Naoko Oda

Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus Gramineus (green habu snake) venom

Two phospholipases A2 named PLA2-III and IV were newly isolated from Trimeresurus gramineus (green habu snake) venom in addition to PLA2-I and II reported previously [ODA et al. (1991) Toxicon 29, 157; Fukagawa et al. (1992) Toxicon 30, 133]. Their isoelectric points were determined to be about 4.5. PLA2-III and IV exhibited almost unchanged lipolytic activity toward egg-yolk when compared with PLA2-I. The amino acid sequences were determined by sequencing the native proteins and the peptides produced by enzymatic (Achromobacter protease I and clostripain) and chemical (hydroxylamine) cleavages of the S-carboxamidomethylated derivative of the proteins. Both proteins consisted of 122 amino acid residues. When compared with PLA2-I, PLA2-III showed only a single amino acid substitution at the N-terminal position; namely from His to Asn. PLA2-IV also showed a single substitution from Ala to Asp at position 72. It was inferred that these amino acid substitutions between PLA2-I and PLA2-III or IV are due to the single base substitution at the corresponding codons of genes, which might be preserved independently. The unique presence of Phe at position 28, where Tyr is commonly located and assumed to be a part of the Ca(2+)-binding loop, was conserved in both PLA2-III and IV as in PLA2-I. There was no significant difference in the dissociation constants (4.3-5.2 x 10(-4) M) for Ca2+ between these PLA2S and Tyr-28-containing PLA2S. These results suggested that the p-hydroxy group of Try-28 does not play a crucial role in binding of PLA2S to Ca2+.

Sequence determination and characterization of a phospholipase A2 isozyme from Trimeresurus gramineus (green habu snake) venom

In addition to phospholipase A2-I (PLA2-I) reported previously (ODA et al., 1991, Toxicon 29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved. Its sequence similarity to PLA2-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA2-I, PLA2-II contains Tyr-28 as seen in most of other PLA2s. There was no significant difference between the dissociation constants of PLA2-I and PLA2-II for Ca2+. Secondary structure compositions of PLA2-II were similar to those of PLA2-I and Crotalus atrox PLA2. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA2-II was over ten times more potent than PLA2-I, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I. The difference in contractile activities of PLA2-I and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLA2-I and Asn for PLA2-II).

Amino acid sequence of a phospholipase A2 from the venom of Trimeresurus gramineus (green habu snake).

Two phospholipases A2, named phospholipases A2-I and A2-II, were purified to homogeneity from the venom of Trimeresurus gramineus (green habu snake). The complete amino acid sequence of phospholipase A2-I was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I, clostripain, and chymotrypsin) and chemical (hydroxylamine) cleavages of the S-pyridylethylated derivative of the protein. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid snakes which belong to the category of Group II. A most striking feature of this protein is that tyrosine at the 28th position which is common in phospholipases A2 and is assumed to be a part of the Ca2(+)-binding loop is replaced by phenylalanine. Such replacement is the first finding in Group II phospholipases A2. Secondary structure compositions of phospholipase A2-I are similar to those of Crotalus atrox phospholipase A2. No appreciable Ca2(+)-induced difference spectrum was observed, due probably to the absence of the effective chromophoric groups in the neighborhood of the Ca2+ binding site although Ca2+ is bound with affinity similar to that for T. flavoviridis phospholipase A2.

Validity of putative calcium binding loops of photoprotein aequorin

Three peptides containing the putative Ca2+ binding loops, I, II and III, respectively, of a photoprotein, aequorin, from jellyfish Aequorea victoria were synthesized by a solid‐phase procedure. The peptides bound Ca2+ with dissociation constants or 10−3 to 10−4 M, providing evidence for the assumption that Ca2+ binding loops are actually responsible for the binding of Ca2+. When the highly conserved 6th glycine residue in the 12‐residue loops was replaced by arginine, no large effect was observed on Ca2+ binding. Exposure to a hydrophobic environment and the binding or Ca2+ brought about conformational changes to the peptides.