Chemistry and Physics of Lipids, 55 (1990) 55--60 Elsevier Scientific Publishers Ireland Ltd.

55

Hydrolysis of supported pyrenephospholipid monolayers by phospholipase A 2 Tom

Thuren*,

Kari

K. Eklund,

Jorma

A.

Virtanen

and

Paavo

K.J.

Kinnunen

Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10, SF-O0170 Helsinki (Finland)

(Received August 6th, 1989; revision received and accepted January 29th,1990) 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-~) were subjected to enzymatic hydrolysis. Snake venom phospholipase A2 could hydrolyze the monolayers almost completely while pancreatic phospholipase As 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 -j C. atrox phospholipase A2 could still exert activity whereas porcine pancreatic phospholipase A2 was inactive. Keywords: phospholipase A2; pyrenephospholipid; monolayers; fluorescence microscopy; Langmuir-Blodgett film.

Introduction Phospholipase A 2 (EC 3.1.1.4, PLA2) hydrolyzes sn-2 ester bonds of sn-3 phospholipids [1]. The best characterized P L A 2 species are f r o m pancreas and snake v e n o m s as they are easily obtained in pure f o r m [1]. The amino acid sequences o f pancreatic and snake v e n o m PLA2s show great structural h o m o l o g y [1,2] yet the catalytic mechanisms m a y differ. A n i m p o r t a n t distinction between snake (Crotalides) and pancreatic PLA2s is the dimerization o f the active v e n o m e n z y m e [1]. N o u n a m b i g u o u s evidence has been published so far on the dimerization o f PLA2s o f pancreatic origin [1,2]. PLA2s from different sources hydrolyze

Correspondence to: P.K.J. Kinnunen. *Present address: Dept. of Biochemistry, Bowman Gray

School of Medicine, Wake Forest University, WinstonSalem, NC, U.S.A. Abbreviatiot~s: PLA2, phospholipase A2; PPHPG, 1-palmitoyl-2-(6(pyren-l-yl) hexanoyl) sn-glycero-3-phosphatidylglycerol; RFI, relative fluorescence intensity.

phospholipid m o n o l a y e r s and bilayers at different rates [3--5]. Snake v e n o m PLA2s are active against phosphocholine substrates over a wide temperature range whereas pancreatic PLA2s can hydrolyze these substrates effectively only at the phospholipid phase transition temperatures [ 6 - 8]. Snake v e n o m PLA2s are able to penetrate into and hydrolyze phosphatidylcholine m o n o layers at surface pressures up to 33--35 m N m -1 whereas the highest surface pressure for pancreatic enzymes to hydrolyze these m o n o l a y e r s is approximately 16 m N m -l [3,5]. Using pyrenephospholipid liposomes and m o n o l a y e r s as substrates for PLA2s we have shown that pancreatic enzymes have highest specific activities towards acidic phospholipid species such as phosphatidylglycerol, whereas snake v e n o m P L A 2 prefers phosphatidylcholine, in agreement with earlier data [9--11]. Pyrenephospholipids have been used in studies on properties o f model m e m b r a n e s [12--17] as well as in studies on lipolytic enzymes [ 1 8 - 23]. Pyrenephospholipids f o r m stable m o n o l a y ers which can be deposited o n t o solid supports

0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

56 with the Langmuir-Blodgett technique, whereafter the spectroscopic properties of these monolayers can be studied [24--26]. Supported monolayers provide a novel approach to study the properties of phospholipid monolayers and their interactions with proteins, ions and other types of ligands. We have reported on the hydrolysis by PLA~ and PLA 2 of phosphatidylglycerol monolayers under an aikylated support [27,28]. The interaction of cholera toxin with ganglioside GM~ receptors in supported monolayer system was studied using fluorescence and electron microscopy [29]. Phase separations and transitions of different lipids in monolayers at an air/water interface have been studied with fluorescence microscopy [30--33]. In this paper we report on the hydrolysis of supported pyrenephosphatidylglycerol monolayers by pancreatic and snake venom PLA 2 studied with fluorescence microscopy. Materials and Methods

1-Palmitoyl-2-(6-(pyren- 1-yl)hexanoyl)-sn-glycero-3-phosphoglycerol (PPHPG) was from KSV Chemicals Corp (Helsinki, Finland). Porcine pancreatic PLA 2 was from Boehringer Mannheim (F.R.G.) and snake venom, Crotalus atrox, PLA 2 from Sigma Co. Other reagents were from E. Merck (Darmstadt, F.R.G.).

Preparation of transferred monolayers KSV-2200 surface barostat interfaced with a Sperry PC was used to deposit PPHPG monolayers onto aikylated [34] quartz glass supports. PPHPG monolayers were formed by injecting the lipid onto a clean air/water interface from a chloroform/methanol (9:1, v/v) solution with a microsyringe. Water was filtered with a Milli RO-Milli Q (Millipore Inc., U.S.A.) system and was purged with argon. Surface pressure was increased to 15 or 31 mN m -z, as indicated, whereafter the monolayer was allowed to stabilize for 10 min before transfer onto the support. Two quartz glass plates (10 × 30 mm) alkylated with octadecylsilane [34] were pressed together and dipped through the monolayer at a speed of 4 mm/min. This results in the transfer of a lipid film on one side of the slide only. After the

glass plates were immersed into the water underneath the monolayer, the remaining lipid was removed from the surface by suction. Thereafter the two plates were separated and placed with the transferred monolayers downwards on plastic support bars glued on the bottom of a Petri dish (diameter 85 mm and volume 50 ml) while keeping the plates with the transferred monolayer in water during this procedure [35]. The fluorescence of the PPHPG monolayer on the alkylated glass plate was then monitored with a fluorescence microscope.

Fluorescence microscopy of transferred monolayers The construction of the equipment has been described in detail elsewhere [33]. Briefly, the monolayer is viewed through a quartz glass window above which the glass plates contained in the Petri dish were placed. Under the quartz window is an Eaiing (36 × ) objective focused by a stepper motor. The transferred film is illuminated through this objective. Light source is a 200 W Hg lamp (Oriel). The excitation light is carried by a quartz fiber bundle on a semitransparent mirror. Visible light is cut off using irises and interference filters (Schott). The pyrene excimer fluorescence (at 470 nm) was selected using suitable filters. The image was collected into a Hamamatsu SIT camera head (C 1966-20) connected to a Hamamatsu image analysis system (C 1966) and a video tape recorder (Sony U-Matic VO 5630). The intensity of each pixel was measured and is given numerically as the sum of the total pyrene excimer intensity of the image. The intensity fractions represent the quantitative distribution of fluorescense intensity in the measured area. Accordingly, a bimodai distribution would suggest microscopic lateral heterogeneity even if the patterns would not be directly evident in the observed images. The images were photographed from the TV display and are considered to be representative of the entire film. The monolayer and fluorescense measurements were carried out in a clean room. Phospholipase A t experiments CaC12 or EDTA was added into the subphase in the Petri dish prior to hydrolysis of the trans-

57

ferred P P H P G monolayer. Thereafter the monolayer was observed for approximately 10 s. Enzymatic hydrolysis was started by adding P L A 2 into the aqueous phase. The water phase was mixed by brief pumping with a 200-/al Finnpipette (Labsystems, Finland) repeated at 2-min intervals. Hydrolysis was then followed by observing the monolayer for 10 s every 5 min. In order to minimize photobleaching, the exciting light was excluded f r o m the monolayer by a shutter between the 10-s illumination periods. All hydrolysis experiments were done in the dark at 23 °C. Photobleaching of pyrene excimer fluorescence and background fluorescence was taken into account when calculating the pyrene excimer emission intensities. The enzyme action was quantitated from the area in the video screen that corresponds to 100 /am × 100 /am area of P P H P G monolayer. Mean molecular area per lipid was obtained from the compression isotherms recorded at an air/water interface. The rate of P L A 2 reaction (expressed in f m o l / m i n ) was estimated from the decrease o f pyrene fluorescence assuming a linear correlation between the two-dimensional lipid concentration given in number of molecules per unit area and the second power of excimer fluorescence intensity [25,33,36]. This activity represents the minimal activity of P L A 2 in these conditions. Results Figure 1 shows the decrement o f pyrene excimer fluorescence of the P P H P G monolayers during 10-s excitation periods repeated at 5-min intervals in the absence of enzyme. The intensity of pyrene excimer emission decreased by 5% in 30 min. The intensity of pyrene fluorescence f r o m P P H P G monolayers collected at 15 m N m -~ during C. a t r o x and porcine pancreatic P L A 2 catalyzed hydrolysis is shown in Fig. 1. C. a t r o x P L A 2 hydrolyzed transferred P P H P G monolayers until 9107o o f pyrene excimer fluorescence emission intensity had disappeared (Fig. 1). The rate of hydrolysis was approximately 1.88 fmol min-L Pancreatic P L A 2 hydrolyzed P P H P G monolayers at a slower rate, 0.70 fmol min -~ (Fig. 1). The activity of pancreatic P L A 2

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Fig. 1. Hydrolysis of transferred PPHPG monolayers by PLA2s. Monolayers were collected at 15 mN M-L Incubation medium was purified water, pH 7.0 containing 4 mM CaCI2. Photobleaching of pyrene excimer fluorescence (13 13) of transferred PPHPG monolayers during 10-sillumination periods repeated at 5-rain intervals. ( • • ) Hydrolysis by 20 ~g of C. atrox PLA2. (1 II) Hydrolysis by 20/ag of porcine pancreatic PLA2. Temperature was 23°C. Addition of enzymestarted the reaction.

decreased slowly during the time course and reached zero after 25 min o f incubation. During pancreatic PLA2-catalyzed hydrolysis approximately 50°7o of the fluorescence emission intensity disappeared. Figure 2 depicts intensity diagrams for the hydrolysis of transferred P P H P G monolayers by pancreatic and snake venom PLA~. Intensity fractions represent the intensity of detected signal from each pixel of video screen correlating to the concentration o f P P H P G in the supported film. As the hydrolysis started the proportion of fractions o f lower intensities increased (Fig. 2A). During C. atrox-catalyzed hydrolysis the highest intensity fractions disappeared almost totally (Fig. 2B). Fluorescence micrographs of transferred P P H P G monolayers before, 5 and 10 min after the initiation of C. a t r o x P L A 2 reaction are illustrated in Fig. 3. Pyrene excimer fluorescence emission decreased as ~he hydrolysis proceeded, evident as increasingly light areas in the black and white reproductions of the original colour prints of the recorded pseudocolour images. Approximately half o f the intensity seen in the transferred P P H P G monolayer disappeared

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Fig. 2. Distribution of pyrene excimer emission intensities during PLA2-catalyzed hydrolysis of transferred PPHPG monolayers. Measurements before, 10 rain and 20 rain after the addition of the enzymes. (A) C. atrox PLA 2. (B) porcine pancreatic PLA:. Experimental conditions as in Fig. 1.

during a 10-min incubation with the enzyme. Careful examination of a large number of photographs taken at the end of hydrolysis sometimes showed very faint stripes at an angle of 120 ° to each other (Fig. 3, panel C). Otherwise the disappearance of pyrene excimer fluorescence showed no clear patterns indicative of microstructures at the resolution level available and the observed heterogeneities are likely to be mainly due to somewhat uneven illumination. As controls we performed hydrolysis experiments with C. atrox P L A 2 in the presence of 2 mM E D T A without added C a 2÷. AS illustrated in

a

b

Fig. 4, pyrene excimer emission remained unaltered if E D T A was present. Figure 4 shows an experiment where the hydrolysis by pancreatic P L A 2 was inhibited after 20 min by adding 6 mM E D T A into the incubation mixture containing 4 mM CaC12. We also deposited P P H P G monolayers onto glass supports at a surface pressure of 31 mN m -]. When these P P H P G monolayers were subjected to enzymatic hydrolysis, C. atrox P L A 2 could still cause the disappearance of pyrene fluorescence whereas pancreatic P L A 2 was inactive (Fig. 5).

c

Fig. 3. Pyrene excimer fluorescence images during hydrolysis of transferred P P H P G monolayers by C. atrox PLA 2. The original pseudocolor images are shown as black and white pictures taken (a) prior to the enzyme addition and (b) 5 rain, and (c) l0 rain after hydrolysis by 20/~g of C. atrox PLA~. Experimental conditions as in Fig. 1.

59

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Fig. 4. Inhibition of hydrolysis of transferred PPHPG monolayers by PLAz in the presence of EDTA. Monolayers were collected at 15 mN m-L (o o) Aqueous medium was purified water, pH 7.0 containing 2 mM EDTA, 20 Mg of C. atrox PLA2 started the reaction. In the second experiment ( • • ) the water phase contained 4 mM CaC12. Reaction was started by adding 20 ~g of pancreatic PLA2. After 20 min EDTA was added to a final concentration of 6 raM. Fig. 5. Hydrolysisof PPHPG monolayerstransferred onto a solid support at 31 mN m'L Experimental conditions as in Fig. 1. For (• • ) 20 Mgof C. atrox PLA~, and for ( • • ) 20/~gof pancreatic PLA2was used.

Discussion We have demonstrated that P L A 2 hydrolyzes short chain phosphatidylglycerol monolayers at the interface between an alkylated support and water as well as pyrenephospholipid monolayers at a free air/water interface [11,27]. These findings suggested that P L A 2 should also be able to hydrolyze pyrenephospholipid films transferred onto a solid support. We used an experimental setup where hydrolysis by P L A 2 o f pyrenephospholipid monolayers transferred on a quartz glass support with the Langmuir-Blodgett technique could be followed by quantitative fluorescence microscopy. To avoid photobleaching of pyrene excimer fluorescence, short illumination times were used and the exposure o f the transferred film to exciting light was minimized. This lowered the decrease o f fluorescence intensity due to photobleaching to reasonable levels (Fig. 1). Accordingly, fluorescence intensity decrement o f transferred P P H P G film due to P L A 2 action could be visualized and quantitated (Figs. 1--3). The experiments with E D T A confirmed that the decrease o f pyrene excimer emission was due to PLA2-catalyzed hydrolysis of P P H P G since E D T A is known to inhibit

completely P L A 2 activity (Fig. 4) [37]. No decrement in fluorescence intensity was seen with either pancreatic or snake venom P L A 2 when E D T A was included in the incubation mixture in agreement with previous findings [11]. Fluorescence microscopy images at different times during P L A 2 hydrolysis showed no clear microstructures in the decrement of fluorescence emission. On the other hand, the possibility cannot be excluded that the dimensions of such microstructures is below the resolution limit of our microscopy system (1/am). We used both pancreatic and snake venom P L A 2 to hydrolyze transferred P P H P G monolayers. Our results showed that snake venom PLA2 hydrolyzed transferred PPHPG monolayers faster and more completely than the pancreatic enzyme. This is in contrast to the earlier observations that pancreatic P L A 2 preferred acidic phospholipids, e.g. phosphatidylglycerol and snake venom P L A 2 phosphatidylcholine as the substrate [11]. However, P P H P G molecules in the transferred monolayer on alkylated glass support may have a different conformation than phospholipids in a monolayer at a free air/water interface, which could result in an inhibition of pancreatic PLA2, analogously with our earlier

60

results from 'vertical compression' o f a m o n o layer inhibiting pancreatic P L A 2 [28]. Snake venom P L A 2 are also k n o w n to be active towards phospholipids present in bilayer structures [6--8] which do resemble the supported monolayers used in the present study. Porcine pancreatic P L A 2 cannot hydrolyze phosphatidylglycerol films at surface pressures higher than 28 m N m -l while snake v e n o m PLA~s can hydrolyze monolayers up to surface pressures o f 33--35 m N m -1 [3,5]. C. atrox P L A 2 hydrolyzed supported P P H P G monolayers at 31 m N m -~ at a faster rate than at 15 m N m -1 agreeing with the high surface pressure optimum o f snake v e n o m PLA2s. Due to the higher penetration efficiency o f snake v e n o m PLAES [3,5] the hydrolysis o f supported monolayers at a surface pressure o f 31 m N m -~ by C. atrox P L A 2 is reasonable. Under similar conditions porcine pancreatic P L A 2 was inactive. To summarize, the present data shows that supported phospholipid monolayers provide a new type o f a substrate for studies on phospholipases.

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