Inhibition of lipoprotein lipase by benzene boronic acid. Effect of apolipoprotein C-II.

The catalytic mechanism of triacylglycerol hydrolysis by lipoprotein lipase was studied. We found lipoprotein lipase to be inhibited by benzene boronic acid, with an apparent Ki of 8.9 micro M at pH 7.4. This indicates the presence of serine and histidine in the active site of the enzyme. Inhibition of lipoprotein lipase by benzene boronic acid is likely to be due to the formation of an inhibitor-enzyme complex having analogous bonding to the active site histidine and serine as the transition-state complex which precedes the formation of an obligatory acyl-enzyme intermediate. The presence of apolipoprotein C-II, the apolipoprotein activator of lipoprotein lipase, partly reverses the inhibition of lipoprotein lipase by benzene boronic acid. This reversal by apolipoprotein C-II has a distinct pH optimum in the range of 8-9.

Lateral organisation of membrane lipids. The superlattice view.

Most biological membranes are extremely complex structures consisting of hundreds or even thousands of different lipid and protein molecules. The prevailing view regarding the organisation of these membranes is based on the fluid-mosaic model proposed by Singer and Nicholson in 1972. According to this model, phospholipids together with some other lipids form a fluid bilayer in which these lipids are diffusing very rapidly laterally. The idea of rapid lateral diffusion implies that, in general, the different lipid species would be randomly distributed in the plain of the membrane. However, there are recent data indicating that the components tend to adopt regular (superlattice-like) distributions in fluid, mixed bilayers. Based on this, a superlattice model of membranes has been proposed. This superlattice model is intriguing because it allows only a limited certain number of 'critical' compositions. These critical compositions could play a key role in the regulation of the lipid compositions of biological membranes. Furthermore, such putative critical compositions could explain how compositionally distinct organelles can exist despite of rapid inter-organelle membrane traffic. In this review, these intriguing predictions are discussed along with the basic principles of the model and the evidence supporting it.

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.

Comparison of the effects of NaCl on the thermotropic behaviour of sn-1' and sn-3' stereoisomers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol.

The phase behaviour of liposomes of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG) and the corresponding sn-3' stereoisomer (3'-DMPG) were studied by DSC as a function of NaCl concentration. The melting of the metastable gel phase to the liquid-crystalline phase was similar for both lipids. However, in the presence of salt and at 6 degrees C (T less than Tp) the gel phase of both stereoisomers of DMPG was shown to be metastable and a new phase nominated here as the highly crystalline phase was formed as the stable state. However, significant differences in the formation and melting of the highly crystalline phase were evident between the two polar headgroup stereoisomers. For 3'-DMPG in the presence of 300 mM NaCl the melting enthalpy of this phase is approx. 82 kJ/mol and the transition temperature about 11 degrees higher (at 33.6 degrees C) than for the gel to liquid-crystalline phase transition (25 kJ/mol at 23.0 degrees C). In the presence of 0.15-1.2 M NaCl at 6 to 10 degrees C the formation of the highly crystalline phase of 3'-DMPG is complete within 2 to 5 days, increasing [NaCl] facilitates the rate. For a 1:1 mixture of 1'- and 3'-DMPG the formation of the highly crystalline phase requires several weeks and melts at about 20 degrees higher than the gel phase (at approx. 40 degrees C). For 1'-DMPG partial conversion into the highly crystalline phase requires several months. For 3'-DMPG several intermediate phases appeared as endothermic peaks between the main phase transition temperature and the melting temperature of the highly crystalline phase. In contrast, for 1'-DMPG and the 1:1 mixture the subgel phase appears to be the only metastable intermediate phase. Different monovalent cations differ in their effect on the metastable behaviour.

Increased plasma phospholipase-A2 activity in schizophrenic patients: reduction after neuroleptic therapy.

Phospholipase-A2 (PLA2) is a key enzyme in the metabolism of phospholipids, and it may play an important role in neuronal function and neuronal plasticity. We determined the activity of PLA2 in the plasma of 20 drug-free schizophrenic patients, 6 nonschizophrenic psychiatric patients, and 21 healthy controls. Schizophrenic patients showed significantly higher plasma PLA2 activity than controls, and higher than our nonschizophrenic patients. Seventy percent of the schizophrenics had enzyme activity higher than the highest value from the control group. The increased plasma PLA2 activity in schizophrenics was reduced to the level of the controls after 3 weeks of neuroleptic treatment. These findings warrant further study for possible implications of this increased PLA2 activity in the etiopathology of schizophrenia.

Estimation of the equilibrium lateral pressure in 1-palmitoyl-2-[6(pyren-1-yl)]hexanoyl-glycerophospholipid liposomes.

Compression isotherms for 1-palmitoyl-2-[6(pyren-1-yl)] hexanoyl-sn-glycero-3-phosphocholine (PPHPC), -ethanolamine (PPHPE), -glycerol (PPHPG), -serine (PPHPS) and -phosphatidic acid monomethylester (PPHPM) were recorded at an argon/water interface. Thereafter, the ratios of pyrene excimer to monomer fluorescence emission intensities (Ie/Im) were determined for liposomes of these lipids and were found to be 20.15, 12.30, 11.80, 10.15 and 6.95 for the ethanolamine, choline, monomethylester, glycerol and serine derivatives, respectively. Assuming Ie/Im to depend on the reciprocal of the mean molecular area of the pyrenelipids in liposomes, equilibrium surface pressure to be the same in liposomes of these lipids regardless of the head group structure and neglecting any possible influence due to differences in the orientation of the pyrene moiety, we sought for that surface pressure value in the compression isotherms where the correlation of the reciprocal of mean molecular area in monolayers to Ie/Im values observed in liposomes was maximal. This treatment results in a value of approximately 12 mN m-1 for the equilibrium surface pressure in 1-palmitoyl-2-[6(pyren-1-yl)]hexanoyl-glycerophospholipid liposomes.

Hydrolysis of supported pyrenephospholipid monolayers by phospholipase A2.

Hydrolysis by pancreatic and snake venom (Crotalus atrox) phospholipase A2 of fluorescent monolayers of pyrene-labelled phosphatidylglycerol on solid support was studied. We used a fluorescence microscope equipped with video camera, video recorder and an image analyzer to monitor changes in fluorescence. Decrease in pyrene excimer emission was evident when pyrene phosphatidylglycerol monolayers transferred onto quartz glass slides (at a surface pressure of 15 mN m-1) were subjected to enzymatic hydrolysis. Snake venom phospholipase A2 could hydrolyze the monolayers almost completely while pancreatic phospholipase A2 could cause only 50% decrease in fluorescence intensity. EDTA totally inhibited the action of both A2 phospholipases. When monolayers were transferred onto solid supports at a surface pressure of 31 mN m-1 C. atrox phospholipase A2 could still exert activity whereas porcine pancreatic phospholipase A2 was inactive.

Fourier transform infrared study on the thermotropic behaviour of fully hydrated 1-palmitoyl-2-[10-(pyren-1-y) decanoyl]-sn-glycero-3-phosphatidylcholine.

Fourier transform infrared (FTIR) spectroscopy was used to study the thermotropic behaviour of fully hydrated 1-palmitoyl-2-[10-(pyren-1-yl)-decanoyl]-sn-glycero-3-phosphatidyl choline (PPDPC) in the temperature range of 3-30 degrees C. Several changes in the spectral features of PPDPC were observed. Major alterations analogous to the gel-to-liquid crystalline phase transition of saturated phosphatidylcholines were evident at approximately 16 degrees C in both the wavenumbers and the halfbandwidths of five different vibrational modes of PPDPC, viz. asymmetric and symmetric CH2 stretching, C = O stretching, and CH2 bending. Also the pyrene ring deformation mode changed at this temperature. Using Fourier self-deconvolution technique we resolved the carbonyl stretching mode into two bands at approx. 1741 and 1726 cm-1. These bands are due to conformational differences in the ester linkages of the two acyl chains, and are further assigned on the basis of literature data to the sn-1 and sn-2 carbonyl groups, respectively. The ratio of the relative intensities of these two bands is shown to depend on the phase state of the phospholipid.

Fluorometric assay for pancreatic cholesterylester hydrolase.

A fluorescent cholesterylester analogue, cholesteryl 6-pyrenylhexanoate (ChPH), was used as a substrate for pancreatic cholesterylester hydrolase (CEH, EC 3.1.1.13). The substrate consisted of ChPH in egg phosphatidylcholine stabilized microemulsion with the aqueous phase containing deoxycholate below its critical micellar concentration. Due to the high local concentration of the pyrene moiety in the ChPH phase the fluorescence emission due to monomeric pyrene (IM) is greatly exceeded by the excimer fluorescence intensity (IE). Upon reacting with CEH 6-pyrenylhexanoic acid and free cholesterol are formed. The fluorescent product, 6-pyrenylhexanoic acid, is transferred into the aqueous phase containing deoxycholate, thus resulting in an enhanced fluorescence due to monomeric pyrene. CEH activity can thus be assessed directly by monitoring IM vs. time without product separation. Useful assay conditions were found to be 10 microM ChPH, 0.1 microM egg phosphatidylcholine, 2 mM sodium deoxycholate at 25 degrees C and pH 6.5-7.0.

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.

Characterization of Langmuir-Blodgett films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphat idylcholine by FTIR-ATR.

Monomolecular films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidylc holine (PPDPC) were transferred from an air/water interface onto a germanium attenuated total reflection crystal by the Langmuir-Blodgett (LB) technique. The assemblies were thereafter investigated by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. To determine the molecular organization in the deposited layers we monitored the CH2 and C = O stretching and the CH2 bending regions of the infrared spectra of these lipids in detail. Using Fourier self-deconvolution technique, the carbonyl stretching mode was resolved into two models corresponding to the conformational differences in the ester linkages of the phospholipid sn-1 and sn-2 acyl chains. By varying the temperature of the subphase and using different surface pressures, we were able to transfer different conformational states of DPPC onto a germanium ATR crystal. Deposition of DPPC at 40 mN m-1 and at 15 degrees C or at 20 mN m-1 and at 35 degrees C results in LB-assemblies in ordered or disordered states, respectively, as judged by the IR spectra. These structures in LB films correspond to the state of DPPC in liposomes below and above the temperature of the order-disorder phase transition. Irrespective of the surface pressure and subphase temperature used during the deposition, an ordering process was found in DPPC films when the number of the transferred layers was increased from one to five. The pyrene-labelled phosphatidylcholine analogue, PPDPC, behaved differently from DPPC. In the case where one to three layers of PPDPC transferred at 35 mN m-1 and at 20 degrees C only conformational structures resembling those in fully hydrated liposomes above the main transition temperature were observed.

Interaction of 7,7,8,8-tetracyanoquinodimethane with diacylphosphatidylcholines and -phosphatidylglycerols. A photoacoustic Fourier transform infrared study.

7,7,8,8-Tetracyanoquinodimethane (TCNQ) was incorporated in fully hydrated liposomes of the following pyrene-containing as well as non-labelled phospholipids: 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatid ylc holine (PPDPC), 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidyl- rac'- glycerol (rac'-PPDPG), 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidyl- sn-3'- glycerol (3'-PPDPG), 1-[10-(pyren-1-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl- sn-3'- glycerol (3'-PDPPG), 1-[10-pyren-1-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl-s n-1'- glycerol (1'-PDPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl-rac'-glycerol (rac'-DPPG). Lyophilized charge-transfer (CT) complexes of TCNQ with phospholipids were examined by Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS). Due to the spectral changes observed in the vibrational bands originating from the CH2 and C = O stretching vibrations, and the bands associated with the polar headgroup of the phospholipids it is evident that TCNQ has only a minor perturbing effect on the hydrocarbon chains. However, the molecular interaction between TCNQ and phospholipids is seen in the polar headgroup region. The donated electrons are most likely located on the oxygens of the phosphate group in the polar head. As judged from the present infrared data interactions of TCNQ with phosphatidylcholines (PC) and phosphatidylglycerols (PG) differ. For PG the complex formation produces a second strong C = O stretching band at approx. 1710 cm-1 in addition to the band at approx. 1735 cm-1 indicating a specific molecular interaction in the interfacial region.

Esterase-type of activity possessed by human plasma apolipoprotein C-II and its synthetic fragments.

Human plasma apolipoproteins apo A-I, A-II, C-I, C-II and C-III (with the exception of apoE), porcine pancreatic colipase and procolipase hydrolyze 4-methylumbelliferyloleate. In all cases, liberation of 4-methylumbelliferone could be inhibited by phenylmethylsulfonyl-fluoride, thus suggesting the involvement of serine residues. To the best of our knowledge this is the first report on the esterase activities of these peptides. Synthetic fragments of the lipoprotein lipase activator, apoC-II, prepared according to the known sequence, also possessed this esterase-type of activity. Furthermore, the esterase-type of activities of the synthetic apoC-II fragments with different chain lengths bore a relatively good correlation to the reported abilities of these peptides to produce activation of lipoprotein lipase. We propose a model for the mechanism of activation of lipoprotein lipase by apolipoprotein C-II. ApoC-II would enhance the apparent catalytic rate constant of lipoprotein lipase by functioning as a specific acyl-enzyme hydrolase. A similar catalytic mechanism is suggested for other protein co-factors of hydrolytic enzymes.

Control of the action of phospholipases A by "vertical compression" of the substrate monolayer.

Monolayers of rac-1,2-didodecanoyl-sn-glycero-3-phosphoglycerol at an air-water interface were "vertically compressed" by substituting an alkylated glass plate for air while maintaining a constant surface pressure of 15 mN m-1. At this surface pressure the overlaying of the lipid film by the alkylated surface resulted in an average increase of 16 A2/molecule in the mean molecular area of those phospholipid molecules residing at the interface between water and the alkylated glass. Subsequently, the activities of phospholipases A1 and A2 toward the monolayers were measured both in the presence and in the absence of the support. While phospholipase A1 activity was increased 4-fold by the support, the activity of phospholipase A2 was reduced to 15% of the activity measured in the absence of the alkylated surface. These findings indicate that such a "vertical compression" of the monolayer is likely to induce a conformational change in the phospholipid molecules, which in turn would cause the above reciprocal changes in the activities of phospholipases A1 and A2. A molecular model accounting to these findings is presented.

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

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 1:1 and 12:1, respectively. Double-reciprocal plots of enzyme activity vs. PPHPG concentration revealed the enhancement to be due to increased apparent Vmax while the apparent Km was slightly increased. In the presence of 4 mM 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 4 mM CaCl2 reversed this inhibition and a twofold activation was observed at 10 microM 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.

Lateral organization of liquid-crystalline cholesterol-dimyristoylphosphatidylcholine bilayers. Evidence for domains with hexagonal and centered rectangular cholesterol superlattices.

The lateral organization of fluid cholesterol-dimyristoylphosphatidylcholine (DMPC) bilayers was studied by measuring the response of fluorescent membrane probes, dipyrenylphosphatidylcholines (diPyrxPCs) or merocyanine 540, to the variation of cholesterol concentration. Parallel absorbance and light-scattering measurements were also carried out. The excimer-to-monomer ratio of diPyrxPCs displayed abrupt deviations at particular cholesterol mole fractions (CMFs). The most notable of these occurred at CMFs of 0.15, 0.33, and 0.67. Deviations were also frequently observed at CMFs of 0.12, 0.20, 0.25, and 0.40. Merocyanine 540 reproducibly reported deviations at CMFs of 0.15 and 0.33 and frequently reported values close to 0.12, 0.20, and 0.25. In absorbance (turbidity) and light scattering versus CMF plots, well-defined kinks were observed at CMFs of 0.16, 0.33, 0.52, and 0.67. The occurrence of kinks or other deviations at those particular CMFs is most readily explained in terms of a superlattice model previously developed to explain the lateral distribution of pyrenylphospholipids in bilayers [Somerharju, et al. (1985) Biochemistry 24, 2773-2781; Virtanen, J. A., et al. (1988) J. Mol. Electron. 4, 233-236]. This model is based on the assumptions that (i) each cholesterol molecule replaces a single acyl chain in a hexagonal lattice, (ii) cholesterol molecules, because of their larger size, perturb the lattice, (iii) this perturbation is minimized when the cholesterol molecules are maximally separated from each other, and (iv) the maximal separation is achieved when the cholesterol molecules form a hexagonal or centered rectangular superlattice. All detected critical CMFs, except that at CMF 0.67, are predicted by the model, thus strongly supporting its validity. The critical CMF at 0.67 is a limiting case, which can be accounted for by assuming that cholesterol and phospholipid molecules form alternating rows, i.e., formation of a cholesterol superlattice with rectangular symmetry. As predicted by the superlattice model, composition-driven order-to-disorder transitions occur between the critical CMFs, as indicated by increased data scatter and sample fluctuations in those regions. Another important prediction of the superlattice model is that domains with different cholesterol superlattices should coexist at most cholesterol concentrations. Such domains do not have to be extensive to account for the critical events observed here; rather, they are expected to be dynamic entities of limited size. It is very likely that such microscopic domains with distinct cholesterol superlattices also coexist in biological membranes. This is expected to have remarkable effects on both the structure and functions of these membranes.

Phospholipid composition of the mammalian red cell membrane can be rationalized by a superlattice model.

Although the phospholipid composition of the erythrocyte membrane has been studied extensively, it remains an enigma as to how the observed composition arises and is maintained. We show here that the phospholipid composition of the human erythrocyte membrane as a whole, as well as the composition of its individual leaflets, is closely predicted by a model proposing that phospholipid head groups tend to adopt regular, superlattice-like lateral distributions. The phospholipid composition of the erythrocyte membrane from most other mammalian species, as well as of the platelet plasma membrane, also agrees closely with the predictions of the superlattice model. Statistical analyses indicate that the agreement between the observed and predicted compositions is highly significant, thus suggesting that head group superlattices may indeed play a central role in the maintenance of the phospholipid composition of the erythrocyte membrane.

Esterase activity of synthetic fragments of human adrenocorticotrophic hormone.

The anterior pituitary hormone adrenocorticotrophin (ACTH) has been extensively studied in terms of structure-function relationships and in vivo and in vitro activities of different synthetic fragments of ACTH have been characterized. Here we describe the ability of synthetic fragments of ACTH to hydrolyze a fluorogenic esterase substrate 4-methylumbelliferyloleate (MUBO). The measured esterase activities (in mumol 4-MU mol-1 s-1) were 79.7 for ACTH1-13, 385.9 for ACTH3-18, 503.0 for ACTH1-19, 1249.9 for ACTH1-24 D-ser3, and 1350 for ACTH1-24. Although the significance of the observed esterase activities in the actual molecular mechanisms of action of ACTH remains to be established it is worth noticing that the esterase activities of the different ACTH fragments closely parallel their reported ability to activate the brain lipase as well as their in vivo ability to induce steroidogenesis in adrenal cortex.

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)

Ca2+-induced lateral phase separation in phosphatidic acid/phosphatidylcholine monolayers as revealed by fluorescence microscopy.

Phase separation in mixed monolayers of phosphatidylcholine (PC) and pyrene-labeled phosphatidic acid (PA) was observed by fluorescence microscopy on an air/water interface as a function of subphase Ca2+ concentration and lateral packing pressure of the film. Below 45 mN m-1 and in the absence of Ca2+ no indications of phase immiscibility were observed. Addition of 1 mM Ca2+ caused extensive phase separation, which was evident immediately after spreading of the film. Further increase in Ca2+ concentration up to 30 mM increased the pyrene excimer intensity of the separated phosphatidic acid enriched domains. In the presence of Ca2+ (1-30 mM) and at surface pressures below 10 mN m-1 phase separation was always evident. However, as surface pressure exceeded 10 mN m-1, mixing of PC and PA occurred. Upon decompression of the film, phase separation reappeared at surface pressures close to 10 mN m-1. The surface textures of the film before and after the compression and subsequent relaxation were different. Inclusion of 30 mol% cholesterol increased the number and decreased the size of the PA domains. In films containing 50 mol% cholesterol no phase separation could be detected at the resolution available.