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Using a novel laser-induced ultrasonic probe, we have examined the bulk viscoelastic properties of fully hydrated dipalmitoylphosphatidylcholine (DPPC) aligned multibilayers in terms of the anisotropic in-plane elastic stiffness (C11) and viscosity (eta 11). Our measurements of C11 are in accord with those reported on Brillouin light scattering on a similar system. Our measurements on viscosity are the first of their kind and are, on the average, a factor of 10 lower than microviscosities estimated by spectroscopic techniques. We report the first comprehensive study of the effects of cholesterol on the bulk mechanical properties of DPPC multibilayers. At temperatures above the phase transition temperature of DPPC (Tc), an increase in both C11 and eta 11 is noticed when cholesterol is incorporated in the multibilayers. However, at temperatures below Tc, no measurable changes are detected in either C11 or eta 11. These results, reflecting changes in the bulk viscoelastic properties of the multibilayers, differ from the changes reported by local fluidity parameters in that the latter indicate a decrease in the bilayer fluidity in the presence of cholesterol above Tc and an increase below Tc ("dual effect" of cholesterol). Our data suggest that the "dual effect" of cholesterol is noticeable only on a molecular scale. Increasing cholesterol concentrations higher than 20 mol % cease to further affect C11 or eta 11 of the DPPC multibilayers. This agrees with various results reported in the literature, by techniques measuring the local effects of cholesterol, and supports the changes in molecular organization postulated to occur when cholesterol concentration reaches 20 mol % in the lipid bilayers.

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A simple kinetic model for the enzymatic activity of surface-active proteins against mixed micelles has been developed. This model uses the Langmuir adsorption isotherm, the classic equation for the binding of gas molecules to metal surfaces, to characterize enzyme adsorption to micelles. The number of available enzyme binding sites is equated with the number of substrate and inhibitor molecules attached to micelies; enzyme molecules are attracted to the micelle due to the affinity ofthe enzyme active site for the molecules in the micelle. Phospholipase C (Bacillus cereus) kinetics in a wide variety of dimyristoyl phosphatidylcholine/detergent micelles are readily explained by this model and the assumption of competitive binding of the detergent at the enzyme active site. Binding of phospholipase C to pure detergent micelles is demonstrated by gel filtration chromatography. Phospholipase action toward phospholipid molecules in a surface is much greater than that toward monomeric substrates ["interfacial activation" (1)]. Enzyme-specific activity also depends on the matrix used to form the surface-i.e., detergent mixed micelles (8), short-chain lecithin micelles (9, 10), bilayers (11), monolayers (12). A variety of kinetic models have been applied to these phenomena. The simplest model, applied to snake venom phospholipase A2 action toward short-chain lecithins, proposes normal Michaelis-Menten kinetics and different Vm and Km values for monomeric and micellar lipid with the monomer as a competitive inhibitor of micellar lecithin (13). Another model, proposed for pancreatic phospholipase A2, accounts for interfacial activation by proposing a second site on the enzyme that "anchors" or "recognizes" surfaces (14). Different surface-active molecules can interact differentially with the two sites and hence modulate the activity. These models have not been extended in a systematic fashion to binary or more complex surfaces except in cases in which the added surface molecule is a substrate analogue. The only detailed binary component kinetic model is that ofDennis and co-workers (15). This "surface as cofactor" model was developed for phospholipase A2 and phospholipase C kinetics using Triton X-100/lecithin micelles as substrates. The model is quite complex, requiring estimation of the surface area/head-group ratio and several assumptions (16) to fit observed activities. It is based on surface association of the enzyme followed by substrate binding in the active site to form the Michaelis complex; i.e., two distinct binding steps are involved.To generalize a kinetic model for surface-active enzymes such as the phospholipases, we have examined the action of phospholipase C (Bacillus cereus) toward dimyristoyl phosphatidylcholine (Myr2PtdCho) in mixed micelles with four different detergents: Triton X-100 (nonionic), Zwittergent 3-14 (zwitterionic), deoxycholate (anionic), and trimethylcetylammonium bromide (Me3CetNBr; cationic). The data are interpreted by using a simple model based on the Langmuir adsorption is...

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