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.     

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.     

PMR relaxation times of micelles of the nonionic surfactant Triton X-100 and mixed micelles with phospholipids.

Previous pmr studies at 220 MHz have led to the suggestion that phosphatidylcholine and the nonionic surfactant Trition-X-100 form mixed micellar structures at high molar ratios of trition to phosphalipid. These mixed micelles provide one form of the phospholipid which the enzyme phospholipase A2 can utilize as substrate. Spin-lattice relaxation times (T1) and spin-spin relaxation times (T2) obtained from line widths for resolvable protons in Triton X-100 micelles and mixed micelles with egg phosphatidycholine and dipalmitoyl phosphatidylcholine are reported. They suggest that the structure of the mixed micelles is generally similar to that of pure Triton X-100 micelles. The T1 values for the phsopholipid in the mixed micelles are found to be similar to those reported for phospholipid in sonicated vesicle preparations which are used as membrane models, but the lines are somewhat sharper suggesting the possibility of less anisotropic motion in the mixed micelles than in the vesicles.     

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetone-extraction or by digestion with phospholipase A₂ or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A₂. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipiddeficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes.These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.     

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.     

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.     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.     

Partial purification and characterization of phosphatidic acid-specific phospholipase A1 in porcine platelet membranes

We have shown previously that the phospholipase A (PLA) activity specific for phosphatidic acid (PA) in porcine platelet membranes is of the A₁ type (PA-PLA₁) [J. Biol. Chem. 259 (1984) 5083]. In the present study, the PA-PLA₁ was solubilized in Triton X-100 from membranes pre-treated with 1 M NaCl, and purified 280-fold from platelet homogenates by sequential chromatography on blue-Toyopearl, red-Toyopearl, DEAE-Toyopearl, green-agarose, brown-agarose, polylysine-agarose, palmitoyl-CoA-agarose and blue-5PW columns. In the presence of 0.1% Triton X-100 in the assay mixture, the partially purified enzyme hydrolyzed the acyl group from the sn-1 position of PA independently of Ca²⁺ and was highly specific for PA; phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) were poor substrates. The enzyme exhibited lysophospholipase activity for l-acyl-lysoPA at 7% of the activity for PA hydrolysis but no lipase activity was observed for triacylglycerol (TG) and diacylglycerol (DG). At 0.025% Triton X-100, the enzyme exhibited the highest activity, and PA was the best substrate, but PE was also hydrolyzed substantially. The partially purified PA-PLA₁ in porcine platelet membranes was shown to be different from previously purified and cloned phospholipases and lipases by comparing the sensitivities to a reducing agent, a serine-esterase inhibitor, a PLA₂ inhibitor, a Ca²⁺-independent phospholipase A₂ inhibitor, and a DG lipase inhibitor.     

Kinetic Analysis in Mixed Micelles of Partially Purified Rat Brain Phospholipase D Activity and its Activation by Phosphatidylinositol 4,5-Bisphosphate

A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of l-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino]caproyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂), and the curve of activation versus PIP₂ molar ratio fitted a Michaelis-Menten equation with a K_act value between molar ratios of 0.001–0.002. Maximal activation was observed at a PIP₂ molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP₂ was shown to elevate by 6–22 fold the specificity constant of PLD towards NBD-PC (K_A, which is proportional to V_max/K_m). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, 1-propanol and 1-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While V_max values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and K_m values for ethanol, 1-propanol and 1-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K_A) towards ethanol, 1-propanol and 1-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. 1-Propanol and 1-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP₂, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.     

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.     

Pancreatic porcine phospholipase A2 catalyzed hydrolysis of phosphatidylcholine in lecithin-bile salt mixed micelles: kinetic studies in a lecithin-sodium cholate system

Pancreatic porcine phospholipase A2 catalyzed hydrolysis of phosphatidylcholine in bile salt lecithin mixed micelles has been studied, utilizing a series of assay mixtures for which the micellar size, weight, and composition had been experimentally determined. Under these conditions the enzymatic hydrolysis is dependent on the phosphatidylcholine-to-sodium cholate molar ratio within the mixed micelle rather than the bulk concentration of the phospholipid in the mixture: at 5 mM phosphatidylcholine, variation of the NPC/NNaCh ratio from 0.2 to 2.0 increases the enzymatic activity from 82 to 933 mumol/min/mg protein. The initial rates are linear throughout the entire series of assay mixtures, the activity vs micellar concentration curves exhibit saturation behavior, and treatment of the data according to the "surface-as-cofactor" theory provides linear double-reciprocal plots which intersect in one point. The assay system should be applicable for detailed kinetic studies of lipolytic enzymes, including mammalian phospholipases which exhibit rather low activities toward lecithin-Triton X-100 mixed micelles. The system should also provide a convenient basis for mechanistic studies involving the use of inhibitory phospholipid substrate analogs.     

Effect of the lipid environment on protein motion and enzymatic activity of sarcoplasmic reticulum calcium ATPase.

In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca2+ -ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components. Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. Solubilization of the lipid-replaced enzyme with Triton X-100 reverses all three of these effects. Ten millimolar CaCl2 added either to the enzyme associated with the endogenous lipids or to the Triton X-100 soulbilized enzyme inhibits both ATPase activity and protein rotational mobility but has no detectable effect on the lipid mobility. These results are consistent with the proposal that both lipid fluidity and protein rotational mobility are essential for enzymatic activity.     

Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells

We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 μM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 μM inhibited 100 % of the nuclear-associated enzyme activity. The K_m for ATP was estimated to be between 36 and 40 μM. The nuclear-associated PI kinase activity was inhibited by both 50 μM ADP and 10 μM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A₂ and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast.     

Role of the carbohydrate moiety of phospholipase B from Penicillium notatum in enzyme activity

Two types of phospholipase B from Penicillium notatum—the native enzyme and enzyme modified by endogenous protease (T. Okumura, S. Kimura, and K. Saito (1980) Biochim. Biophys. Acta, 617, 264–273)—were treated with endoglycosidase H (endo-β-N-acetylglucosaminidase H, Streptomyces griseus) to investigate the orientational change of the sugar chains associated with the lower activity of the modified enzyme. On measurement of release of sugar chains, by periodic acid-Schiff staining of endoglycosidase H-treated phospholipase B on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by direct sugar analysis of the isolated endoglycosidase H-treated phospholipase B, distinct curves were obtained for release of sugar chains from the native and modified enzymes with ultimately loss of about 30 and 55%, respectively, of the carbohydrate. Removal of sugar chains from the two enzymes resulted in similar increases in phospholipase B activity (phosphatidylcholine hydrolysis) and their phospholipase A₁ and A₂ activities in the presence of Triton X-100, but no change of lysophospholipase activity (lysophosphatidylcholine hydrolysis). The three former activities of the native and modified enzymes increased to almost 170 and 350%, respectively, of their initial values. However, little increase in phospholipase B activity was observed when the activity was assayed in the absence of Triton X-100, and none when it was assayed in the presence of sodium taurocholate. These findings suggest that the carbohydrate moiety of phospholipase B greatly influence the phospholipase B activity, especially in the presence of Triton X-100, and that the low phospholipase B activity of the modified enzyme is due to excess exposure of sugar chains on the surface of the molecule as a result of protease attack.     

Effect of substrate properties on the activity of lysosomal cholesteryl ester hydrolase.

The effects of the substrate properties on the catalytic activity of lysosomal cholesteryl ester hydrolase from rat liver have been examined with three standard substrate types: vesicle, micelle and emulsion. The pH optimum of the enzyme coincided to 4.5--5.0 with the substrate types employed. The apparent Km values were 15.3, 14.3 and 7.3 microM for vesicle, micelle and emulsion substrates, respectively. In the systems used in this study reaction products, cholesterol and oleic acid, and the nonionic surfactant Tween 80 and Triton X-100 Had an inhibitory effect. The emulsifier phosphatidylcholine and the charged phospholipid phosphatidic acid stimulated the activity. The mixed micelle of sodium taurocholate and phosphatidylcholine was the most potent substrate vehicle. With dipalmitoyl phosphatidylcholine vesicles the enzyme showed maximal activity at the gel-liquid-crystalline transition temperature of the phospholipid. The possible physiological significance of the lysosomal cholesteryl ester hydrolase is discussed with special reference to the form of the substrate.     

尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征

将尖吻蝮蛇毒碱性磷脂酶A₂（A.aBPLA₂）基因克隆至温敏表达载体pBLMVL2,在大肠杆菌RR1中成功诱导表达.表达产物A.aBPLA₂约占细菌蛋白质总量的20％,并以包涵体的形式存在.纯化包涵体后,将产物变性、复性,然后用FPLC Superose^TM12纯化,产物经过SDS-聚丙烯酰胺凝胶电泳检测只有单一条带.对纯化后的表达A.aBPLA₂进行了酶活性、抑制血小板聚集活性和溶血活性的测定.结果显示,表达A.aBPLA₂的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A₂酶活性相近,具有类似变性后复性江浙蝮蛇碱性磷脂酶A₂的溶血活性,没有抑制血小板聚集活性.最后对磷脂酶A₂的结构与这些活性的关系进行了讨论.     

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.     

Partial purification and kinetic characterization of the microsomal phospholipase A2 from thermally acclimated rainbow trout (Salmo garidneri)

Summary Phospholipase A₂ (PLA₂) was extracted from liver microsomal membranes of both 5 and 20°C-acclimated rainbow trout (Salmo gairdneri), using the non-ionic detergent, Triton X-100. Further purification was achieved by precipitation with 35–65% ammonium sulfate followed by gel filtration chromatography in the presence of 0.1% Triton X-100 on Sephadex G-200. These procedures resulted in a 30-fold purification and the removal of all traces of phospholipid from the enzyme of both warm-and cold-acclimated trout. Column elution profiles were similar for both acclimation groups, yielding a molecular weight estimate for the trout liver enzyme of 73,000. Comparisons of activity levels and kinetic parameters of PLA₂ from warm-and cold-acclimated fish, indicated that compensation for temperature at nonsaturating substrate concentrations was an attribute of both the particulate (microsomal) enzyme and the lipid-free protein. Cold acclimation resulted in higher activity belowV _max due primarily to decreased apparentK _m values. These adaptations to temperature could not be attributed to the interaction of the enzyme with the membrane lipids, but were due to qualitative changes in the enzyme that resulted from acclimation. Other adaptive qualities of PLA₂, such as reducedK _m in response to acute decreases in temperature in warm-acclimated fish, were only apparent in particulate preparations, and thus were a function of the protein-lipid complex. These data suggest that an acclimation-induced increase in the activity of PLA₂ may result in the activation of a deacylation-reacylation cycle at cold temperatures.Abbreviations PAGE polyacrylamide gel electrophoresis - PC phosphatidylcholine - PLA ₂ phospholipase A₂ - SDS sodium dodecylsulfate     

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.     

Kinetic analysis of Arabidopsis phospholipase Ddelta. Substrate preference and mechanism of activation by Ca2+ and phosphatidylinositol 4,5-biphosphate

Phospholipase D (PLD) is a major plant phospholipase family involved in many cellular processes such as signal transduction, membrane remodeling, and lipid degradation. Five classes of PLDs have been identified in Arabidopsis thaliana, and Ca(2+) and polyphosphoinositides have been suggested as key regulators for these enzymes. To investigate the catalysis and regulation mechanism of individual PLDs, surface-dilution kinetics studies were carried out on the newly identified PLDdelta from Arabidopsis. PLDdelta activity was dependent on both bulk concentration and surface concentration of substrate phospholipids in the Triton X-100/phospholipid mixed micelles. V(max), K(s)(A), and K(m)(B) values for PLDdelta toward phosphatidylcholine or phosphatidylethanolamine were determined; phosphatidylethanolamine was the preferred substrate. PLDdelta activity was stimulated greatly by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Maximal activation was observed at a PIP(2) molar ratio around 0.01. Kinetic analysis indicates that PIP(2) activates PLD by promoting substrate binding to the enzyme, without altering the bulk binding of the enzyme to the micelle surface. Ca(2+) is required for PLDdelta activity, and it significantly decreased the interfacial Michaelis constant K(m)(B). This indicates that Ca(2+) activates PLD by promoting the binding of phospholipid substrate to the catalytic site of the enzyme.