The structural events associated with the binding of divalent cations to beta-bungarotoxin.
In order to address the mechanism why the Ca2+ was crucial for the manifestation of the phospholipase A2 (PLA2) activity of beta-bungarotoxin (beta-BuTx), four divalent cations were used to assess their influences on the catalytic activity and the fine structures of beta-BuTx. Substitution Mg2+ or Sr2+ for Ca2+ in the substrate solution was found to cause a decrease in the PLA2 activity to approximately 15 or 6% of that in the presence of Ca2+. However, only marginally detectable PLA2 activity was observed with the addition of Ba2+. The nonpolarity of 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of beta-BuTx markedly increased with the binding of cations to beta-BuTx. The negative ellipticity noted with the CD spectra of beta-BuTx increased upon the binding of cations too. With the exception of Ba2+, the order of the ability of cations to enhance the intensity of ANS fluorescence or increase the increment of negative ellipticity was Sr2+ > Ca2+ > Mg2+, which was the same order as the increase in their atomic radii. However, the energy transfer from Trp fluorescence emission to ANS was most effective upon the addition of Ca2+. Moreover, the extent of glutaraldehyde crosslinking between A chain and B chain decreased in the presence of cations. Nevertheless, the binding affinities of beta-BuTx for the four cations were similar. These results, together with the findings that the ANS molecule binds at the active site of the A chain in beta-BuTx, suggest that the binding of Ca2+ to beta-BuTx induces subtly conformational changes occurred at the active site for exerting the activity of beta-BuTx. Moreover, the change in the gross conformation induced by the binding of Ca2+ may affect the interaction between A chain and B chain, and consequently the activity of beta-BuTx as well.
Chu
Yuan-Ping
YP
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan, ROC.
Cheng
Yun-Ching
YC
Yang
Chen-Chung
CC
Chang
Long-Sen
LS
D001464:Barium / Q000378:metabolism; D002038:Bungarotoxins / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism; D054467:Phospholipases A2; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013324:Strontium / Q000378:metabolism
D002038:Bungarotoxins; D002413:Cations, Divalent; D010713:Phosphatidylcholines; D001464:Barium; D010741:Phospholipases A; D054467:Phospholipases A2; D008274:Magnesium; D002118:Calcium; D013324:Strontium