Abstract
Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate depletion becomes significant. The reaction can be divided phenomenologically into two phases: a latency phase where the amount of product formed is a square function of time (P(t) = At2) and a phase distinguished by a sudden increase in activity. The parameter A, which describes the activation rate of the enzyme during the initial phase in a quantitative fashion, increases with increasing [PLA2], decreasing [PC], decreasing vesicle size, and increasing relative cholate content of the vesicles. The effect of [PLA2] and [PC] on the hydrolysis reaction is similar to that found with pure DPPC unilamellar vesicles in their thermotropic phase transition region. The effect of cholate on the hydrolysis reaction is similar to that of temperature variation within the phase transition of temperature variation within the phase transition of DPPC. These results are consistent with our previously proposed model, which postulates that activation of PLA2 involves dimerization of the enzyme on the substrate surface and that the rate of activation is directly proportional to the magnitude of lipid structural fluctuations. It is suggested that large structural fluctuations, which exist in the pure lipid system in the phase transition range, are introduced into liquid crystalline vesicles by the presence of cholate and thus promote activation of the enzyme.
Highlights
0.27 is similar to that of the mixed micelles
Pancreatic phospholipase AP is a water-soluble enzyme responsible for the hydrolysis of water-insoluble phospholipids that exist as micelles, bilesalt-containing lipid bilayers, or triglyceride-containing emulsion particles in the gastrointestinal tract
P, appears to increase slightly with increasing vesicle size (Fig. 5 B ) while the maximal rate of hydrolysis remains unchanged. These results are consistent with the observation that gel state small unilamellar vesicles (E, = 11 nm) made of DPPC activate PLA2 very rapidly whereas large unilamellar vesicles ( R h = 35 nm) in the gel state do not activatePLAz within any reasonable time
Summary
Where K is the partition coefficient of cholate between the vesicles P L A 2 Hydrolysis of Micelles-Hydrolysis of PC in mixed and theaqueous medium. The initial rate of hydrolysis is a. In the range of Re values between 0.30 and 0.40 vesicles and micelles coexist. All mixed micelle and vesicle dispersions were made from mixedmicellar stock solutions containing 100 mM PC and 90.5-105.5 mM cholate. These stock solutions were prepared from solutions of the appropriate amount of cholate and PC in a mixture of chloroform monotonicallyincreasingfunction of [PLAz] and [PC] as shown in Fig. The initial rate of hydrolysis appears t o be a decreasing function of the effective ratio of cholate toPC. For example the initial rateobtained at Re = 2.50 is onlya third of the rate obtained at Re values inthe range of 0.60 t o 1.00(Fig. IC).
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