Inactivation of phospholipase A2 by manoalide. Localization of the manoalide binding site on bee venom phospholipase A2.
The marine natural product manoalide (MLD), a potent inhibitor of phospholipases, completely inactivates bee venom phospholipase A2 (PLA2) by an irreversible mechanism. It has been proposed [K. B. Glaser and R. S. Jacobs, Biochem. Pharmac. 36, 2079 (1987)] that the reaction of MLD with PLA2 may involve the selective reactivity of MLD to a peptide sequence, possibly a Lys-X-X-Lys peptide. Localization of the MLD binding site on bee venom PLA2 demonstrated that upon MLD modification of bee venom PLA2 the only change in amino acid content was an apparent loss of Lys, corresponding to approximately three of the eleven Lys residues present. Selective chemical modification of Lys residues with [14C]maleic anhydride demonstrated that all eleven Lys residues on bee venom PLA2 were accessible to this reagent (11.6 mol maleyl group incorporated/mol of PLA2). Pretreatment of PLA2 with MLD (less than 0.7% residual activity) resulted in a molar ratio of 8.7, also consistent with the loss of three Lys residues upon modification by MLD. Reverse phase high performance liquid chromatography (RP-HPLC) of the cyanogen bromide (CNBr) digestion product of MLD-treated PLA2 produced three peaks (A280). The second peak showed the most intense absorbance at 434 nm. This material corresponded to residues 81-128, as determined by gas-phase microsequence analysis. Sequencing failure was observed at Lys-88 in the MLD-treated fragment. The control carboxymethylated-PLA2 fragment corresponding to residues 81-128 sequenced beyond Lys-88 without significant change in the expected yield. These data suggest that Lys-88 may correspond to one of the three MLD-modified Lys residues. The minor absorbance at 434 nm of the CNBr fragments containing residues 42-80 and 1-36 as compared to the fragment of residues 81-128 suggests that the major MLD binding fragment residues in residues 81-128.
Glaser
K B
KB
Department of Biological Sciences, University of California, Santa Barbara 93106.
Vedvick
T S
TS
Jacobs
R S
RS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001514:Bee Venoms / Q000032:analysis*; D001665:Binding Sites; D008969:Molecular Sequence Data; D010740:Phospholipases / Q000037:antagonists & inhibitors*; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054467:Phospholipases A2; D013729:Terpenes / Q000378:metabolism / Q000494:pharmacology*
D000596:Amino Acids; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001514:Bee Venoms; D013729:Terpenes; C045673:manoalide; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2