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Small bilayer particles form spontaneously from gel-state long-chain phospholipids such as dipalmitoylphosphatidylcholine and 0.2 mol fraction short-chain lecithins (e.g., diheptanoyl-phosphatidylcholine). When the particles are incubated at temperatures greater than the Tm of the long-chain phosphatidylcholine (PC), the particles rapidly fuse (from 90-A to greater than or equal to 5000-A radius); this transition is reversible. A possible explanation for this behavior involves patching or phase separation of the short-chain component within the gel-state particle and randomization of both lipid species above Tm. Differential scanning calorimetry, 1H T1 values of proteodiheptanoyl-PC in diheptanoyl-PC-d26/dipalmitoyl-PC-d62 matrices of varying deuterium content, solid-state 2H NMR spectroscopy as a function of temperature, and fluorescence pyrene excimer-to-monomer ratios as a function of mole fraction diheptanoyl-PC provide evidence that such phase separation must occur. These results are used to construct a phase diagram for the diheptanoyl-PC/dipalmitoyl-PC system, to propose detailed geometric models for the different lipid particles involved, and to understand phospholipase kinetics toward the different aggregates.

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Several short-chain asymmetric lecithins with a total of 14 carbons in the acyl chains (ranging from 1-lauroyl-2-acetylphosphatidylcholine to 1-hexanoyl-2-octanoylphosphatidylcholine) have been synthesized and characterized. The specific activities of phospholipase A2 from cobra venom, phospholipase A2 from porcine pancreas, and phospholipase C from Bacillus cereus toward these lecithins as micelles have been determined. The results of these kinetic studies allow the definition of hydrophobic binding requirements in the active sites of these water-soluble phospholipases. For phospholipase C, with the exception of monomyristoylphosphatidylcholine, each of the asymmetric short-chain lecithins exhibits high activity, comparable to the 14-carbon symmetric short-chain species, diheptanoylphosphatidylcholine. Therefore, for phospholipase C, in addition to the acyl linkages, only a certain degree of hydrophobicity in the fatty acyl chains is requisite for substrate binding and appreciable hydrolysis; there is no chain specificity. The activity of phospholipase A2 from cobra venom toward the same asymmetric lecithins is quite different. As the sn-2 chain lengthens, activity is increased to a maximum for diheptanoyl-PC. Further increase in the number of carbons in the sn-2 chain has no effect on hydrolysis rates. For this enzyme, seven carbons in the sn-2 chain are necessary for optimal activity. In contrast, porcine pancreatic phospholipase A2 activity shows very little dependence on sn-2 chain length.

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