ISSN:
1573-4943
Schlagwort(e):
snake venom
;
phospholipase A2
;
tyrosine modification of phospholipase A2
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Chemie und Pharmazie
Notizen:
Abstract Two phospholipases A2 (PLA2) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA2 were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca 2+; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca2+. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA2 could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity. Tyrosine-63-modifiedN. naja atra PLA2 exhibited the same Ca2+-induced difference spectra as that of native PLA2, indicating that the Ca2+-binding ability of Tyr-63-modifiedN. naja atra PLA2 was not impaired. However, Tyr-3-modified PLA2 and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca2+, revealing that the Ca2+-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca2+ binding. AtpH 8.0, both native PLA2 enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca2+, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/BF01025370
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