Tom Thuren

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.

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)

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.

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.

A model for the molecular mechanism of interfacial activation of phospholipase A2 supporting the substrate theory

Changes occurring in the activity of porcine pancreatic phospholipase A2 upon formation of mixed micelles of sodium cholate and the fluorescent phosphocholines 1,2‐di [6‐(pyren‐1‐yl)butanoyl]‐sn‐glycero‐3‐phosphocholine or 1‐[6‐(pyren‐1‐yl)butanoyl]‐2‐[6‐(pyren‐1‐yl)hexanoyl]‐sn ‐glycero‐3‐phosphocholine were studied. A 2‐fold enhancement was observed in the activity of phospholipase A2 towards both pyrene phospholipids upon exceeding the critical micellar concentration of the system. Changes in the pyrene excimer/monomer fluorescence emission intensity ratio coincide with the enhancement of phospholipase A2 activity at the critical micellar concentration. Due to the different effects of micellization on the alignment of the pyrene in the two fluorescent probes conformational changes could be assessed. A model describing possible conformations of these pyrene phospholipid molecules below and above the critical micellar concentration is presented and correlated with the interfacial activation of phospholipase A2.

Hydrolysis of phospholipid monolayers by human spermatozoa. Inhibition by male contraceptive gossypol.

Monomolecular films of phospholipid were used to study the interaction of intact human spermatozoa with model membranes. Exclusively with negatively charged phosphatidylglycerol monolayers rapid penetration of spermatozoa into the monolayer with subsequent hydrolysis of the lipid was triggered by the addition of 5 mM calcium into the medium. The results suggest the localization of a calcium-dependent phospholipase A2 at the outer acrosomal or plasma membrane of human spermatozoa with its active site exposed to the external environment. Preincubation of the cells with 100 microM gossypol completely abolished the ability of human spermatozoa to hydrolyze or penetrate monolayers of phosphatidylglycerol. The inhibition of the phospholipase activity by gossypol may contribute to the unknown contraceptive mechanisms of this non-steroidal male antifertility agent.

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.

Phospholipid-platelet activating factor interactions probed by monolayers, pyrene fluorescence and phospholipase A2

Abstract The interactions of synthetic platelet activating factor with phosphatidylcholine and -glycerol in monolayers as well as in liposomes and the effects of platelet activating factor on phospholipase A 2 -catalyzed hydrolysis of fluorescent phospholipid analogs were studied. Pure platelet activating factor formed stable monolayers at an air-water interface with a collapse pressure at 34.8 mN · m −1 . The isotherm of platelet activating factor was continuous with a lift-off point of 150 A 2 /molecule. Isotherms of mixed platelet activating factor/dilauroylphosphatidylcholine or -glycerol monolayers were continuous and no signs of phase separation were observed. using a stop-flow apparatus, the mixing in liposomes of platelet activating factor and 1-palmitoyl-2-pyrenedecanoylphosphatidylcholine and 1-palmitoyl-2-pyrenedecanoylphophatidylglycerol was observed to be complete within approximately 4.5 and 2.5 s, respectively, when assessed according to fluorescence changes. Increasing amounts of platelet activating factor mixed with 1-palmitoyl-2-pyrenehexanoylphosphatidylcholine decreased the excimer to monomer fluorescence emission intensity ratio indicating the dilution of the local concentration of pyrene in liposomes. Similar experiments with pyrenephosphatidylglycerol revealed a discontinuity in pyrene excimer to monomer emission intensity ratio at a molar ratio of 1:2 of platelet activating factor to phosphatidylglycerol. When dipyrenephosphatidylglycerol was mixed with platelet activating factor, an increase in pyrene excimer to monomer emission intensity ratio was observed between platelet activating factor/phospholipid molar ratios of 1:2 and 3:4 suggesting an alteration in the physicochemical state of the phospholipid analog. In the absence of added CaCl 2 , platelet activating factor could activate the hydrolysis of 1-palmitoyl-2-pyrenehexanoylphosphatidylglycerol by pancreatic phospholipase. A 2 4.5-fold whereas in the presence of added CaCl 2 , platelet activating factor had no effect on the hydrolysis. The hydrolysis of phosphatidylcholine by phospholipase A 2 was inhibited by platelet activating factor regardless of the presence or absence of added Ca 2+ . In the absence of added CaCl 2 , the hydrolysis of two other acidic phospholipid species, phosphatidylserine and phosphatidylinositol, was activated 1.7- and 5.7-fold, respectively, by platelet activating factor. These results suggest that the activation of phospholipase A 2 by platelet activating factor is substrate dependent and probably is due to an altered “quality” of the substrate interface, i.e., the physical state of the substrate.

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.