Role of the N-terminal region of phospholipase A2 subunit of beta 1-bungarotoxin in the toxin-Ca2+ complex-formation.
beta 1-Bungarotoxin consists of a phospholipase A2 subunit and a non-phospholipase A2 subunit. Modification of beta 1-bungarotoxin with CNBr resulted in cleavage at Met-6 and Met-8 of its phospholipase A2 subunit. Analysis of the fluorescence data of both the toxin-Ca2+ complex at 300-350 nm and the toxin-Tb3+ complex at 450-650 nm showed the existence of two binding sites for both metal ions on the different domains of the toxin molecule. At pH 7.6 the association constants for the high-affinity and low-affinity sites of the toxin-Ca2+ complex were determined to be 2.79 x 10(3) +/- 0.21 x 10(3) M-1 and 0.47 x 10(3) +/- 0.06 x 10(3) M-1 respectively. For the toxin-Tb3+ complex the association constant for the high-affinity site was 2.95 x 10(3) +/- 0.43 x 10(3) M-1 and that for the low-affinity site was 0.11 x 10(3) +/- 0.03 x 10(3) M-1. Removal of the N-terminal octapeptide of the phospholipase A2 subunit from the toxin molecule caused disintegration of the low-affinity site but did not disrupt the high-affinity site. This might accompany a change in the configuration around His-48 of the phospholipase A2 subunit. Between pH 6 and 8 the binding of metal ions to the high-affinity site increased but that to the low-affinity site did not change with increasing pH. The neurotoxicity and enzymic activity of the toxin were lost on removal of the low-affinity site.
Chu
S T
ST
Institute of Biochemical Science, College of Science, National Taiwan University, Taipei.
Chen
Y H
YH
D000595:Amino Acid Sequence; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism
D002038:Bungarotoxins; D014364:Tryptophan; D010741:Phospholipases A; D054467:Phospholipases A2; D003488:Cyanogen Bromide; D002118:Calcium