]PLA 2 that has much stronger lipolytic activity than BPII, failed to induce cell death. BPIItreated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPIIinduced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive pbromophenacylated BPII induced cell death, BPIIinduced apoptotic cell death is independent of PLA 2 lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.Key words: Protobothrops flavoviridis; [Lys 49 ]phospholipase A 2 ; caspase-independent cell death Snake venoms are complex mixtures of a number of physiologically active proteins including metalloproteases, 1,2) thrombin-like enzymes, 3,4) complement activating enzyme, 5) phospholipases A 2 (PLA 2 s) [EC 3.1.1.4] 6,7) and so on. PLA 2 catalyzes the hydrolysis of 1,2-diacyl-3-sn-phosphoglycerides at the C2-position with a requirement for Ca 2þ . 8) PLA 2 s are abundant in the mammalian pancreas and also in reptile and insect venoms. Snake venoms contain plural PLA 2 isozymes, which show a wide variety of physiological activities, including induction of edema, 9) neurotoxicity, 10) and myotoxic activity.11) Snake-venom PLA 2 isozymes are classified into groups I and II based on the mode of disulfide pairing. 13,14) It is well established that these PLA 2 isozymes have the same scaffold. 15,16) Protobothrops (formerly Trimeresurus) flavoviridis (Habu snake, Crotalinae) inhabit southwestern islands of Japan, Amami-Oshima, Tokunoshima, and Okinawa. Five PLA 2 isozymes have been isolated from P. flavoviridis (Amami-Oshima Island, Japan) venom. [17][18][19][20][21] They all consist of 122 amino acid residues and have been well studied: 22) It is known that BPII is 10-100 times more active than BPI in contracting activity in the guinea pig ileum and artery. 23) In the study reported here, we examined the cell death activity of P. flavoviridis venom PLA 2 isozymes BPII and PLA2. It was found that BPII can induce caspaseindependent cell death in human leukemia cells regardless of its depressed enzymatic activity, but PLA2, much more active and the most abundant [Asp 49 ]PLA 2 in P. flavoviridis venom, does not induce cell death. The characteristics and mechanism of cell death induced by BPII were investigated.y To whom correspondence should be addressed. Fax: +81-96-326-5048; E-mail: naoko@ph.sojo-u.ac.jp Abbreviations: Ac, acetyl; AcD, actinomycin D; AMC, 7-amino-4-methylcoumarin; pBPB, p-bromophenacyl bromide; CK, creatine kinase; CM, S-carboxymethyl; FITC, fluorescein isothiocyanate; fmk, fluoromethyl ketone; HRP, horseradish peroxidase; FSC, forward scatter; LDH, lactate dehydrogenase; OMe, methoxy; PARP, poly(ADP-ribo...