Jorma A. Virtanen

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.

Characteristics of Pyrene Phospholipid/γ-Cyclodextrin Complex

Recently, it was demonstrated that gamma-cyclodextrins (gamma-CDs) greatly accelerates transfer of hydrophobic pyrene-labeled and other fluorescent phospholipid derivatives from vesicles to cells in culture (). To understand better the characteristics of this process, we studied the interaction of gamma-CD with pyrene-labeled phosphatidylcholines (PyrPCs) using a variety of physical methods. Either one or both of the acyl chains of PC was labeled with a pyrene moiety (monoPyrPCs and diPyrPCs, respectively), and the length of the labeled chain(s) varied from 4 to 14 carbons. Fluorescent binding assays showed that the association constant decreases strongly with increasing acyl chain length. PyrPC/gamma-CD stoichiometry was 1:2 for the shorter chain species, but changed to 1:3 when the acyl chain length exceeded 8 (diPyrPCs) or 10 (monoPyrPCs) carbons. The activation energy for the formation of diPyr(10)PC/gamma-CD complex was high, i.e., +92 kJ/mol, indicating that the phospholipid molecule has to fully emerge from the bilayer before complex formation can take place. The free energy, enthalpy, and entropy of transfer of monoPyrPC from bilayer to gamma-CD complex were close to zero. The absorption, Fourier transform infrared, and fluorescence spectral measurements and lifetime analysis indicated that the pyrene moiety lies inside the CD cavity and is conformationally restricted, particularly when the labeled chain is short. The acyl chains of a PyrPC molecule seem to share a CD cavity rather than occupy different ones. The present data provide strong evidence that the ability of gamma-CD to enhance intermembrane transfer of pyrene-labeled phospholipids is based on the formation of stoichiometric complexes in the aqueous phase. This information should help in designing CD derivatives that are more efficient lipid carriers then those available at present.

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)

Pyrene-fatty acid-containing phospholipid analogues: Characterization of monolayers and Langmuir-Blodgett assemblies.

Abstract The following pyrene-containing phospholipid analogues have been studied: 1-palmitoyl-2(pyren-1-yl)decanoyl-sn-glycero-3-phosphatidylcholine§ (PPDPC), -glycerol (PPDPG), and -PE (PPDPE), 1-palmitoyl-2(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatidylmethanol (PPHPC), 1-octacosanyl-2(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatidylmethanol (C-28-O-PHPM) and 1-octacosanyl-2(pyren-1-yl)octanoyl-sn-glycero-3-phosphatidylmethanol (C-28-O-POPM). These compounds were first characterized by compression isotherms and surface potential measured at an air/water interface, after which conditions for their transfer onto Cd arachidate-coated quartz glass slides were sought. The transferred layers were investigated for their absorption in the range of 300–650 nm. All compounds revealed spectra characteristic for pyrene. It was further found that with the slide at an angle of 45° to the beam, the ratio of absorption of light polarized parallel to that polarized perpendicular to the layer plane, A∥/A⊥, was strongly dependent on the chemical structure of the lipid derivative; however, the surface pressure at which the film was maintained during the deposition had less effect. In fluorescence emission the excimer fluorescence intensity (Ie) exceeded by several times the intensity of emission from the monomeric pyrene species (Im). The most significant new findings of the present study came from preliminary fluorescence decay measurements of transferred layers of PPDPG. These were obtained using time-resolved photon counting with the sample held in vacuo and at a temperature of 10, 200, 250, 280, or 300 K. The observed rapid non-exponential decay of monomer fluorescence at 380 nm was rather insensitive to temperature. The decay of excimer emission at 380 K appeared to exhibit three components of approximate lifetimes of (a) below 0.1 ns, (b) 43 ns and (c) 83 ns. The relative proportions of the component lifetimes were sensitive to temperature. Notably, in the temperature range of 10–300 K the decay of excimer emission was observed immediately following the nanosecond flash, i.e. the kinetics of excimer formation from monomeric pyrene could not be resolved with the instrument used. Therefore, we conclude that the organization of pyrene in the phospholipid liquid crystal monolayer on quartz to be such that redistribution of electronic excitation to yield excimers is allowed within less than 0.1 ns following the formation of the excited monomeric species. This indicates the very close proximity of the pyrene moieties, yet without significant ground state interaction, as suggested by the similarity to the absorption spectra of pyrene in organic solvents.

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.

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.

Characteristics of excimer formation in langmuir-blodgett assemblies of 1-palmitoyl-2-pyrenedecanoylphosphatidylcholine and dipalmitoylphosphatidylcholine

Abstract Langmuir-Blodgett film alloys of PPDPC (1-palmitoyl-2-[10-(pyren-1-yl)]decanoyl- sn -glycero-3-phosphocholine) and DPPC (1,2-dipalmitoyl- sn -glycero-3-phosphatidylcholine) were assembled in varying molar proportions on quartz glass slides. The transferred layers were then characterized according to their pyrene excimer to monomer fluorescence intensity ratio i E / i M and fluorescence quantum yields as a function of film composition. The observed deviation from non-ideal mixing is considered to be due to the formation of regular distribution patterns of PPDPC in a DPPC lattice. The observed critical mole fractions of PPDPC evident as steps in I E / I M versus X PPDPC plots can be accounted for by a model involving a trigonal distribution of pyrenedecanoyl chains in the phospholipid acyl chain lattice. The implications of the distribution of PPDPC as superlattices to the excimer formation mechanism are discussed.

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-phosphatidylcholine 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.

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

SummaryConditions 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 activityvs. PPHPG concentration revealed the enhancement to be due to increased apparentVmax while the apparentKm 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 μm 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.