title: Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus gramineus (green habu snake) venom.
abstract: 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+.
authors.affiliation: Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Fukagawa
authors.forename: T
authors.initials: T
authors.lastname: Nose
authors.forename: Y
authors.initials: Y
authors.lastname: Shimohigashi
authors.forename: T
authors.initials: T
authors.lastname: Ogawa
authors.forename: N
authors.initials: N
authors.lastname: Oda
authors.forename: K
authors.initials: K
authors.lastname: Nakashima
authors.forename: C C
authors.initials: CC
authors.lastname: Chang
authors.forename: M
authors.initials: M
authors.lastname: Ohno
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003435:Crotalid Venoms / Q000737:chemistry*; D054501:Group II Phospholipases A2; D054513:Group IV Phospholipases A2; D006868:Hydrolysis / Q000187:drug effects; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D030162:Reptilian Proteins; D012910:Snake Venoms; D017841:Trimeresurus
chemical_list: D003435:Crotalid Venoms; D010743:Phospholipids; D030162:Reptilian Proteins; D012910:Snake Venoms; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2; C484164:phospholipase A2 III protein, Colubroidea; D002118:Calcium