Role of the N-terminal region in phospholipases A2 from Naja naja atra (Taiwan cobra) and Naja nigricollis (spitting cobra) venoms.
The N-terminal alpha-amino groups of two phospholipases A2 (PLA2) from Naja naja atra and Naja nigricollis venoms were selectively modified with trinitrobenzene sulfonic acid, and the modified derivatives were separated by high performance liquid chromatography (HPLC). Trinitrophenylated (TNP) derivatives contained only one TNP group in the alpha-amino group of Asn-1 and showed a marked decrease in enzymatic activity. PLA2 enzymes were cleaved with CNBr, and the N-terminal octapeptide was separated from the large C-terminal fragment by HPLC. Removal of the N-terminal octapeptide from PLA2 enzymes caused a precipitous decrease in enzymatic activity. Enzyme immunoassay and double immunodiffusion revealed that the N-terminal octapeptide is one of the antigenic determinants of PLA2 enzymes. The presence of dihexanoyllecithin influenced the interaction between PLA2 enzymes and 8-anilinonaphthalene sulfonate (ANS), indicating that ANS-binding site of PLA2 enzymes is at or near the substrate binding site. Modification of the N-terminal region perturbed the substrate binding and the binding ability for ANS. The modified derivatives retained their affinity for Ca2+, indicating that the N-terminal region is not involved in Ca2+-binding. A fluorescence study revealed that the alpha-amino group is near Trp residue(s) and that the N-terminal region is important for stabilizing the architectural environment of the Trp residue(s). The results, together with the proposal that Trp residues in PLA2 enzymes are involved in substrate binding, suggest that the N-terminal region of PLA2 enzymes is involved in substrate binding and in maintaining a functional active site.
Yang
C C
CC
Institute of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan, R.O.C.
Chang
L S
LS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004546:Elapid Venoms / Q000633:toxicity; D010740:Phospholipases / Q000633:toxicity; D010741:Phospholipases A / Q000633:toxicity; D054467:Phospholipases A2; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D004546:Elapid Venoms; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2