Substrate level modulation of the activity of phospholipase A2 in vitro by 12-O-tetradecanoylphorbol-13-acetate.

The action of porcine pancreatic phospholipase A2 towards fluorescent phospholipid analogs is either enhanced or suppressed by 4 beta-12-O-tetradecanoylphorbol-13- acetate (TPA), depending on the chemical structure of the substrate and the concentration of Ca2+. In the presence of nmolar Ca2+ concentrations increasing [TPA] enhanced by approx. 5-fold the rate of hydrolysis of the pyrene-labelled acidic alkyl-acyl phospholipid, 1-octacosanyl-2-[6- (pyrene-1-yl)] hexanoyl-sn-glycero-3-phosphatidylmethanol. Maximal effect was obtained at high TPA/substrate molar ratios approaching 1:2. In the presence of 4 mM CaCl2 maximal activation was reduced to approximately 1.5-fold. With the corresponding phosphatidylcholine derivative as a substrate increasing [TPA] reduced fatty acid release maximally by 90% both at low [Ca2+] as well as in the presence of 4 mM CaCl2. Essentially identical results were obtained using 4 alpha-TPA, a stereoisomer which does not activate protein kinase C.     

Hydrolysis of 1-palmitoyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero- 3-phospholipids by phospholipase A2: effect of the polar head-group

The effect of the phospholipid polar head-group on the porcine pancreatic phospholipase A2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) reaction was studied using 1-palmitoyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3- phosphatidylcholine, -ethanolamine, -glycerol, -monomethylester and -serine as substrates. Except for the monomethylester analogue, which was maximally activated by 3.5 mM CaCl2, maximal enhancement of hydrolysis of the other pyrenephospholipids was obtained at 2 mM Ca2+. Sodium cholate inhibited hydrolysis of the ethanolamine and serine lipids, whereas a slight (1.4-2.0-fold) activation was observed for the -choline, -glycerol and -monomethylester derivatives. Arrhenius plots of hydrolysis of pyrenephospholipids by porcine pancreatic phospholipase A2 revealed no discontinuities, thus indicating the absence of phase transition for these lipids in the temperature range 15-45 degrees C. Specific activities of porcine and bovine pancreatic, porcine intestinal and snake venom (Crotalus atrox) phospholipases A2 towards pyrenephospholipid liposomes were then compared. Whereas the snake venom phospholipase A2 preferred phosphatidylcholine as a substrate, the other phospholipases A2 preferred acidic phospholipids in the order monomethylester greater than or equal to glycerol greater than or equal to serine.     

Hydrolysis of 1-triacontanoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine by human pancreatic phospholipase A2

A novel fluorescent phospholipid analogue, 1-triacontanoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (C30PHPC) was employed as a substrate for human pancreatic phospholipase A2. C30PHPC has a main endothermic phase transition with Tm at 46 degrees C as determined by differential scanning calorimetry (DSC). For an aqueous dispersion of C30PHPC the ratio of the intensities of pyrene excimer and monomer fluorescence emission, (IE/IM) has a maximum between 32 and 36 degrees C. The excimer emission intensity (at 480 nm) exceeds the monomer emission intensity (at 400 nm) 6.5-fold thus indicating a close packing of the phospholipid pyrene moieties in the lipid phase. C30PHPC has a limiting mean molecular area of 37 A2 at surface pressure 35 dyn cm-1 as judged by the compression isotherm at an air-water interphase. The hydrolysis of C30PHPC by human pancreatic phospholipase A2 was followed by monitoring the increase in the pyrene monomer fluorescence emission intensity occurring as a consequence of transfer of the reaction product, pyren-1-yl hexanoic acid into the aqueous phase. The enzyme reaction exhibited an apparent Km of 2.0 microM substrate. Calcium at a concentration of 0.2 mM activated the enzyme 4-fold. Maximal hydrolytic rates were obtained at 45 degrees C and at pH between 5.5 and 6.5. The enzyme reaction could be inhibited by 5 mM EDTA, confirming the absolute requirement for Ca2+ of this enzyme. The present fluorimetric assay easily detects hydrolysis of C30PHPC in the pmol min-1 range. Accordingly, less than nanogram levels of human pancreatic phospholipase A2 can be detected.     

Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family

Human placental anticoagulant protein-I (PAP-I) is a member of the lipocortin/calpactin/annexin family of Ca2+-dependent phospholipid binding proteins. PAP-I was labeled with fluorescein 5-isothiocyanate (1 mol/mol); this derivative had anticoagulant activity identical to the unlabeled protein and could be used to measure Ca2+-dependent binding to phospholipid vesicles through changes in fluorescence quenching. At 1.2 mM Ca2+, 0.50 M ionic strength, pH 7.4, 25 degrees C, fluorescein-labeled PAP-I bound to phospholipid vesicles containing 80% phosphatidylcholine, 20% phosphatidylserine with a Kd of 1.2 +/- 0.2 nM (mean +/- S.D.). At an ionic strength of 0.15 M, the Kd decreased to less than 0.1 nM. Prothrombin and factor Xa both competed with fluorescein-labeled PAP-I for binding to anionic phospholipid vesicles, but with affinities at least 1000-fold weaker than PAP-I. PAP-I bound only weakly (Kd greater than 2 x 10(-5) M) to neutral or anionic phospholipid monomers, and this binding was not calcium-dependent. These results show that the affinity of PAP-I for anionic phospholipid surfaces is sufficient to explain its potency as an in vitro anticoagulant.     

Interfacial activation of porcine pancreatic phospholipase A(2) studied with 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled lipids

The interfacial activation of porcine pancreatic phospholipase A(2) (PLA(2)) during the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes at different temperatures has been monitored by fluorescence changes of the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) lipid derivatives 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (C(12)-NBD-PC) and 12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)]dodecanoic acid (C(12)-NBD-FA) inserted in the substrate vesicles. These long-chain monitors, in contrast to the previously used C(6)-NBD-PC, detect latency times of PLA(2) action, similar to those measured by the classic titrimetric, pH-stat method. Interestingly, hydrolysis of the host vesicles results in a decrease in fluorescence not only of C(12)-NBD-PC, a substrate analog, but also of product derivative C(12)-NBD-FA. Ultrafiltration experiments show that C(12)-NBD-FA does not migrate to the aqueous phase upon hydrolysis of the host liposomes. Besides, in a simulated hydrolysis experiment in which increasing proportions of palmitic acid and 1-palmitoyl-sn-glycero-3-phosphocholine were cosonicated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C(12)-NBD-PC fluorescence was insensitive to products, whereas C(12)-NBD-FA did show a decreased emission intensity as in the actual hydrolysis experiments. The phenomenon is triggered above a critical concentration of products (10 mol%) suggesting that cosegregation of NBD-FA (either added as such or generated by hydrolysis of C(12)-NBD-PC) and products may be related to the decrease in fluorescence. Phase separation should create microdomains of increased C(12)-NBD-FA surface density and cause concentration quenching. In addition, and taking into account that the NBD group may be located near the interfacial region, it is possible that in segregating with products, the fluorescent moiety of C(12)-NBD-FA becomes exposed to microenvironments of higher surface polarity, which further decreases its quantum yield.     

Reversible, nonionic, and pH-dependent association of cytochrome c with cardiolipin-phosphatidylcholine liposomes.

Membrane association of cytochrome c (cyt c) was monitored by the efficiency of resonance energy transfer from a pyrene-fatty acid containing phospholipid derivative (1-palmitoyl-2[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphocholine (PPHPC)) to the heme of cyt c. Liposomes consisted of 85 mol% egg phosphatidylcholine (egg PC), 10 mol% cardiolipin, and 5 mol% PPHPC. Cardiolipin was necessary for the membrane binding of cyt c over the pH range studied, from 4 to 7. In accordance with the electrostatic nature of the membrane association of cyt c at neutral pH both 2 mM MgCl2 and 80 mM NaCl dissociated cyt c from the vesicles completely. At neutral pH also adenine nucleotides in millimolar concentrations were able to displace cyt c from liposomes, their efficiency decreasing in the sequence ATP > ADP > AMP. In addition, both CTP and GTP were equally effective as ATP. The detachment of cyt c from liposomes by nucleotides is likely to result from a competition between cardiolipin and the nucleotides for a common binding site in cyt c. When pH was decreased to 4 there was a small yet significant increase in the apparent affinity of cyt c to cardiolipin containing liposomes. Notably, at pH 4 the above nucleotides as well as NaCl and MgCl2 were no longer able to dissociate cyt c and, on the contrary, they slightly enhanced the quenching of pyrene fluorescence by cyt c. The above results do suggest that the membrane association of cyt c at acidic pH was non-ionic and presumably due to hydrogen bonding. The pH-dependent binding of cyt c to membranes was fully reversible. Accordingly, in the presence of sufficient concentrations of either nucleotides or salts rapid detachment and membrane association of cyt c could be induced by varying pH between neutral and acidic values, respectively.     

Pyrene-labeled gangliosides: micelle formation in aqueous solution, lateral diffusion, and thermotropic behavior in phosphatidylcholine bilayers

By use of the excimer technique, the formation in aqueous solution of pyrene-labeled ganglioside micelles and their lateral diffusion and distribution in phosphatidylcholine membranes were investigated. For these studies 12-(1-pyrenyl)dodecanoic acid was covalently attached to the ceramide part of lysogangliosides GM1, GM2, GM3, GD1a, and GD1b. The 12-(1-pyrenyl)dodecanoic acid substitute of phosphatidylcholine was used for comparison. All pyrene-labeled gangliosides were present in aqueous solution in a predominantly micellar form down to 2 X 10(-8) M, which is the technical limit of this method. The tendency to aggregate is highest for PyGD1a and PyGD1b. In fluid dipalmitoylphosphatidylcholine bilayers the excimer-to-monomer fluorescence intensity ratio of pyrene-labeled gangliosides PyGM1, PyGM2, PyGM3, PyGD1a, and PyGD1b increases linearly with ganglioside concentration. The calculated diffusion coefficients for gangliosides are comparable to 1.6 X 10(-7) cm2/s, which is the diffusion coefficient of pyrene-labeled phosphatidylcholine [Galla, H.-J., & Hartmann, W. (1980) Chem. Phys. Lipids 27, 199-219]. In comparison to phosphatidylcholine, the diffusion of monosialogangliosides is slightly increased, with that diffusion of disialogangliosides being slightly decreased. Ca2+ ions up to 200 mM do not affect ganglioside diffusion significantly. The shape of the lipid phase transition curves obtained by the excimer technique yields information on the lateral distribution of the tested probe molecules. Pyrene-labeled phosphatidylcholine was taken as reference for a system with complete miscibility but nonideal mixing. 1-Acyl-2-[10-(1-pyrenyl)decanoyl]-sn-glycero-3-phosphocholine (PyPC) is known to be randomly distributed in the gel and in the fluid-crystalline lipid phase of dipalmitoylphosphatidylcholine bilayer membranes. It distributes preferentially into the fluid phase in the phase-transition region. In comparison, PyPC in dimyristoylphosphatidylcholine membranes is an example of a system with nearly ideal mixing [Hresko, R. C., Sugar, J. P., Barenholz, Y., & Thompson, T. E. (1986) Biochemistry 25, 3813-3828]. Phase-transition curves of pyrene-labeled gangliosides exemplify a nearly ideal mixing system with PyGD1a or PyGD1b producing best effects. The monosialogangliosides, however, exhibit less ideality of mixing, the deviation from an ideal mixing behavior increasing with decreasing number of both neutral sugar residues and sialic acid groups. Addition of Ca2+ triggers a tightening of the phosphatidylcholine bilayer and thus induces a change in the lateral distribution of the gangliosides at the phase transition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels

A sensitive method which utilizes fluorescence energy transfer to assay Ca2+ -or Mg2+ -mediated fusion of phospholipid vesicles is reported. More than 85% quenching results when phosphatidylserine vesicles labelled with dansyl phosphatidylethanolamine (donor) are fused with vesicles labelled with rhodamine phosphatidylethanolamine (acceptor) in the presence of 5 mM CaCl2 or 10 mM MgCl2. Higher concentrations of divalent cations are required to obtain maximal quenching when phosphatidylserine is partially replaced with phosphatidylethanolamine or phosphatidylcholine. The rate of vesicle fusion is dependent upon the concentrations of both cation and vesicles. Maximum quenching occurs within 5 min using phosphatidylserine vesicles and 5 mM Ca2+, but quenching is incomplete even after 20 h with 0.8--2 mM Ca2+. This probably reflects the heterogeneous size distribution of these vesicles, since the extent of fusion was found to correlated with vesicle size. Binding of antibody to membrane-localized phenobarbital hapten effectively blocks Ca2+ -mediated vesicle fusion. This effect can be inhibited by preincubation of the antibody with phenobarbital. Leakage of tempocholine from intact vesicles induced by 5 mM Ca2+ occurs even when fusion is prevented by bound antibody. This demonstrates that fusion is not a necessary requirement for Ca2+ -induced leakage.     

Polyamine-phospholipid interaction probed by the accessibility of the phospholipidsn-2 ester bond to the action of phospholipase A2

Summary Conditions were used where the action of porcine pancreatic phospholipase A2 on phospholipids can be followed in the absence of added calcium and the catalytic activity is supported by the calcium brought with the nanomolar enzyme. Therefore, alterations in the enzyme velocity resulting from the presence of spermine or spermidine could be specifically studied using 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) and 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) as substrates. Both spermine and spermidine activated the hydrolysis of PPHPG fourfold at polyamine/phospholipid molar ratios of approximately 11 and 121, respectively. Double-reciprocal plots of enzyme activityvs. PPHPG concentration revealed the enhancement to be due to increased apparentV _max while the apparentK _m was slightly increased. In the presence of 4mm CaCl₂ inhibition by polyamines of PPHPG hydrolysis by phospholipase A2 was observed. Using synthetic diamines we could further demonstrate that two primary amino groups are required for the activation. In the absence of exogenous CaCl₂ polyamines inhibited the hydrolysis of PPHPC by phospholipase A2. The presence of 4mm CaCl₂ reversed this inhibition and a twofold activation was observed at 10 m spermine. The results obtained indicate that the activation of PLA2 by spermine and spermidine is produced at the level of the substrate, PPHPG. This implies the formation of complexes of phosphatidylglycerol and polyamines with defined stoichiometries.     

Phospholipid lateral organization in synthetic membranes as monitored by pyrene-labeled phospholipids: effects of temperature and prothrombin fragment 1 binding

Pyrene-labeled phospholipids have been used to test for the existence of lateral domains due to temperature-induced phase separations and binding of prothrombin fragment 1 to charged lipid vesicles. When in close proximity, pyrene-containing probes can exchange excited-state energy to form excimers; the ratio of the excimer to monomer fluorescence intensity (E/M) is proportional to the local concentration of probe in the membranes, as well as to the excimer lifetime and the probe's lateral diffusion coefficient. The ability of the pyrene-labeled phospholipids to quantitatively report the coexistence of multiple environments was demonstrated in dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylcholine multilamellar vesicle preparations of varying compositions, each of which contained coexisting fluid and gel phases. In this system, pyrene-labeled phosphatidylcholine was found to favor the fluid relative to the gel phase with a partition coefficient of 7. At 37 degrees C, in dioleoylphosphatidylglycerol (DOPG)/palmitoyloleoylphosphatidylcholine (POPC) large, unilamellar vesicles containing either pyrene-labeled phosphatidylglycerol (py-PG) or pyrene-labeled phosphatidylcholine (py-PC), the excimer lifetime (37 ns) and the lateral diffusion constant of the probe (5.8 X 10(-8) cm2/s) were independent of the membrane composition and the presence of fragment 1 and Ca2+. Consequently, E/M was directly proportional to only the local concentration of the py-PG or py-PC probes. When saturating amounts of fragment 1 and 5 mM Ca2+ were added to DOPG/POPC vesicles that contained either probe, no change in E/M and hence the local probe concentration was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational basis of the phospholipid requirement for the activity of SR Ca(2+)-ATPase

The delipidated sarcoplasmic reticulum (SR) Ca(2+)-ATPase was reconstituted into proteoliposomes containing different phospholipids. The result demonstrated the necessity of phosphatidylcholine (PC) for optimal ATPase activity and phosphatidylethanolamine (PE) for the optimal calcium transport activity. Fluorescence intensity of Fluorescein 5-isothiocyanate (FITC)-labeled enzyme at Lys515 as well as the measurement of the distance between 5-((2-[(iodoacetyl) amino] ethyl) amino)naphthalene-1-sulphonic acid (IAEDANS) label sites (Cys674/670) and Pr3+ demonstrated a conformational change of cytoplasmic domain, consequently, leading to the variation of the enzyme function with the proteoliposomes composition. Both the intrinsic fluorescence of Trp and its dynamic quenching by HB decreased with increasing PE content, revealing the conformational change of transmembrane domain. Time-resolved fluorescence study characterized three classes of Trp residues, which showed distinctive variation with the change in phospholipid composition. The phospholipid headgroup size caused the conformational change of SR Ca(2+)-ATPase, subsequent the ATPase activity and Ca2+ uptake.     

Binding of adriamycin to liposomes as a probe for membrane lateral organization.

A stopped-flow spectrofluorometer equipped with a rapid scanning emission monochromator was utilized to monitor the binding of adriamycin to phospholipid liposomes. The latter process is evident as a decrease in fluorescence emission from a trace amount of a pyrene-labeled phospholipid analog (PPDPG, 1-palmitoyl-2-[(6-pyren-1-yl)]decanoyl-sn-glycero-3-phospho-rac-++ +glyce rol) used as a donor for resonance energy transfer to adriamycin. For zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes, fluorescence decay was slow, with a half-time t1/2 of approximately 2 s. When the mole fraction of the acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), was increased to XPG >/= 0.04, the decay of fluorescence became double exponential, and an additional, significantly faster process with t1/2 in the range between 2 and 4 ms was observed. Subsequently, as XPG was increased further, the amplitude of the fast process increased, whereas the slower process was attenuated, its t1/2 increasing to 20 s. Increasing [NaCl] above 50 mM or [CaCl2] above 150 microM abolished the fast component, thus confirming this interaction to be electrostatic. The critical dependence of the fast component on XPG allows the use of this process to probe the organization of acidic phospholipids in liposomes. This was demonstrated with 1, 2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes incorporating PPDPG (XPPDPG = 0.03), i.e., conditions where XPG in fluid bilayers is below the required threshold yielding the fast component. In keeping with the presence of clusters of PPDPG, the fast component was observed for gel-state liposomes. At approximately 34 degreesC (i.e., 6 degrees below Tm), the slower fluorescence decay also appeared, and it was seen throughout the main phase transition region as well as in the liquid-crystalline state. The fluorescence decay behavior at temperatures below, above, and at the main phase transition temperature is interpreted in terms of thermal density fluctuations and an intermediate state between gel and liquid-crystalline states being involved in the phospholipid main phase transition. This is the first observation of a cluster constituted by acidic phospholipids controlling the membrane association of a drug.     

Incorporation of (1-14C)palmitoyl-CoA into phosphatidylcholine by plasma membranes of rat submaxillary glands in vitro.

1. On incubation with the isolated rat submaxillary gland plasma membranes, [1-14C]palmitoyl-CoA was incorporated mainly into phosphatidylcholine and hydrolysed to [1-14C]palmitic acid and CoASH. 2. The addition of lysophosphatidylcholine enhanced the incorporation into phosphatidylcholine and lowered the hydrolysis of palmitoyl-CoA markedly. 3. In the presence of lysophosphatidylcholine, palmitoyl-CoA incorporation into phosphatidylcholine was maximum at 0.1 mM palmitoyl-CoA, 0.5 mM lysophosphatidylcholine and between pH 7.0 and 9.0. 4. The incorporation into phosphatidylcholine was stimulated by Na+, K+ and K-, inhibited by Ca2+ and Mg2+ and unaffected by sodium deoxycholate and ATP. 5. Epinephrine inhibited the incorporation of palmitoyl-CoA into phosphatidylcholine in the presence or absence of ATP, the inhibition being more in the presence of ATP than in its absence. Dibutyryl adenosine 3':5'-monophosphate mimicked the inhibitory effect of epinephrine.     

Reconstitution of rabbit thrombomodulin into phospholipid vesicles

The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C.     

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.     

Phospholipase A2 assay using an intramolecularly quenched pyrene-labeled phospholipid analog as a substrate

A phospholipid analog 1-palmitoyl-2-6(pyren-1-yl)hexanoyl-sn-glycero-3-phospho-N- (trinitrophenyl)aminoethanol (PPHTE) in which pyrene fluorescence is intramolecularly quenched by the trinitrophenyl group was used as a substrate for pancreatic phospholipase A2. Upon phospholipase A2 catalyzed hydrolysis of this molecule pyrene monomer fluorescence emission intensity increased as a result of the transfer of the pyrene fatty acid to the aqueous phase. Optimal conditions for phospholipase A2 hydrolysis of PPHTE were similar to those observed earlier for other pyrenephospholipids (T. Thuren, J. A. Virtanen, R. Verger, and P. K. J. Kinnunen (1987) Biochim. Biophys. Acta 917, 411-417). Although differential scanning calorimetry revealed no thermal phase transitions for PPHTE between +5 and +60 degrees C the Arrhenius plot of the enzymatic hydrolysis of the lipid showed a discontinuity at 30 degrees C. The molecular origin of this discontinuity remains at present unknown. To study the effects of dimyristoylphosphatidylcholine (DMPC) phase transition at 23.9 degrees C on phospholipase A2 reaction PPHTE was mixed with DMPC in a molar ratio of 1:200 in small unilamellar vesicles. The hydrolysis of DMPC-PPHTE vesicles was measured by following the increase in pyrene monomer fluorescence emission due to phospholipase A2 action on PPHTE. Below the phase transition of DMPC the enzymatic reaction exhibited a hyperbolic behavior. At the transition as well as at slightly higher temperatures a lag period was observed. The longest lag period was approximately 20 min. Above 26 degrees C no lag time could be observed. However, the reaction rates were slower than below the phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution and interface aggregation states of Crotalus atrox venom phospholipase A2 by two-photon excitation fluorescence correlation spectroscopy

The dimeric Crotalus atrox venom PLA2 is part of the secreted phospholipase A2 (PLA2) enzyme family that interacts at the lipid-solution interface to hydrolyze the sn-2 acyl ester bond of phospholipids. We have employed fluorescence correlation spectroscopy (FCS) to study the monomer-dimer equilibrium of the C. atrox venom PLA2 in solution, in the presence of urea, and in the presence of monomeric and micellar n-dodecylphosphocholine (C12-PN), a phosphatidylcholine analogue. Dilution experiments show that PLA2 is an extremely tight dimer, Kd < or = 0.01 nM, in solution. Urea was introduced to weaken the subunit's association, and an estimate for the PLA(2) dimer dissociation constant in buffer was obtained by linear extrapolation. The derived dissociation constant was at least several orders of magnitude greater than that suggested from the dilution experiments, indicating a complex interaction between urea and the PLA2 dimer. FCS data indicate that the PLA2 dimer begins to dissociate at 10 mM C12-PN in 10 mM Ca2+ and at 5 mM C12-PN in 1 mM EDTA. The PLA2 tryptophan fluorescence displayed spectral shifts and intensity changes upon interacting with C12-PN. On the basis of the FCS and tryptophan fluorescence results, we postulate an intermediate state where the two monomers are in loose interaction within a protein-lipid comicelle. As the concentration of C12-PN was increased, complete dissociation of the dimer was observed, inferred from the doubling of the particle number, and the average diffusion constant decreased to approximately 60 microm2/s, consistent with PLA2 associated with a C12-PN micelle. The presence of Ca2+ makes the comicelle intermediate more stable, retarding the separation of the monomers in the micellar suspension. Our data clearly indicate that PLA2, though a strong dimer in the absence of lipids, is dissociated by micellar C12-PN and supports the monomer hypothesis for PLA2 action.     

Phospholipase C from Clostridium novyi type A. II. Factors influencing the enzyme activity

The apparent activity of phospholipase C[EC 3.1.4.3] of Clostridium novyi type A toward phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine increased in the presence of sodium deoxycholate (SDC). The effects of divalent cations on phospholipase C activity were examined in detail at various concentrations of these cations. These effects varied with substrate. Hydrolysis of phosphatidylcholine by this enzyme significantly increased in the presence of Mg2+ or Ca2+. Hydrolysis of sphingomyelin was inhibited by Ca2+, but increased in the presence of Mg2+. Phosphatidylethanolamine-hydrolyzing activity increased only slightly in the presence of Mg2+ and Ca2+. Zn2+ rather inhibited hydrolysis of these substrates. The effects of divalent cations and detergent appear to be directly related to the physical state of the phospholipid micelles used as substrates. When phosphatidylcholine, sphingomyelin, or phosphatidylethanolamine was used as a substrate, phospholipase C activity was completely inhibited by 2.5 mM EDTA or o-phenanthroline (concentration in the final incubation mixture: 0.5 mM), and was fully restored by Zn2+ alone. Both Ca2+ and Mg2+ were ineffective for reactivation. The isoelectric point of the enzyme was 7.1 +/- 0.1.     

Evidence for the activation of phospholipases during acrosome reaction of human sperm elicited by calcium ionophore A23187

Incubation of washed human sperm with [3H]- or [14C]arachidonic acid allowed a major incorporation of the label into phospholipids, provided that the final concentration of the fatty acid did not exceed 20 microM. A further challenge with calcium ionophore A23187 of spermatozoa suspended in a calcium-containing medium led to phospholipid hydrolysis, which could account for 10-12% of total cell radioactivity. Degradation products were identified as free, unconverted arachidonic acid, occurring with some diacylglycerol. Phospholipid hydrolysis was significant after 15 min of incubation and became maximal after 120 min. It was found to be calcium dependent, diacylglycerol and free arachidonate production occurring maximally at 2 mM and 5 mM CaCl2, respectively. Phosphatidylcholine and phosphatidylinositol were the most significantly degraded phospholipids after 60 min of incubation. Similar incubations conducted with 32P-labeled sperm confirmed the selective hydrolysis of phosphatidylcholine and revealed an increase production of phosphatidic acid probably due to a phosphorylation of diacylglycerol. Under the same conditions, one third of the cells remained motile and electron microscopy revealed that acrosome reaction was completed in 40% of the cells and displayed an intermediary state in 40-50% of the spermatozoa. Furthermore, a good parallelism was observed between the extent of the acrosome reaction and the extent of phospholipid hydrolysis promoted by increasing concentrations of A23187. It is concluded that calcium entry into the cells activates both a phospholipase A2 and a phospholipase C, leading to the production of substances, like lysophospholipid, diacylglycerol or phosphatidic acid, which may or may not be involved in acrosome reaction.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.