PURIFICATION AND PROPERTIES OF PHOSPHOLIPASES A(2) FROM THE CROWN-OF-THORNS STARFISH (ACANTHASTER PLANCI) VENOM

Two phospholipases A < inf2 (named AP-PLA(2)-I and II) were purified f rom the crown-of-thorns starfish (Acanthaster planci) venom. Both enzy mes were confirmed to be PLA(2)s, based on the results that they showe d hemolytic activity only in the presence of phosphatidylcholine (PC) and also released fluorescent fatty acids from PC with labeled fatty a cids at the sn-2 position. The enzyme activity of both PLA(2)s was enh anced by Ca2+ but reduced by Cu2+ and Zn2+. The molecular mass of AP-P LA(2)-I was estimated to be 28 kDa by gel filtration and 15 kDa by SDS -PAGE, indicating that AP-PLA(2)-I is a dimer composed of the same sub unit. In contrast, AP-PLA(2)-II was judged to be a monomer with a mole cular mass of 12 kDa (gel filtration) or 15 kDa (SDS-PAGE). The amino acid compositions of the two enzymes were comparable to each other: As x, Glx and Gly were rich in both molecules, while Met, His and Trp wer e poor. Analyses by a sequencer determined the first 62 amino acid res idues for both PLA(2)s. III the AP-PLA(2)-I preparation, minor amino a cids were additionally found at 17 positions, suggesting the coexisten ce of another PLA(2)-component. As compared to the N-terminal sequence s of the known PLA(2)s, both AP-PLA(2)-I and II were identified as cla ss I enzymes not only because they have Cys-11 and lack Cys-51 but als o because they contain the elapid loop in the region 53-61. (C) 1998 E lsevier Science Ltd. All rights reserved.