Characterization of phospholipases A2 of Naja melanoleuca snake venom modified at the N-terminal region.
In phospholipase A2 from Naja melanoleuca snake venom all four lysines were converted into the epsilon-amidinated derivatives without reaction of the alpha-amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity. Starting from AMPA, chemical modification reactions were carried out at the alpha-amino function. This group was blocked with a tert-butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N-terminal asparagine, resulting in the formation of des-Asn1-AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine residue. Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N-terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N-terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid-bound enzyme.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002853:Chromatography, Liquid; D004546:Elapid Venoms / Q000032:analysis*; D008055:Lipids / Q000302:isolation & purification; D008239:Lysine / Q000302:isolation & purification; D008823:Micelles; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000302:isolation & purification*; D054467:Phospholipases A2; D012911:Snakes; D013056:Spectrophotometry, Ultraviolet
D004546:Elapid Venoms; D008055:Lipids; D008823:Micelles; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine; D002118:Calcium