|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1992 : ¥1,300,000 (Direct Cost : ¥1,300,000)
1. Freshly prepared extracts of bovine aorta contained a minimal phospholipase A (PLA) activity towards either various combinations of different phospholipids and detergents or phospholipid dispersions. However, incubation of the extracts at 30ﾟC under sterile conditions led to a gradual increase in Ca^<2+>-dependent PLA activity towards phosphatidylethanolamine (PE) dispersions after a 6-hour lag, and it reached a maximum after 48h. This activation of PLA activity absolutely required the presence of Ca^<2+> ions. Other divalent cations, Sr^<2+> and Mg^<2+> also were effective in this order, but to a less extent than Ca^<2+> ions. Guanine nucleotides, GTP, GMP, cGMP, and guanosine, enhanced the activation rate, whereas ATP and cAMP suppressed it. Interestingly, NO-forming reagents, NaN_3, NaNO_2, and isosorbide dinitrate, inhibited the activation. Serine protease inhibitors markedly inhibited the activation, suggesting the activation mechanism through the limited proteolysis of an inactive prophospholipase A.
2. We purified to near homogeneity bovine aorta PLA, which had been previously activated at 4ﾟC, through the sequential use of Q-Sepharose, phenyl-Sepharose, Sepharose 4B, and Cosmosil 5C_8-300 chromatographies. The purified enzyme was characterized as follows : a, The enzyme prefer PE dispersions over other classes of phospholipid. Anionic detergent inhibited the enzyme activity. : b, The molecular mass of the enzyme was found to be about 30 kDa. c, Calcium ions were essential for activity. d, Fatty acids were released from both sn-1 and sn-2 positions of mixed acyl phospholipids by the enzyme action. e, Bovine aorta PLA was immunochemically distinct from group I, and II phospholipases A_2.