Reversibility of the activation of soluble phospholipase A2 on lipid bilayers: implications for the activation mechanism.
The time-courses of hydrolysis of large vesicles of dipalmitoylphosphatidylcholine were compared using four species of phospholipase A2 (Agkistrodon piscivorus piscivorus, Crotalus adamanteus and Naja naja venoms and porcine pancreatic). In all four cases, the hydrolysis rate suddenly increases 10 to 100-fold at the time (tau) when a specific mole fraction of reaction products has accumulated. The intrinsic fluorescence emission of the three venom enzymes also increases suddenly at time tau. Both the activation and the fluorescence change are reversible with a half-time of about 50 s for the activity and 2 to 6 s for the fluorescence. These reversal rates and the vesicle concentration dependence of tau are considered for monomer and dimer enzyme activation models. Apparently, at least three states of the enzyme exist beyond the initial unbound state: (1) inactive and bound, (2) inactive with high fluorescence and (3) active. The dimer model already contains the necessary number of states but requires that the activation rate be much lower than the reversal rate to account for the vesicle concentration dependence of tau. Success of the monomer model requires an enzyme state additional to those proposed previously. Although these results do not exclude either the monomer or dimer models conclusively, they do impose important constraints on each model.
Bell
J D
JD
Department of Zoology, Brigham Young University, Provo, UT 84602.
Brown
S D
SD
Baker
B L
BL
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000818:Animals; D003435:Crotalid Venoms / Q000201:enzymology; D004546:Elapid Venoms / Q000201:enzymology; D004789:Enzyme Activation; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008433:Mathematics; D008956:Models, Chemical; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D012995:Solubility; D013552:Swine
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D004546:Elapid Venoms; D008051:Lipid Bilayers; D015060:1,2-Dipalmitoylphosphatidylcholine; D010741:Phospholipases A; D054467:Phospholipases A2