Faculty Bibliography

The enzyme phospholipase A2 (PLA2), which catalyzes the hydrolysis of an ester bond at the sn-2 position of 1,2-sn-diacylglycerols, has been suggested to play an important role in regulating cellular functions. Although ethanol (EtOH)-induced activation of PLA2 activity was reported previously by us in mouse brain (Hungund et al., Neurochem Int 25: 321-325, 1994), its subcellular localization and biochemical properties have not been investigated. Therefore, in the present study, we examined the subcellular localization and characterization of EtOH-activated PLA2 activity in mouse brain. The results indicated that EtOH treatment decreased the specific activity of PLA2 for the first 48 hr, and then the activity increased and reached a peak level in both cytosol (1.6-fold) and membrane (1.7-fold) fractions at 96 hr of exposure. Specific activity was found to be higher in the membrane fraction than in the cytosol. Using differential density gradient centrifugation, subcellular localization of the membrane-associated PLA2 revealed that most of the EtOH-activated PLA2 specific activity was associated with the synaptic membrane (44%) followed by the nuclear membrane (13%). No significant increase in the PLA2 specific activity of mitochondrial and microsomal membranes was observed. No activity was detected in the myelin membrane. PLA2 specific activity of membranes from control and EtOH-exposed mouse brain exhibited preference for arachidonic acid over linoleic acid at the sn-2 position of glycero-3-phosphocholine (PC). No detectable PLA2 specific activity was found when PC containing oleic acid at the sn-2 position was used as a substrate. The present results also indicated that the PLA2 specific activity of membrane from control and EtOH-exposed mouse brain was insensitive to dithiothreitol, strongly stimulated by Ca2+, enhanced by glycerol, and inhibited by the cytosolic PLA2 (cPLA2) inhibitor methyl arachidonyl fluorophosphonate with an IC50 value of 3.33 microM. In summary, results suggest that the properties of EtOH-activated PLA2 activity found in mouse brain membrane fraction are similar to those of cPLA2 found in variety of cells, including mammalian brain

Human neuroblastoma cells were exposed to ethanol (EtOH; 100 mM) in culture for various time periods. It was found that chronic EtOH exposure increased the arachidonyl-specific phospholipase A2 (PLA2) activity significantly in both cytosol (1.6-fold) and membrane (2.2-fold) fractions when 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine was used as a substrate. This arachidonyl-specific PLA2 activity progressively increased with increasing duration of EtOH exposure and reached peak level at 72-hr EtOH exposure (chronic). A significant amount of the PLA2 activity was associated with the membrane fraction. No significant difference in PLA2 activity was observed when 1-palmitoyl-2 oleoyl or linoleoyl-sn-glycero-3-phosphocholine was used as a substrate. It was also found that co-treatment of neuroblastoma cells with ganglioside GM1 reduced the EtOH-induced activation of arachidonyl-specific PLA2 activity. The present results indicate that arachidonic acid-specific PLA2 may play a role in adaptation mechanisms to chronic EtOH in cultured neuroblastoma cells. Ganglioside GM1, in part, may exert its neuroprotective effects by modulating arachidonyl-specific PLA2 activity in chronic EtOH-exposed neuroblastoma cells

Rabbit antibodies were prepared against purified phospholipase A2 (NN-XIa-PLA2) from Indian cobra (Naja naja naja) venom. The PLA2 has haemolytic, neurotoxic, myotoxic, cytotoxic and oedema-inducing activities apart from the catalytic activity. The immunological cross-reactivity of structurally similar neurotoxic PLA2s was investigated using enzyme-linked immunosorbent assay (ELISA) and immunodiffusion. Anti-NN-XIa-PLA2 IgG cross-reacted with other purified neurotoxic PLA2s from the same venom. Immunochemical cross-reactions of anti-NN-XIa-PLA2 IgG with NN-XIa-PLA2, NN-XIb-PLA2, NN-XIII-PLA2, NN-IVb1-PLA2 and NN-Vb-PLA2 were shown by a very high ELISA titre and a single precipitin band on double immunodiffusion agarose plates. The catalytic activity of these PLA2s was inhibited dose-dependently by anti-NN-XIa-PLA2 IgG but was unable to neutralize lethality and neurotoxic symptoms in experimental animals injected with neurotoxic PLA2. Anti-NN-XIa-PLA2 IgG fails to neutralize myotoxicity and oedema-inducing activities of NN-XIa-PLA2 and NN-XIII-PLA2. Anti-NN-XIa-PLA2 IgG inhibited cytotoxic effects of NN-XIa-PLA2 dose-dependently, but failed to inhibit NN-XIII-PLA2-induced cytotoxicity. Direct haemolytic activity of NN-XIa-PLA2 and NN-XIII-PLA2 was inhibited dose-dependently by these antibodies. The results indicate the presence of separate catalytic and pharmacologic site(s).

Indian cobra venom contains many phospholipase A2 (PLA2) toxins. In the present study two toxic PLA2s have been purified from the Indian cobra (Naja naja naja) venom by column chromatography. The NN-XIa-and NN-XIb-PLA2s have mol. wts between 10,700 and 15,000. The NN-XIa-PLA2 induces myotoxic effects, oedema and neurotoxicity in mice and has an i.p. LD50 of 8.5 mg/kg body weight. The NN-XIa-PLA2 is also cytotoxic to Ehrlich ascites tumour cells. The other PLA2, NN-XIb, in contrast has an i.p. LD50 of 0.22 mg/kg body weight, and it induces acute neurotoxicity. The NN-XIb-PLA2 is devoid of the other biological activities which are exhibited by NN-XIa-PLA2.