, [15-N6-acetyl-lysine]-, [27-N6-acetyl-lysine]-, [47-N6-propionyl-lysine]- and [51-N6-acetyl-lysine]-erabutoxin b were at pH 9.52, 9.31, 9.45, 9.22 and 9.09 respectively, being definitely different from each other and lower than the value for the unmodified molecule. The isoelectric point of erabutoxin c, which is [51-asparagine]-erabutoxin b, was the same as that of [51-N6-acetyl-lysine]erabutoxin b. Assuming that no change in pK occurs on monoacylation, the pK values of amino groups in erabutoxin b were calculated from the isoelectric-point data. It is indicated that the pK values of zeta-amino groups differ markedly from each other and that the value of alpha-amino group is anomalously high.

Amino Acids↗
PubMed1982

Isolation and properties of lysophospholipases from the venom of an Australian elapid snake, Pseudechis australis.

Two lysophospholipases were isolated from the venom of an Australian elapid snake (subfamily Acanthophiinae), Pseudechis australis, by sequential chromatography on CM-52 cellulose, Sephadex G-75 and DE-52 cellulose columns. They were very similar to each other. One of them, lysophospholipase I, was obtained as a homodimer, the monomer of which consisted of 123 amino acid residues with seven disulphide bridges. The amino acid composition and the N-terminal amino acid sequence of the enzyme were similar to those of phospholipase A2, Ca2+ was required for its activity and the maximum activity was attained at 2 mM-CaCl2 in the presence of 1 mM-EDTA. The optimum pH was 7.5. Lysophospholipase I hydrolysed lysophosphatidylcholine more rapidly than lysophosphatidylethanolamine. It did not hydrolyse, however, phosphatidylcholine, 1-palmitoylglycerol, tripalmitoylglycerol or p-nitrophenyl acetate. Modification of the enzyme with p-bromophenacyl bromide or 2-nitrophenylsulphenyl chloride suppressed the activity. A strong direct haemolytic activity was exhibited when the lysophospholipase was present together with phospholipase A2.

Acetophenones↗
PubMed1980

Electronic spectroscopy and deuteration kinetics of tyrosine and tryptophan residues: an application to the study of erabutoxin b.

The fluorescence increase on the deuterium oxide addition to the solvent medium was studied in various tryptophan- (or indole-) and/or tyrosine-containing model compounds. It was shown how the rates of the deuteration at the indole NH group of tryptophan and at the OH group of tyrosine could be followed independently of each other. The method was applied to a study of erabutoxin b molecule, a neurotoxic protein from a sea snake, to analyze the microenvironments of its single tryptophan and tyrosine residues. It was shown that the "functionally invariant" single tryptophan residue was exposed to the solvent in the surface of the molecule and that the "structurally conserved" single tyrosine residue was buried in the molecule. The rate of deuteration of the tyrosine residue (80 s(-1) at pH 6.3 and 33 degrees C) was 1/20 of that of an exposed tyrosine. It was also found that the amino group of Lys-27 quenched the fluorescence of Trp-29 but its deuteration had no effect on the fluorescence.

Deuterium↗