Immunochemical properties of Naja naja atra (Taiwan cobra) phospholipase A2 using polyclonal and monoclonal antibodies.
The immuno-chemical properties of Naja naja atra phospholipase A2 (NNA-PLA2) were studied by using the chemically modified PLA2 derivatives and the PLA2 homologues toward anti-NNA-PLA2 polyclonal and monoclonal antibodies. Anti-NNA-PLA2 polyclonal antibodies inhibited the enzymatic activity of NNA-PLA2 and Hemachatus haemachatus DE-I by 87% and 68%, respectively. However, the enzymatic activities of Naja nigricollis CMS-9 and notexin were not significantly affected by the polyclonal antibodies. Competitive enzyme immunoassay revealed that the affinity of NNA-PLA2 for polyclonal antibodies was 330-fold higher than that of Hemachatus haemachatus DE-I. Naja nigricollis CMS-9 and notexin failed to inhibit the binding of NNA-PLA2 to polyclonal antibodies. This implies that the epitope(s) of NNA-PLA2 might comprise some substituted residues in the sequence of PLA2 homologues. Three monoclonal antibodies against NNA-PLA2 were prepared by a hybridoma technique. Two of these monoclonal antibodies inhibited the enzymatic activity of NNA-PLA2, but the other did not. Removal of the N-terminal octapeptide affected the epitope interacting with these monoclonal antibodies. Selective modification of tyrosine residues at positions 3 and 63 or lysine residues at positions 6 and 65 induced a substantial reduction in affinity of NNA-PLA2 for polyclonal and monoclonal antibodies. The three monoclonal antibodies failed to recognize PLA2 homologues. The comparison of the sequence of NNA-PLA2 to those of PLA2 homologues showed that most of the amino acid substitutions of PLA2 homologues occur in the spatially nearby region of the N-terminal region and residues at positions 63 and 65.(ABSTRACT TRUNCATED AT 250 WORDS)
Yang
C C
CC
Institute of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Chang
L S
LS
Ong
P L
PL
Tung
T H
TH
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D004546:Elapid Venoms / Q000201:enzymology* / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D007124:Immunoenzyme Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D010741:Phospholipases A / Q000737:chemistry / Q000276:immunology*; D054467:Phospholipases A2; D011487:Protein Conformation; D011863:Radioimmunoassay
D000906:Antibodies; D000911:Antibodies, Monoclonal; D004546:Elapid Venoms; D000939:Epitopes; D010741:Phospholipases A; D054467:Phospholipases A2