Proton magnetic resonance relaxation studies on the structure of mixed micelles of Triton X-100 and dimyristoylphosphatidylcholine.

Proton magnetic resonance and gel chromatographic studies on mixtures of phospholipid and the nonionic surfactant Triton X-200 have shown that at temperatures above the thermotropic phase transition of the phospholipid and below the cloud point of Triton, mixed micelles are present at molar ratios above about 2:1 Triton/phospholipid. Proton T1 and T2 (from line widths) relaxation times are reported for protons in Triton micelles and in mixed micelles of Triton and dimyristoylphosphatidylcholine at a molar ratio of 3:1 Triton/phospholipid. The T1 values and their temperature dependence and the activation energies of the various Triton proton groups appear to reflect internal motions of the Triton molecules in the micelle. Measurements of the T1/T2 ratio and frequency dependence (55-220 MHz) suggest that the hydrophobic tert-butyl group in Triton is observed under extreme narrowing conditions. The T1 and T2 values of Triton are unchanged in the presence of phosphatidylcholine. The T1 values of various protons of dimyristoylphosphatidylcholine in mixed micelles are similar to those reported for the phospholipid in sonicated vesicles, which are used as membrane models, and presumably the same coupled trans-gauche motions dominate. The T2 values for the terminal methyl and choline methyl protons in the phospholipid are longer than those reported for these groups in vesicles. Hence, the motion of the phospholipid in the mixed micelles appears to be less restricted than in vesicles. T1 measurements in H20/D20 mixtures are consistent with the idea that water does not penetrate the hydrophobic core of the mixed micelles, while water does solvate the polar oxyethylene and choline methyl groups. Titration with Mn2+ confirms that the oxyethylene and choline methyl groups are on the exterior of the mixed micelle while the hydrophobic groups are located in the micellar interior.     

Formation and characterization of mixed micelles of the nonionic surfactant Triton X-100 with egg, dipalmitoyl, and dimyristoyl phosphatidylcholines

Mixed micelles of the nonionic surfactant Triton X-100 and egg phosphatidylcholine were isolated by column chromatography on 6% agarose and by centrifugation at 35,000g. It was found that egg phosphatidylcholine bilayers are able to incorporate Triton X-100 at molar ratios of Triton to phospholipid below about 1:1, whereas above a molar ratio of about 2:1 Triton/phospholipid all of the phospholipid is converted into mixed micelles. Mixed micelles at a molar ratio of about 10:1 Triton/phospholipid were found to be in the same size range as pure micelles of Triton X-100. The formation of mixed micelles with dipalmitoyl phosphatidylcholine at room temperature, when the phospholipid is below its thermotropic phase transition, is shown to require relatively high concentrations of Triton X-100. The point at which dimyristoyl phosphatidylcholine bilayers are converted to mixed micelles was found to be less clear cut than with egg phosphatidylcholine, but above a molar ratio of about 2:1 Triton/phospholipid, all of this phospholipid is also in mixed micelles. The relevance of these results to the solubilization of membrane-bound proteins with Triton X-100 and the action of phospholipase A₂, which hydrolyzes phosphatidylcholine when it is in mixed micelles with Triton X-100, is discussed.     

Phospholipase A2 activity towards phosphatidylcholine in mixed micelles: surface dilution kinetics and the effect of thermotropic phase transitions

Phospholipase A₂ will act on dipalmitoyl phosphatidylcholine as substrate when the phospholipid is part of a mixed micelle with Triton X-100 at a molar ratio of Triton to phospholipid of 2:1 or greater. Kinetic studies at high molar ratios of Triton X-100 to phospholipid are reported and show that the binding of phospholipase A₂ to substrate depends on the total concentration of Triton X-100 and phospholipid, but that the rate of enzymatic catalysis decreases proportionally to the Triton X-100 concentration. These results are interpreted in terms of a model involving surface dilution kinetics. The relationship of this model to that of competitive inhibition is discussed. In addition, the activity of phospholipase A₂ towards dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine at different temperatures is reported, and the results show a direct effect of the thermotropic phase transition of dipalmitoyl phosphatidylcholine on enzymatic activity.     

Studies on mixed micelles of triton X–100 and phosphatidylcholine using nuclear magnetic resonance techniques

The addition of the nonionic detergent Triton X-100 to aqueous phosphatidyl-choline dispersions converts the bilayer structures to mixed micellar structures containing Triton X-100. High-resolution nuclear magnetic resonance spectroscopy at 220 MHz was used to follow this conversion, and the general spectral characteristics of the mixed micelles are presented. The results are discussed in terms of the precise change in structure which occurs as Triton is mixed with the phospholipid bilayers, and it is concluded that, above a molar ratio of about 2:1 Triton to phospholipid, most or all of the phospholipid is in mixed micelles. The relevance of these results to the study of enzymes which require substrate in the form of micelles is discussed.     

Mixed micelles of sphingomyelin and phosphatidylcholine with nonionic surfactants. Effect of temperature and surfactant polydispersity.

Mixed micelle formation of the polydisperse nonionic surfactant Triton X-100 as well as its homogeneous analogue, p-(1,1,3,3-tetramethylbutyl)-phenoxynonaoxyethylene glycol (OPE-9), with bovine brain sphingomyelin or dipalmitoyl phosphatidylcholine has been characterized by column chromatography on 6% agarose. At 40 degrees C, mixtures of OPE-9 and either sphingomyelin or dipalmitoyl phosphatidylcholine give a narrow size distribution for mixed micelles. A this temperature the size distribution of Triton X-100-containing mixed micelles is complicated because of the polydispersity of the oxyethylene chains. At 20 degrees C narrow size distributions are observed for mixed micelles of sphingomyelin/Triton X-100 and sphingomyelin/OPE-9 up to at least 0.06 mol fraction of lipid. For dipalmitoyl phosphatidylcholine this is observed only with OPE-9. At intermediate mol fractions of lipid (around 0.25), two populations of mixed micelles exist for sphingomyelin/Trition X-100, sphingomyelin/OPE-9, and dipalmitoyl phosphatidylcholine/OPE-9. At high mol fractions of lipid only one population of mixed micelles again exists. At 20 degrees C, sphingoymelin forms a clear solution with Triton X-100 and OPE-9 to a lipid mol fraction of at least 0.46 and 0.67, respectively. Dipalmitoyl phosphatidylcholine forms a clear solution with OPE-9 to a lipid mol fraction of at least 0.57 at the same temperature. Triton X-100 and dipalmitoyl phosphatidylcholine do not form stable, clear solutions at 20 degrees C unless the lipid mol fraction is extremely low. These results show that surfactant polydispersity and temperature are important determinants in the solubilization of lipids by nonionic surfactants. It is also shown that pure surfactant micelles and lipid/surfactant mixed micelles do not co-exist in the same solution.     

Lipid exchange between mixed micelles of phospholipid and triton X-100

If phospholipase catalyzed hydrolysis of phospholipid dissolved in a detergent mixed micelle is limited to the phospholipid carried by a single micelle, then hydrolysis ceases upon exhaustion of that pool. However, if the rate of phospholipid exchange between micelles exceeds the catalytic rate then all of the phospholipid is available for hydrolysis. To determine phospholipid availability we studied the exchange of 1,2-dioleoyl-sn-glycero-3-phosphocholine between mixed micelles of phospholipid and non-ionic Triton detergents by both stopped-flow fluorescence-recovery and nuclear magnetic resonance-relaxation techniques. Stopped-flow analysis was performed by combining mixed micelles of Triton and phospholipid with mixed micelles that contained the fluorescent phospholipid 1-palmitoyl-2-(12-[{7-nitro-2-1, 3-benzoxadiazo-4-yl}amino]dodecanoyl)-sn-glycero-3-phosphocholine (P-2-NBD-PC). The concentration dependence of fluorescence recovery suggested a second-order exchange mechanism that was saturable. The true second-order rate constant depends on the specific mechanism for exchange, which was not determined in this study, but the rate constant will be on the order of 106 to 107 M-1s-1. Incorporation of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine into micelles increased the rate of proton relaxation and gave a limiting relaxation time of 1.3 ms. The results demonstrate that phospholipid exchange was rapid and that the phospholipid content of a single micelle did not limit the rate of phospholipid hydrolysis by phospholipases.     

Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine

A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.     

1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine, a highly specific chromogenic substrate of phospholipase A2

1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine (2-thioPC), a structurally modified phospholipid analog is specifically hydrolyzed by phospholipase A2 to liberate 2-thiolysophosphatidylcholine and palmitic acid. The sulfhydryl group of the product is readily trapped by 5,5'-dithiobis (2-nitrobenzoic acid) allowing continuous spectrophotometric monitoring of the enzymatic reaction. The rates of hydrolysis by bee-venom phospholipase A2 have been determined in a series of Triton X-100 containing mixed micelles. At 1 mM 2-thioPC increasing the concentration of Triton X-100 from 4 to 16 mM changes the specific activity of bee-venom phospholipase A2 from 96.9 to 17.9 mumol/min/mg, about one order of magnitude lower than dipalmitoyl phosphatidylcholine hydrolysis in micelles of similar composition. The chromogenic substrate is the first phospholipid analog exhibiting absolute specificity for phospholipase A2 and should be applicable to spectrophotometric detection and kinetic characterization of both water soluble and membrane-bound forms.     

Interaction with phospholipids of a membrane thiol peptidase that is essential for the signal transduction of mating pheromone in Rhodosporidium toruloides

Interaction with phospholipids of a membrane thiol peptidase [referred to as trigger peptidase (TPase), T. Miyakawa et al. (1987) J. Bacteriol. 169, 1626-1631] that plays a key role in the signalling of a lipopeptidyl mating pheromone at the cell surface of pheromone-target cell (mating type a) of Rhodosporidium toruloides was studied. The activity of highly purified TPase which requires phospholipids was restored by reconstitution of the enzyme into liposomes prepared with phospholipids extracted from the yeast cell. The presence of Ca2+ was essential for both the reconstitution process and the catalytic reaction of TPase. Triton X-100 mixed micelles containing phospholipids also activated the enzyme. The specificity and stoichiometry of activation by phospholipids was investigated by determination of TPase in the presence of mixed micelles that contained defined classes and numbers of phospholipid molecules in the Triton X-100 micelles. It was demonstrated that TPase is activated by mixed micelles containing 2-6 molecules of phosphatidylserine or phosphatidylethanolamine. Other phospholipids of the membranes of this organism, such as phosphatidylcholine and phosphatidylglycerol, had little effect on activation, indicating that the amino group of the phospholipids may be required for the function of TPase. Direct evidence for the interaction of TPase and Triton X-100/phosphatidylserine mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data established that a phospholipid bilayer is not a requirement for TPase activation, and that the purified enzyme can be activated by a relatively small number of phospholipid molecules of specific classes.     

Kinetic analysis of the dual phospholipid model for phospholipase A2 action

A kinetic scheme is proposed for the action of cobra venom phospholipase A2 on mixed micelles of phospholipid and the nonionic detergent Triton X-100, based on the "dual phospholipid model." (formula; see text) The water-soluble enzyme binds initially to a phospholipid molecule in the micelle interface. This is followed by binding to additional phospholipid in the interface and then catalytic hydrolysis. A kinetic equation was derived for this process and tested under three experimental conditions: (i) the mole fraction of substrate held constant and the bulk substrate concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied (surface concentration of substrate varied); and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. The substrates used were chiral dithiol ester analogs of phosphatidylcholine (thio-PC) and phosphatidylethanolamine (thio-PE), and the reactions were followed by reaction of the liberated thiol with a colorimetric thiol reagent. The initial binding (Ks = k1/k-1) was apparently similar for thio-PC and thio-PE (between 0.1 and 0.2 mM) as were the apparent Michaelis constants (Km = (k-2 + k3)/k2) (about 0.1 mol fraction). The Vmax values for thio-PC and thio-PE were 440 and 89 mumol min-1 mg-1, respectively. The preference of cobra venom phospholipase A2 for PC over PE in Triton X-100 mixed micelles appears to be an effect on k3 (catalytic rate) rather than an effect on the apparent binding of phospholipid in either step of the reaction.     

Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles

Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear. Tryptophanyl fluorescence quenching by acrylamide shows that a significant fraction of the Trp residues sensing the quencher become less readily available to it in the absence of surfactant. The temperature dependence of enzyme activity (expressed in the form of Arrhenius plots) is also different in the presence and absence of detergent. The effects of surfactant removal do not appear to be readily reversible upon readdition of Triton X-100.     

Hydrolysis of lipid mixtures by rat hepatic lipase

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of phospholipase D in reverse micelles of Triton X-100 and phosphatidylcholine in diethyl ether

Summary Phospholipase D, from cabbage, is active in reverse micelles formed from its substrate phosphatidylcholine and Triton X-100 in diethyl ether. The activity is optimum at w₀=12.5. The increase of the molar ratio of Triton X-100/substrate from 1:4 to 2:1 results in an activity decrease by 25 %. At 136 mM Triton X-100 the K_M value in reverse micelles is 136 mM, whereas it is 0.40 mM in the aqueous system containing SDS.     

Specificity reversal in phospholipase A2 hydrolysis of lipid mixtures

The activity and specificity of phospholipase A₂ from cobra venom ($\text{Naja naja naja}$) toward binary mixtures of phosphatidylcholine and phosphatidylethanolamine in mixed micelles with the nonionic surfactant Triton X-100 were examined. In mixtures containing 5–50 mol % phosphatidylcholine, the rate for phosphatidylethanolamine hydrolysis was enhanced greatly over that for phosphatidylcholine. This is in marked contrast to previous studies with individual phospholipid species in mixed micelles where phosphatidylcholine was found to be the preferred substrate and phosphatidylethanolamine was found to be a very poor substrate. Possible explanations for this specificity reversal are considered.     

Deuterium nuclear magnetic resonance studies of bile salt/phosphatidylcholine mixed micelles

Mixed micelles of deoxycholate (DOC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) have been prepared in which the POPC was specifically deuterated in the 2-, 6-, 10-, or 16-position of the palmitoyl chain or in the N-methyl position of the choline head group. The deuterium nuclear magnetic resonance (2H NMR) spectrum of each of these specifically deuterated mixed micelles consists of a singlet whose line width depends upon the position of deuteration. Spin-spin relaxation times indicate a gradient of mobility along the POPC palmitoyl chain in the mixed micelle, with a large increase in mobility on going from the 10- to the 16-position. Spin-lattice relaxation times (T1's) demonstrate a similar gradient of mobility. Both trends in NMR relaxation behavior are consistent with a bilayer arrangement for the solubilized POPC. 2H T1 times for DOC/POPC micelles are significantly shorter than those measured in other bilayer systems, indicating unusually tight phospholipid acyl chain packing in the mixed micelle.     

Glycosyl-phosphatidylinositol anchored acetylcholinesterase as substrate for phosphatidylinositol-specific phospholipase C from Bacillus cereus.

We investigated the enzymatic properties of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus towards glycosyl-phosphatidylinositol anchored acetylcholinesterase (AChE) from bovine erythrocytes and Torpedo electric organ as substrate. The conversion of membrane from AChE to soluble AChE by PI-PLC depended on the presence of a detergent and of phosphatidylcholine. In presence of mixed micelles containing Triton X-100 (0.05%) and phosphatidylcholine (0.5 mg/ml) the rate of AChE conversion was about 3 times higher than in presence of Triton X-100 alone. Furthermore, inhibition of PI-PLC occurring at Triton X-100 concentrations higher than 0.01% could be prevented by addition of phosphatidylcholine. Ca2+, Mg2+ and sodium chloride had no effect on PI-PLC activity in presence of phosphatidylcholine and Triton X-100, whereas in presence of Triton X-100 alone sodium chloride largely increased the rate of AChE conversion. Determination of kinetic parameters with three different substrates gave Km-values of 7 microM, 17 microM and 2 mM and Vmax-values of 0.095 microM.min-1, 0.325 microM.min-1 and 56 microM.min-1 for Torpedo AChE, bovine erythrocyte AChE and phosphatidylinositol, respectively. The low Km-values for both forms of AChE indicated that PI-PLC not only recognized the phosphatidylinositol moiety of the anchor but also other components thereof.     

GM1-ganglioside-triton X-100 mixed micelles: Changes of micellar properties studied by laser-light scattering and enzymatic methods

The micellar properties of mixtures of GM1 ganglioside and the non-ionic amphiphile Triton X-100 in 25 mM Na phosphate-5 mM di Na EDTA buffer (pH = 7.0) were investigated by quasielastic light scattering in a wide range of Triton/GM1 molar ratios and in the temperature range 15–37°C. These measurements: (a) provided evidence for the formation of mixed micelles; (b) allowed the determination of such parameters as the molecular weight and the hydrodynamic radius of the mixed micelles; (c) showed the occurrence of statistical aggregates of micelles with increasing temperature and micelle concentration. Galactose oxidase was chosen for studying the relation between enzyme activity and micellar properties. The action of the enzyme on GM1 was found to be strongly dependent on the micellar structure. In particular: (a) galactose oxidase acted very poorly on homogeneous GM1 micelles, while affecting mixed GM1/Triton X-100 micelles; (b) at fixed GM1 concentration the oxidation rate increased by enhancing Triton X-100 concentration and followed a biphasic kinetics with a break at a certain Triton X-100 concentration; (c) the formation of statistical micelle aggregates was followed by inhibition of the enzyme activity.     

Characterization of mixed micelles of phospholipids of various classes and a synthetic,homogeneous analogue of the nonionic detergent triton X-100 containing nine oxyethylene groups

The synthesis and high-pressure liquid chromatographic purification of the homogeneous nonionic surfactant $\text{p-}\text{(1,1,3,3-tetramethylbutyl)phenoxynonaoxyethylene}$ glycol (OPE-9) in quantities suitable for membrane solubilization studies is reported. Micelles of OPE-9 and mixed micelles of OPE-9 with dimyristoyl and dipalmitoyl phosphatidylcholine as well as phosphatidylserine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, and palmitic acid were characterized by column chromatography on 6% agarose. It was found that at 28°C OPE-9 micelles have a Stokes' radius of 32 Å, giving a molecular weight for a spherical micelle of about half that of micelles of the polydisperse nonionic surfactant Triton X-100 under the same conditions. The micelle size is temperature dependent: at 40°C the OPE-9 micelles have a Stokes' radius of 44 Å, giving a molecular weight for a spherical micelle of about twice that of the OPE-9 micelles at 28°C. The size of the mixed micelles varies linearly (as measured by $\text{K}{}_{\mathrm{<mtext>av</mtext>}}$) with the mole fraction of phospholipid. The mixed micelle size was found to be relatively independent of the absolute concentration of surfactant over a four-fold range if the mole fraction of phospholipid is kept constant. The usefulness of the OPE-9/phospholipid mixed micelle system for lipolytic enzyme substrates and membrane-related studies is considered.     

Analysis of phospholipase C (Bacillus cereus) action toward mixed micelles of phospholipid and surfactant.

The action of phospholipase C (Bacillus cereus) toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 is analyzed according to the “surfaceas-cofactor” kinetic scheme recently proposed for characterizing the action of lipolytic enzymes [Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem.250, 9013–9020]. According to this scheme, the enzyme first associates with the surface or mixed micelles, where the dissociation constant is K_s^A. The enzyme, now part of the mixed micelle surface, then binds the substrate phospholipid molecule in its active site and this binding is related to the Michaelis constant, K_m^B. The surface, or mixed micelles in this scheme, behaves kinetically as a cofactor in that, under initial rate conditions, the surface properties of the mixed micelles are virtually unchanged after catalysis. For phospholipase C with egg phosphatidylcholine as substrate, it was found that at pH 6.4 (the pH optimum for the enzyme) and 40 °C, V is about 2 × 10³ μmol min^?1 (mg of protein)^?1. K_s^A is about 2 mm and K_m^B is 1 to 2 × 10^?10 mol cm^?2. The kinetic constants for phospholipase C are compared with those previously reported for phospholipase A₂ and the membrane-bound enzyme phosphatidylserine decarboxylase determined under similar conditions.     

Protein kinase C activation in mixed micelles. Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies

The phospholipid, sn-1,2-diacylglycerol, and calcium dependencies of rat brain protein kinase C were investigated with a mixed micellar assay (Hannun, Y., Loomis, C., and Bell, R.M. (1985) J. Biol. Chem. 260, 10039-10043). Protein kinase C activity was independent of the number of Triton X-100, phosphatidylserine (PS), and sn-1,2-dioleoylglycerol (diC18:1) mixed micelles. Activation was strongly dependent on the mole per cent of PS and diC18:1. Activity of protein kinase C was dependent on PS, diC18:1, and calcium in mixed micelles prepared from detergents other than Triton X-100. This is consistent with the micelle providing an inert surface into which the lipid cofactors partition. Molecular sieve chromatography provided direct evidence for the homogeneity of Triton X-100, PS, and diC18:1 mixed micelles. Mixing studies and surface dilution studies indicated that PS and diC18:1 rapidly equilibrate among the mixed micelles. At saturating calcium, the diC18:1 dependence was strongly dependent on the mole per cent PS present. At 10 mol % PS, 0.25 mol % diC18:1 gave maximal activity whereas 6 mol % PS and 6 mol % diC18:1 did not give maximal activity. diC18:1 dependencies were hyperbolic at all PS levels tested. The data support the conclusion that a single molecule of diC18:1/micelle is sufficient to activate monomeric protein kinase C. The mole per cent PS required for maximal activation was reduced markedly as the mole per cent diC18:1 increased. Under all conditions tested, the PS dependence of protein kinase C activation lagged until greater than 3 mol % PS was present. Then activation occurred in a cooperative manner with Hill numbers near 4. These data indicate that 4 or more molecules of PS are required to activate monomeric protein kinase C. PS was the most effective of all the phospholipids tested in the mixed micelle assay. diC18:1 was found to modulate the amount of calcium required for maximal activity. As the level of Ca2+ increased, the mole per cent PS required reached a limiting value of 3 mol %. A number of sn-1,2-diacylglycerols containing short chain fatty acids activated protein kinase C in a saturable manner in mixed micelles. The data are discussed in relation to a model for protein kinase activation.