Interaction of enzymes with mixed micelles of phospholipid and detergent: Analysis of the phospholipase A₂–dipalmitoyl phosphatidylcholine–triton X 100 system

Abstract

Physical studies on the formation and structure of mixed micelles of the nonionic surfactant Triton X-100 and phospholipids and enzymatic studies on the action of phospholipase A, toward these mixed micelles are presented. Results of nmr intensity, line width, and T I determinations, as well as gel chromatography and centrifugation experiments on the interaction of Triton X-100 with egg and dipalmitoyl phosphatidylcholine, are presented and discussed. The structure of mixed micelles is discussed in terms of a working schematic model which is consistent with the experimental results. Kinetic studies on phospholipase A, (Naja naja) action are then analyzed in terms of this model. The temperature dependence of phospholipase A, action toward dipalmitoyl phosphatidylcholine is considered in terms of the effect of thermotropic phase transitions on mixed micelle formation. The phospholipase A, -dipalmitoyl phosphatidylcholine-Triton X-I00 system is then considered as an artificial model system for studying the effect of lipid phase separations on biological activity.For the last few years, our laboratory has been studying the action of phospholipase A, toward phosphatidylcholine as substrate in mixed micelles with the surfactant Triton X-100, employing largely kinetic techniques to characterize the enzyme (1, 2 ) and nuclear magnetic resonance (nmr) techniques t o characterize the substrate (3-5). Our orientation has been that of attempting t o develop a suitable in vitro system for studying the detailed mechanism of action of enzymes of lipid metabolism; these enzymes normally act on substrates that are part of complex structures rather than ones which are molecularly dispersed (6-8). In addition, these studies provide: a) information on the use of and possible effect of Triton and other surfactants (often referred t o as "detergents") in the assay of enzymes that act on or require lipids (9), b) information about the process of protein solubilization, since mixed micelles are formed when surfactants are employed 682 0 1974 Alan R. Liss, Inc.,

Cobra venom phospholipase A2: A review of its action toward lipid/water interfaces

“…in which the phospholipid is imbedded and this allows one to vary the concentration of phospholipid in a lipid-water interface by varying the surfactant to phospholipid ratio (27). The activity of the phospholipase depends directly on this concentration and this should occur if the enzyme binds to the interface in some manner (28) before binding its substrate phospholipid.…”

Investigation on electron transfer from spinach thylakoids to modified electrodes in a photosynthetic electrochemical cell

“…~10nm absorbance blue-shift shows the unbound light harvesting antennas. This observation is in accordance with previous studies that reports the thylakoids stability upon incubation with ≤ 7x10 -3 % (w/w) TX-100 [168,170,171].…”

Interaction and charge transfer between isolated thylakoids and multi-walled carbon nanotubes

“…This observation is in accordance with previous studies that report thylakoid stability upon incubation with r7 Â 10 À3 % (w/w) TX-100. [34][35][36] The dispersed MWCNT absorbance is indicated by gradual absorbance decrease from the UV spectrum toward relatively flat absorbance in the visible spectrum. 37,38 The preserved 680 nm peak on both thylakoids and MWCNTs absorbance profiles suggests that the protein complexes and light harvesting antennas in the thylakoid membrane remain intact upon direct interaction with MWCNTs (Fig.…”