From the Department of Zoology, Brigham Young University, Provo, Utah 84602
Received for publication, December 1, 2000, and in revised form, April 2, 2001
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ABSTRACT |
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Artificial membranes may be resistant or
susceptible to catalytic attack by secretory phospholipase
A2 (sPLA2) depending on the physical
properties of the membrane. Living cells are normally resistant but
become susceptible during trauma, apoptosis, and/or a significant
elevation of intracellular calcium. Intact erythrocytes and ghosts were
studied to determine whether the principles learned from artificial
systems apply to biological membranes. Membrane properties such as
phospholipid and/or protein composition, morphology, and microscopic
characteristics (e.g. fluidity) were manipulated by
preparing ghosts under different experimental conditions such as in the
presence or absence of divalent cations with or without ATP. The
properties of each membrane preparation were assessed by biochemical
and physical means (fluorescence spectroscopy and electron and
two-photon microscopy using the membrane probes bis-pyrene and laurdan)
and compared with sPLA2 activity. The properties that
appeared most relevant were the degree of phosphatidylserine exposure
on the outer face of the membrane and changes to the membrane physical
state detected by bis-pyrene and laurdan. Specifically, vulnerability
to hydrolysis by sPLA2 was associated with an increase in
bilayer order apparently reflective of expansion of membrane regions of
diminished fluidity. These results argue that the general principles
identified from studies with artificial membranes apply to biological systems.
Secretory phospholipase A2
(sPLA2)1 is an
enzyme that hydrolyzes the acyl bond at the sn-2 position of
glycerophospholipids, releasing free fatty acids and lysophospholipids.
These hydrolysis by-products are precursors for a variety of chemical
messengers involved in metabolism, host defense, and signal
transduction (1). Secretory PLA2 is of further interest
because of its involvement in a number of disease states. A few
examples of conditions that correlate with an increase in
sPLA2 concentration are inflammation (2-4), sepsis (5),
and cancer (6). Secretory PLA2 plays a partially unknown,
but potentially important role in these diseases. Healthy cells are
resistant to hydrolysis by sPLA2. However, under circumstances involving alterations in membrane structure, cells become
susceptible to sPLA2. The mechanisms that promote changes in the physical state of the membrane and eventually lead to hydrolysis by sPLA2 are not well understood.
Much of the present knowledge involving the induction of hydrolysis by
sPLA2 has been learned from studies using artificial membranes. However, studies with artificial membranes are potentially limiting because they may lack applicability to biological systems. Nevertheless, experiments using vesicles have contributed a number of
insights into the possible physical changes needed for induction of
sPLA2 hydrolysis. For example, it has been hypothesized
that initiation of hydrolysis by sPLA2 consists of two
steps (7-13). The first involves adsorption of sPLA2 to
the membrane and the second requires movement of phospholipids from
their position in the membrane to the active site of the enzyme. Both
of these steps are facilitated by certain alterations in the membrane
including changes that increase negative charge on the outer leaflet,
increase bilayer curvature, diminish phospholipid/neighbor
interactions, and/or promote microheterogeneities in the organization
of membrane lipids (8-10, 13-17). It is reasonable to suppose that
when a biological cell becomes susceptible to sPLA2 it is
because of changes in these properties.
Experiments with cultured cells have led to some information concerning
mechanisms that govern susceptibility. However, there are limitations
to the analysis that can be performed on the data because of the
complexity of cultured cells. Erythrocytes are an intermediate between
artificial membranes and biological systems. They lack the
complications arising from multiple intracellular membranes and
signaling pathways but retain properties generally common to cell
membranes such as a diversity of lipid species, asymmetry between the
bilayer leaflets, membrane proteins, and a cytoskeleton.
Intact erythrocytes are naturally resistant to hydrolysis by
sPLA2. However, microvesicles released by erythrocytes upon
addition of a calcium ionophore are immediately hydrolyzed by
sPLA2 (18). Bilayer curvature obviously distinguishes
erythrocytes and microvesicles, but it is probable that lipid
composition, transbilayer asymmetry, protein distribution, cytoskeleton
content, and other physical properties vary as well (19-24).
Therefore, one or more of these characteristics probably influence
susceptibility. Erythrocytes ghosts represent a model in which several
of these parameters can be manipulated by preparing the ghosts in the
presence of different ions with or without ATP. The purpose of this
study was to use such manipulations to determine whether information concerning induction of susceptibility obtained from studies of artificial membranes pertains to biological membranes.
Materials--
Snake venom sPLA2 (monomeric
aspartate 49 (AppD49) from the venom of Agkistrodon piscivorus
piscivorus) was isolated according to published procedures (25).
The protein was stored as a lyophilized powder at
Acrylodan-labeled fatty acid-binding protein (ADIFAB), bis-pyrene, and
laurdan were obtained from Molecular Probes (Eugene, OR). Factor
Va, factor Xa, and prothrombin were purchased
from Hematologic Technologies, Inc. (Essex Junction, VT). Thrombin substrate II was obtained from Calbiochem (La Jolla, CA).
Glutaraldehyde and osmium tetroxide were procured from Ted Pella, Inc.
(Redding, CA). All other reagents were from standard sources.
Erythrocyte Ghost Preparation--
Blood samples were collected
and stored in EDTA Vacutainers from the Brigham Young University
Student Health Center. These samples were anonymous surpluses from
healthy patients undergoing routine physical exams. Samples were stored
overnight at 4 °C and used the next day. Erythrocytes were washed
three times in phosphate-buffered saline solution (PBS: 137 mM NaCl, 2.7 mM KCl, 10.6 mM
Na2HPO4, 8.5 mM
KH2PO4, pH 7.4), suspended to their original hematocrit (0.75 ml) in MBSS (134 mM NaCl, 6.2 mM KCl, 1.6 mM CaCl2, 1.2 mM MgCl2, 18.0 mM Hepes, and 13.6 mM glucose, pH 7.4, 37 °C) and used for ghost
preparation or stored at 4 °C overnight for experiments.
Washed erythrocytes (1.5 ml) were suspended in 10 ml of one of the
following buffers: PBS diluted to 1/25 in water (PBS ghosts), 1/25 PBS
containing 1 mM MgCl2 (Mg2+
ghosts), 1/25 PBS containing 1 mM MgCl2 and 1 mM ATP (Mg2+-ATP ghosts), or PBS diluted 2/5 in
water with 1 mM CaCl2 (Ca2+
ghosts). After 30 min at 0 °C, 5-fold concentrated PBS alone or
containing 1 mM MgCl2, 1 mM
MgCl2-ATP, or 1 mM CaCl2 was added to the corresponding ghost preparations to restore isotonicity. Next,
suspensions were incubated for 45 min at 37 °C to reseal the
membrane, and sealed ghosts were collected by centrifugation at
2,500 × g for 10 min. The ghosts were washed in PBS
until the supernatant appeared free from hemoglobin. The PBS ghosts,
Mg2+ ghosts, and Mg2+-ATP ghosts were then
resuspended in MBSS, and the Ca2+ ghosts were resuspended
in MBSS without Mg2+ (26) and stored at 4 °C overnight.
To determine membrane protein content, samples were lysed by freezing
in liquid nitrogen, and membranes were isolated by rapid centrifugation
in a microcentrifuge. Membranes were then diluted 1:1 in 0.4 N NaOH and warmed for 30 min at 37 °C. Protein
concentration was determined by the method of Bradford (27).
Acetylcholinesterase Activity--
Membrane sidedness was
determined by acetylcholinesterase activity using an adaptation of the
method of Steck and Kant (28). Red blood cell or ghost samples (10 µg
of membrane protein) were suspended in 600 µl of 100 mM
sodium phosphate (pH 7.5) with enough 5 mM sodium phosphate
(pH 8.0) or 0.2% Triton X-100 to adjust the volume to 700 µl.
Acetylcholine chloride (0.78 mM) and
5,5'-dithiobis-(2-nitrobenzioc acid) (0.63 mM) were added
to the mixture, and the reaction optical density was monitored at 412 nm for 3 min at room temperature. Sidedness was assessed by comparing
acetylcholinesterase activity in the absence of Triton X-100 to that
observed in its presence.
Glyceraldehyde-3-phosphate Dehydrogenase
Accessibility--
Sealing of the membranes was verified by assaying
glyceraldehyde-3-phosphate dehydrogenase activity (28). Red blood cell or ghost samples (10 µg of membrane protein) were suspended to 720 µl of sodium pyrophosphate (30 mM) with 4 mM
cysteine. The volume was adjusted to 820 µl in either 5 mM sodium phosphate (pH 8.0) or 0.2% Triton X-100.
DL-Glyceraldehyde 3-phosphate (1.5 mM) and
sodium arsenate (12 mM) were added to the mixture, and the
spectrophotometer was zeroed. Following subsequent addition of
Phosphatidylserine (PS) Exposure Assayed by Prothrombinase
Activity--
Exposure of PS was measured using a variation of the
method of de Jong and Ott (29). Membrane preparations or erythrocytes were diluted to 3 × 108 cells or ghosts/ml with
either MBSS or hypotonic MBSS (10% MBSS diluted in water). The
preparations suspended in hypotonic MBSS were then frozen in liquid
nitrogen and thawed immediately to lyse the samples before addition to
the reaction mixture. These lysed samples served as a standard
representing 100% PS exposure. To quantify the amount of PS exposed in
the various samples, factor Va (6 nM final),
factor Xa (3 nM final), and 3 × 105 cells were added to 25 µl of buffer containing 10 mM Tris-HCl, 136 mM NaCl, 2.7 mM
KCl, 4 mM CaCl2, and 0.5 mg/ml bovine serum albumin at pH 7.9. This reaction mixture was allowed to incubate for 2 min at 37 °C, after which prothrombin (4 µM final)
suspended in a solution containing 5.6 mM CaCl2
and 0.5 mg/ml bovine serum albumin was added (final volume = 30 µl). The mixture was incubated 5 min at 37 °C, and the reaction
was then stopped by adding the entire mixture to 920 µl of pre-warmed
buffer (50 mM Tris-HCl, 120 mM NaCl, 2 mM EDTA, pH 7.5) in a spectrophotometer cuvette. The
spectrophotometer was zeroed and 50 µl of thrombin substrate (final = 100 µM) were added. The absorbance was
monitored at 405 nm for 6 min. The absorbance of each membrane
preparation was divided by the absorbance of the lysed control to
determine the proportion of PS exposed (i.e. Figs.
1A and 4c).
Fluorescence Spectroscopy--
Membrane preparations were
suspended in 2 ml of MBSS in a fluorometer sample cell to a final
density of about 2 × 106 cells or ghosts/ml. Steady
state fluorescence of laurdan and bis-pyrene was measured using a
Fluoromax (Spex Industries) photon-counting spectrofluorometer. Band
pass was set at 4.25 nm for both monochromators in these experiments.
Simultaneous assessment of fluorescence intensity at multiple
excitation and emission wavelengths was obtained by rapid sluing of
monochromator mirrors using control software provided with the
instrument. Laurdan anisotropy measurements were obtained in the
L-format using a PC1 fluorometer from ISS (Urbana, Il) equipped with
Glan-Thompson polarizers and 16-nm band pass on both monochromators.
Temperature was maintained at 37 °C in all experiments using
circulating water baths. Continuous gentle magnetic stirring preserved
sample homogeneity in both instruments.
Light Scattering--
The amount of light scattering was
assessed as a function of wavelength for each preparation by
synchronous scanning of excitation and emission wavelengths from 250 to
700 nm with a 0-nm offset and a 0.4-nm band pass. This procedure
allowed us to distinguish between variations in size, shape, and
concentration. Control experiments with microscopic beads of uniform
size indicated that the light scattering intensity below 280 nm
normalized to that at 300-350 nm was most useful for determining ghost
size. Relative hemoglobin concentration of each preparation was
determined by comparing the average normalized light scattering between
408 and 416 nm.
Hydrolysis by sPLA2--
Release of fatty acids from
cells was assayed with ADIFAB (65 nM final,
excitation = 390 nm, emission = 432 and 505 nm; Ref. 30 and
31). The results were quantified by calculation of the generalized
polarization (GP) as described (31, 32) and fit to a double exponential
equation by nonlinear regression. The amount of hydrolysis at 100 s after addition of sPLA2 (1 µg/ml) was calculated using
parameter values from the nonlinear regression results. This value was
chosen as a parameter for comparison with the various physical
parameters because it provided information related to both the extent
and rate of membrane hydrolysis.
Membrane Fluidity--
The fluidity of the membrane was
determined with the use of laurdan and bis-pyrene (32, 33). Background
contributed by light scattering of the individual preparations was
subtracted prior to analysis. Laurdan fluorescence (2.5 µM final, excitation = 350 nm, emission = 435 and 500 nm) was used to monitor the overall movement and/or amount of
water in the membrane. Laurdan fluorescence was monitored as a function
of time after addition of the probe and quantified by calculation of GP
(32). Kinetic parameters describing laurdan equilibration with various
environments in the membrane were estimated by fitting the GP values to
a single exponential (see Equation 1 under "Results") by nonlinear
regression. Laurdan anisotropy at 435 nm was used to infer the relative
ability of laurdan to rotate in the membrane as described (8).
Bis-pyrene monomer emission intensity (1.25 µM final,
excitation = 344 nm, emission = 377 nm) was divided by the
intensity of bis-pyrene in water to quantify membrane viscosity.
Two-photon Excitation Scanning Microscopy--
The distribution
of laurdan GP values on ghost membranes was visualized using two-photon
microscopy (34). The two-photon excitation images were collected on an
Axiovert 35 inverted microscope (Zeiss, Thornwood, NY), with a Zeiss
20X LD-Achroplan (0.4 N.A., air) using a titanium-sapphire laser
excitation source (Coherent, Palo Alto, CA) tuned to 770 nm and pumped
by a frequency doubled Nd:Vanadate laser (Coherent, Palo Alto, CA). The
laser was guided by a galvanometer-driven x-y
scanner (Cambridge Technology Watertown, MA) to achieve beam scanning
in both x and y directions. A frequency synthesizer (Hewlett-Packard, Santa Clara, CA) controlled the scanning
rate of 9 s to acquire a 256 × 256 pixel frame that covered approximately a 60 × 60-µm region. Dual images were
collected simultaneously using a beam-splitter, two emission short-pass filters (centered at about 450 and 500 nm), and two detectors for
calculation of GP (32). Laurdan was added to a suspension of ghost
preparations as described above. After equilibration, a two-photon
image was obtained. Secretory PLA2 was then added, and
two-photon micrographs of the same field were acquired at several time
intervals thereafter.
Scanning Electron Microscopy--
Samples were prepared for
scanning electron microscopy by a modification of Schneider's method
(35). Briefly, the preparations were washed in PBS at pH 7.4. Ten ml of
4 × 106 cells/ml were incubated in a jar having a
5.5-cm diameter and allowed to settle onto
poly-L-lysine-coated cover glasses at 4 °C overnight.
Samples were then fixed in 2% glutaraldehyde for 2.5 h. Following
fixation, the cells were washed six times in PBS, fixed in 2% osmium
tetroxide for 2 h at 23 °C, and washed six more times in PBS.
Samples were dehydrated through a graded series of ethanol solutions
(10, 30, 50, 70, 95, and 100%) for 10 min each then washed three times
in acetone. The slides were then subjected to critical point drying,
using liquid carbon dioxide. Finally, samples were sputter coated with
gold for 2 min. Images were obtained on a JEOL JSM 840A scanning
electron microscope.
The electron micrographs were analyzed using three parameters.
These parameters were the membrane size, surface texture, and shape
distortion from the original biconcave disc of erythrocytes. Images
were arranged randomly and labels removed so that scoring of these
parameters could be done without bias. Four raters then visually scored
each image for these parameters. The size of the membrane was measured
in millimeters along the largest diameter and normalized by the
magnification of the picture. The surface texture and the shape
distortion were rated on a scale of 1-5 (5 high). The values obtained
from each of the raters were averaged. Correlations in scores among
raters ranged from 0.72 to 0.99.
Electrophoresis--
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was performed using 10% pre-cast gels in a mini
electrophoresis unit (Mini-PROTEAN 3, Bio-Rad). Buffers and protein
samples from ghost membranes were prepared by the methods of Laemmli
(36) according to instructions provided with the unit. To each well, 20 µg of protein were added. Proteins were separated at 120 V until the
lowest molecular mass marker (29 kDa) reached the bottom of the
gel. Bands were visualized by silver staining.
The gels were photographed using FOTO/Analyst software. A spectrum of
the optical density of the bands along each lane was created with
Bandleader software. Background intensity was subtracted from each
spectrum, and spectrum peaks were aligned by proportional expansion or
contraction of the spectrum for each sample and normalized. The
resultant spectra were divided into 250 equal segments. To avoid
possible bias based on the choice of the segment of the gels used for
normalization, the procedure was repeated four more times using the
intensity of a different segment in each case as the denominator for normalization.
Statistical Analysis--
The relationships between
susceptibility to sPLA2 and the various physical properties
were quantified by linear regression. When relationships were
significant (i.e. p Erythrocyte ghosts were prepared in which membrane properties
hypothesized to be responsible for the level of susceptibility to
sPLA2 were varied. For example, we manufactured ghosts by
including either Mg2+ or Mg2+-ATP in the lysing
and resealing media in order to control the degree to which the normal
phospholipid asymmetry of the bilayer was maintained. The inclusion of
ATP would be expected to support the activity of aminophospholipid
translocase and thus maintain the asymmetry (19). As shown in Fig.
1A, these protocols generated ghosts that differed in the amount of PS exposed on the outer leaflet
of the membrane. In an attempt to obtain additional variation in
membrane properties, we experimented with other preparation conditions
such as the absence of divalent cations or with Ca2+
instead of Mg2+. Fig. 2
demonstrates that these conditions produced different morphological
characteristics among the ghost preparations. Importantly, they also
generated a series of membrane preparations for which the variation in
susceptibility to sPLA2 was high. Fig. 1B
displays the time course of hydrolysis of intact erythrocytes and two
example ghost preparations by sPLA2. Fig. 1C
summarizes the degree of variation in susceptibility among all of the
ghost preparations. Not only did the different types of ghosts differ
from intact erythrocytes in their susceptibility to sPLA2
(p = 0.0005 by analysis of variance), the individual
preparations within each category varied substantially from each other.
Rather than discard preparations that behaved differently from the norm
for a given type of ghost, we elected instead to make use of the
variability by comparing membrane properties with susceptibility for
each preparation independently. In essence, this choice provided a
broad continuum allowing a more complete analysis in which 30 sets of
properties could be compared instead of multiple replicates of only
five sets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Stock
solutions were suspended at a concentration of 100 µg/ml in 50 mM KCl with 3 mM NaN3 as a
preservative and stored at 4 °C.
-nicotinamide adenine dinucleotide (1 mM), the optical
density was monitored at 340 nm for 3 min. Membrane sealing was
verified by comparing glyceraldehyde-3-phosphate dehydrogenase activity in the absence of Triton X-100 to that observed in its presence.
0.05), the analysis was repeated
with removal of data points at the extremes to ensure that overall
trends represented real covariance rather than the influence of
possible outliers. Also, the analysis was repeated again with only the
data from ghosts (no erythrocytes) included. This manipulation ensured
that the relationship observed represented true covariance between the
property in question and susceptibility rather than a simple difference
between erythrocytes and ghosts in general. Only those relationships
that remained significant after these manipulations are reported as
such under "Results." Quantitative comparisons among ghost
preparations and intact erythrocytes were accomplished by one-way
analysis of variance or Student's t test as appropriate.
The combined contribution of more than one physical property to
susceptibility was assessed by multiple regression.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Variability of PS exposure and susceptibility
of erythrocytes and ghosts to hydrolysis by sPLA2.
Panel A, the proportion of PS exposed on the outer bilayer
leaflet for multiple samples of erythrocytes (n = 10),
Mg2+ ghosts (n = 5), and
Mg2+-ATP ghosts (n = 5). Data were obtained
and calculated as explained under "Experimental Procedures." Data
are expressed as the mean ± S.E. The groups were significantly
different by analysis of variance (p < 0.0001).
Individual differences were identified by a Bonferoni post-test
comparing all three permutations of groups (p < 0.01 in each case). Panel B, erythrocytes (solid
squares), Mg2+ ghosts (solid triangles),
and PBS ghosts (open circles) were mixed with
sPLA2, the time courses of fatty acid release were
monitored using ADIFAB, and the data fit by nonlinear regression as
described under "Experimental Procedures." Panel C, the
amount of fatty acid produced at 100 s (calculated as described
under "Experimental Procedures") for multiple samples of each
membrane preparation (n = 10 for erythrocytes,
n = 5 for each of the ghost preparations).
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Fig. 2.
Scanning electron micrograph images
representative of the various preparations. Panel A,
intact erythrocyte; panel B, Ca2+ ghosts;
panel C, Mg2+ ghosts; panel D, PBS
ghosts; and panel E, Mg2+-ATP ghosts. The images
are shown on the same scale.
A series of control experiments were conducted to determine
whether the exposure to PS and/or susceptibility to sPLA2
might be attributed to inside-out or leaky ghosts. Acetylcholinesterase activity was used to judge whether the ghosts were oriented outside out. Fig. 3A displays the
results of acetylcholinesterase assays on each of the ghost and
erythrocyte preparations considered in Fig. 1C. As shown by
the small standard errors, the ghost preparations differed little in
the amount of activity present. Furthermore, they were identical to
native erythrocytes in the level of acetylcholinesterase exposed to the
extracellular medium. This result suggested that the ghosts were
oriented outside-out.
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A possible alternative interpretation of the data in Fig. 3A is that the ghosts were permeable to the assay reagents used. To test this possibility and assess any relationship between membrane leakiness and susceptibility to sPLA2, the integrity of the ghost membranes was assessed by several means. First, examples of each type of ghost preparation were tested for their permeability to hemoglobin (by absorbance of samples and supernatant at 412 nm). The amount of hemoglobin released from the ghosts following >24 h incubation was 3.5 ± 0.4% (mean ± S.E., n = 8). Experiments were completed within a couple of days of preparation of the ghosts. Nonetheless, ghosts retained their integrity much longer in that hemoglobin retention was stable for more than 1 month. Rarely we did observe that ghosts become leaky after extended storage (i.e. >1 month); however, the tendency to eventually lose membrane integrity was not predictable based on the original level susceptibility to sPLA2. Second, ghosts were loaded with propidium iodide during preparation, washed, and incubated overnight. The amount of propidium iodide retained by the ghosts was then assessed by incubation with extracellular DNA before and after membrane lysis by freeze-thaw. For each type of ghost preparation, the transmembrane propidium iodide gradient was maintained during overnight incubation. Third, ghosts prepared in Mg2+ were stored overnight in buffer with or without Ca2+ to identify whether possible permeability to Ca2+ might influence the behavior of the ghosts toward sPLA2. The average levels of susceptibility in these experiments were 0.10 ± 0.02 ADIFAB GP units for ghosts incubated without Ca2+ added to the storage buffer and 0.11 ± 0.02 ADIFAB GP units for ghosts stored in 1.6 mM Ca2+ (mean ± S.E., p = 0.77, n = 3 and 7). Therefore, storage with or without Ca2+ had no influence on the susceptibility of the ghosts. Fourth, the integrity of all of the ghost preparations used in Fig. 1C and the other experiments reported in this paper was assessed by measuring the activity of an intracellular enzyme, glyceraldehyde-3-phosphate dehydrogenase, before and after membrane lysis. The results of these experiments are shown in Fig. 3B. As with the acetylcholinesterase assay, the data for the various ghost preparations were indistinguishable from those obtained for native intact erythrocytes. Nevertheless, since some variation was observed among the samples, we tested whether the observed values for these assays could account for differences in susceptibility to sPLA2 or exposure of PS among the preparations. No correlation was observed for either susceptibility or the fraction of PS exposed (p > 0.15 in each case, n = 30). Therefore, we were confident that observed variations in susceptibility among the ghost preparations were due to factors other than membrane sidedness or leakiness.
Membrane morphology was considered since bilayer curvature has been shown to be a critical factor in determining whether artificial membranes are resistant or susceptible to catalysis by sPLA2. For example, small vesicles composed of phosphatidylcholine are immediately hydrolyzed upon addition of sPLA2, while larger vesicles are only vulnerable after perturbation of the membrane with contaminants (8, 9, 14, 15, 37). Both gross (size and distortion of shape) and fine (surface texture) morphology were scored from micrographs such as those shown in Fig. 2 and compared with the level of susceptibility to sPLA2 for the various preparations. None of these properties predicted the level of hydrolytic attack upon addition of sPLA2 (p = 0.81, 0.84, and 0.52 for size, surface texture, and shape distortion). Size was additionally assessed by light scattering. Comparison of those values with the level of susceptibility verified the result from electron microscopy (p = 0.1).
One obvious distinction between the artificial membranes used
previously to study sPLA2 and biological systems is the
presence of membrane proteins and cytoskeleton. It is therefore
possible that differences in the vulnerability of the various ghost
preparations to sPLA2 are attributable to variations in
protein content. In order to explore this possibility, membrane and
cytoskeleton proteins were isolated from each ghost preparation and
separated by SDS-polyacrylamide gel electrophoresis. In
preliminary experiments, Coomassie Blue staining of the gels reveals
the usual pattern of erythrocyte membrane proteins (spectrin, ankyrin,
bands 3-7). No obvious differences in the content of these proteins
were observed among the preparations. Nevertheless, many proteins do
not stain well with Coomassie Blue due to either sensitivity or heavy
glycosylation. Since it seemed important to consider all membrane
proteins, we chose to focus our analysis on gels that had been
developed by silver staining. Densitometric profiles of sample gels
from ghost preparations (curves a-d) and erythrocyte
membranes (curve e) are displayed in Fig.
4. Due to the large number of proteins
visible with silver staining, the individual bands apparent with
Coomassie Blue staining are more difficult to discern. The approximate
locations of some of those proteins along the gels (based on Coomassie
Blue staining) are labeled on the figure.
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Each preparation displayed a somewhat different pattern of protein bands on the gels (Fig. 4). To identify which, if any, of these differences could account for the differences in susceptibility, the staining intensity of each of 250 equal segments of the gel was compared with the susceptibility of the corresponding preparation to sPLA2. In general, no consistent relationships among the 20 samples of ghosts were identified. In a few cases, correlations that appeared significant statistically were observed, but for each of these, the significance was abolished by removal of a single point from the data set. Thus, all apparent relationships observed were excluded based on the criteria explained under "Experimental Procedures." To avoid possible bias based on the choice of the segment of the gels used for normalization, the procedure was repeated four more times using the intensity of a different segment in each case as the denominator for normalization. This repetition validated the interpretation that protein content of the various preparations was not a determinant of susceptibility. A similar result was found when comparing the hemoglobin content (assessed from light scattering spectra) to susceptibility. A significant correlation was identified, but it was abolished when erythrocyte samples were excluded from the analysis (see "Experimental Procedures").
It is clear from studies with artificial membranes that microscopic physical properties of the membrane are the major factors responsible for the level of susceptibility to hydrolytic attack (8-10, 13-17, 37). Prominent among these properties is membrane surface charge (15, 16). Specifically, membranes that contain excess negative charge are more susceptible to attack because of important structural relationships between the membrane surface and the interfacial recognition region of the enzyme (38-40). Additional properties shown to be relevant include the membrane phase state and dynamics, the strength of phospholipid/neighbor interactions, and the presence of compositional microheterogeneities (8-10, 14, 16, 17, 37). Fluorescent probes such as laurdan and pyrene probes have been useful in delineating some of these relationships (8, 9, 15).
To investigate the possibility that changes in membrane surface charge
are involved in determining the susceptibility of red blood cell
membranes to sPLA2, we assayed the degree of exposure of
the anionic phospholipid, PS on the membrane surface for erythrocytes and the various ghost preparations using an assay for
PS-dependent conversion of prothrombin to thrombin. Figs.
5, A and B,
demonstrate that the assay successfully distinguished PS exposed on the
outer leaflet from total PS exposed after lysis of the cells by
freeze-thaw in hypotonic medium. The degree of exposure was a
significant predictor of the level of susceptibility of each
corresponding preparation to sPLA2 (Fig.
5C).
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Additional microscopic membrane properties were investigated using the fluorescent probes laurdan and bis-pyrene. Laurdan emission at two wavelengths (435 and 500 nm) was observed as a function of time after addition of the probe. The data were quantified by calculating the generalized polarization value (32). The GP measurement is a function of the degree of solvent relaxation experienced by the excited fluorophore. In general, an increase in the value of GP represents a diminution in the degree and/or rate of solvent relaxation suggesting a decrement in membrane water content and/or membrane fluidity (32). These measurements also allowed us to assess the dynamics of interaction of these probes with the membranes. Membrane order was further assessed by laurdan anisotropy and by the intensity of bis-pyrene monomer emission (33). Possible formation of phospholipid microheterogeneities in the membrane was examined by two-photon excitation scanning microscopy with laurdan as the probe.
The value of laurdan GP displayed time-dependent changes
reflecting the interaction of the probe with erythrocyte or ghost membranes (Fig. 6). This slow
equilibration of the probe with the membranes appeared to follow
first-order kinetics in that it was well fit to the following
equation.
![]() |
(Eq. 1) |
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The coefficient represents the magnitude of change in GP during
binding. Using the value of
and the information described above, we
calculated the value of GP for laurdan bound to the membrane
(GPm) with Equation 2.
![]() |
(Eq. 2) |
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The positive relationship between laurdan GPm and
susceptibility suggested that an increase in membrane molecular viscosity could be important for rendering the membrane receptive to
attack by sPLA2. To explore this possibility further, we
also compared the value of two other parameters sensitive to membrane order: laurdan anisotropy and normalized bis-pyrene monomer
fluorescence. As shown in Fig. 8, both
measurements also suggested a positive relationship between membrane
order and susceptibility to sPLA2. Interestingly, it
appeared that bis-pyrene and laurdan fluorescence reported
distinguishable properties of the membrane since the values obtained
with each probe did not correlate with each other.
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Two-photon microscopy allowed direct confirmation of the steady state
fluorescence results obtained with laurdan (i.e. Fig. 7).
Figs. 9, A and B,
display two-photon images of erythrocytes and Mg2+ ghosts
as examples of preparations with low and high susceptibility. Histograms representing the frequency distribution of GP values for
multiple images such as those shown in Fig. 9, A and
B, are exhibited in Fig. 9C. The absolute values
of GP were not the same when obtained with the two-photon technique
compared with the results shown in Figs. 6 and 7. This is due to
differences in the optics of the two detection systems. However, the
increase in average GP between erythrocytes and susceptible ghosts was quantitatively similar for both methods.
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The two-photon technique also allows one to identify changes in the
microheterogeneity of the membrane. Such changes appear as alterations
in the number and/or size of domains with distinct physical properties
illustrated by the spatial distribution of GP values (34). Inspection
of images such as those shown in Fig. 9 reveals that the value of GP
was not homogeneous across the surface of the cells and ghosts. This
microheterogeneity is verified quantitatively by the breadth of the
frequency distribution of GP values (Fig. 9C). Comparison of
the frequency distribution did not indicate the presence of gross
differences in the degree of membrane heterogeneity between resistant
and susceptible membranes. However, repeated imaging of the same cells
or ghosts before and after mixing with sPLA2 suggested that
membrane heterogeneity may be an important factor in determining loci
of membrane attack by the enzyme. Fig.
10 displays such a series of images of
the same ghost. As illustrated by the arrows, regions of the
membrane with higher GP values expanded systematically and became more ordered following sPLA2 addition. Inspection of multiple
images such as these confirmed this observation.
|
Based on the data obtained, three distinguishable factors appeared
relevant as determinants of membrane susceptibility to sPLA2: the amount of PS exposed on the outer leaflet, the
order of the membrane assessed by bis-pyrene, and the value of laurdan GPm. We used multiple regression analysis to determine whether
these factors were sufficient to account for the variation in the
degree of membrane hydrolysis catalyzed by sPLA2 among the
different preparations and to identify the relative contribution of
each. These three parameters were considered together as well as the
all the permutations of pairs. Incorporating all three parameters
provided the best fit, as one would expect (Table
I). The fact that all three parameters
contributed significantly to the fit suggested that all three were
relevant. The values of the F-statistic and correlation coefficient
indicate that these three variables accounted well for the observed
data. After adjusting for the number of independent variables, the
value of the square of the correlation coefficient was 0.79, suggesting
that only 21% of the variation among samples was due to experimental
error or unexplained factors. Hence, the p value for the
regression was extremely low (1 × 109). Since the
range of measured susceptibility was 0.2 units (see Fig. 5, for
example), the 21% unexplained in our analysis would represent about
0.04 units. The best estimate of the range of random population
variation was about 0.02 units based on data from native erythrocyte.
Thus, at least half of the 0.04 units for which our model could not
account was probably a result of individual and unidentified
differences among donors and/or experimental error. In terms of
relative contributions to the regression slope, GPm was the
major determinant in that it accounted for almost twice as much of the
variation as either of the other two factors.
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DISCUSSION |
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Much of the understanding of relationships between membrane properties and susceptibility to sPLA2 has been obtained using artificial membranes of defined composition as models. These numerous studies have identified a broad range of phenomenology about which a few key generalizations can be made. First, for the enzyme to be active on the surface of a membrane, the enzyme must first adsorb to the surface, and phospholipids within the bilayer must then migrate up into the active site of the bound enzyme (7-9, 12-15, 42). Second, changes in membrane physical properties that promote one or both of these events render the membrane more susceptible to catalysis (8-10, 14-17). The properties found to be important for inducing susceptibility can be summarized in two categories: those that promote negative charge at the bilayer surface and those that perturb the interactions among phospholipid molecules (9, 10, 13-17).
The purpose of this study was to use erythrocyte ghosts as simple models to determine whether these same principles apply to biological membranes. Properties that distinguish biological membranes from artificial bilayers were manipulated and compared systematically to the susceptibility of the corresponding membranes to sPLA2. Among the properties evaluated, two appeared related to the level of susceptibility. They were membrane order and exposure of the anionic phospholipid, PS, on the cell exterior. As discussed below, these characteristics correspond to some of those previously found to promote hydrolysis of artificial membranes by sPLA2. Other major properties that distinguish artificial from biological membranes appeared not to be important, i.e. membrane morphology and cytoskeletal and membrane protein content and composition.
The presence of negative charge on the outer membrane leaflet is important for the induction of hydrolysis of artificial vesicles by sPLA2 and appeared to be relevant to hydrolysis of ghost membranes (Refs. 16 and 38-40 and Fig. 5). Under physiological conditions, erythrocytes have an asymmetrical distribution of phospholipids with zwitterionic lipids, phosphatidylcholine and sphingomyelin, constituting the majority of phospholipids on the outer leaflet and phosphatidylserine and phosphatidylethanolamine concentrated on the inner leaflet (43). Under some circumstances, such as an increase in intracellular Ca2+, during normal cell aging, or in sickle cell anemia, PS and phosphatidylethanolamine migrate to the outer monolayer (44-48). Exposure of PS presumably could promote susceptibility under these conditions by enhancing the negative charge of the cell surface.
The conclusion that changes in membrane order play an important role in the susceptibility of the ghosts was based on three types of fluorescence measurements: laurdan GPm, laurdan anisotropy, and normalized bis-pyrene monomer fluorescence. The increased value of laurdan GPm observed with increased susceptibility suggests a decrease in the mobility of membrane water molecules and/or a decrement in the amount of water in the membrane, either of which may be caused by an increase in bilayer viscosity. This interpretation seemed to be validated by anisotropy measurements assessing rotational motion of laurdan in the membrane and by bis-pyrene monomer fluorescence which is sensitive to the translational movement of molecules at the level of the phospholipid acyl chains (Figs. 7 and 8, Ref. 33).
Closer scrutiny of the results with laurdan and bis-pyrene argued that the two probes were not reporting identical phenomena. The correlation in values between the two was very poor (r = 0.1, p = 0.6) suggesting that the two observations were independent. In agreement with that assessment, both laurdan GPm and bis-pyrene monomer fluorescence contributed significantly in the multiple regression analysis, which would not be expected if they were redundant measurements (Table I). Independence of results with these two probes is not unprecedented (8). The basis for the independence of these measurements probably lies in one or both of two distinctions between the biophysics of the probes. First, they assay the ability of molecules to diffuse on two different scales; bis-pyrene assesses diffusion of molecules the size of phospholipids, whereas laurdan assays water movement. Second, the vertical depth reported by the two probes is different with bis-pyrene probably located deep in the region of the phospholipid acyl chains (33) and laurdan positioned more superficially (49). Laurdan anisotropy correlated better with bis-pyrene fluorescence (r = 0.5, p = 0.005) than with GPm (r = 0.37, p = 0.04). Based on this result, it seems likely that the relevant distinction between bis-pyrene fluorescence and GPm is not the depth of the probe in the membrane. Thus, it appears that one must subdivide the effect of membrane order on susceptibility into two categories representing fluidity of membrane molecules and accessibility of the bilayer to water.
It may seem counterintuitive that a membrane in a state of increased order would be more vulnerable to attack by sPLA2. However, the assumption that fluid lipids would be hydrolyzed more readily than ordered lipids is an oversimplification not supported by data obtained with artificial membranes. For example, bilayers composed of pure phosphatidylcholine are most vulnerable to attack when the membrane is in a state intermediate between fluid and ordered, such as that observed at the membrane phase transition temperature (9, 14, 17). Therefore, if the membrane is fluid, lowering the temperature to make it more ordered can improve susceptibility. For example, small unilamellar vesicles composed of saturated phosphatidylcholine are much more susceptible to sPLA2 when in the ordered gel phase than in the fluid liquid crystalline phase (11, 37). Artificial membranes can also be made more susceptible by adding certain lipophilic contaminants to the bilayer. Two examples are fatty acid and diacylglycerol. In both cases, the addition of the contaminant causes the membrane to become more ordered under conditions at which hydrolysis is promoted (9, 14, 15). It could be argued that the effect of the fatty acid to promote hydrolysis is due to its negative charge; however, it is equally or more effective at making the membrane susceptible when in the protonated (non-charged) form (15). Finally, microscopic images of hydrolysis of monolayers composed of coexisting solid-phase and liquid-phase phospholipid domains revealed that sPLA2 preferentially attacks the more ordered solid-phase domains (50). The generalization that emerged from the studies with artificial membranes was that it is not the fluidity of the membrane per se that determines susceptibility, but rather it is the organization of lipids within the bilayer that appears critical. Perturbations that promote heterogeneity of structure either through compositional domains or dynamic diversity of physical state create the environment optimal for sPLA2 activity (9, 10, 14-17). It is thought that this heterogeneity produces defects and/or fluctuations in the membrane surface structure that promote increased binding of sPLA2, activation of the enzyme, and/or facilitation of phospholipid movement into the active site of the enzyme (8-10, 13-17, 42).
The two-photon micrographs in Fig. 10 suggest that hydrolysis of biological membranes may be sensitive to boundaries around ordered membrane domains as it is in artificial membranes (50). The data do not distinguish whether sPLA2 attacked regions of lower GP causing them to shrink or regions of higher GP causing them to expand. Resolution of this issue will require future studies. Nevertheless, the results support the interpretation that the capacity of the membrane for enzymatic attack by sPLA2 depends on changes to the membrane structure that involve reorganization and/or altered physical properties of the molecular environment. From this perspective, the similarity in behavior to that observed with artificial systems is striking (8-17, 37, 50).
In summary, we have provided evidence supporting the conclusion that
the general biophysical principles that govern the susceptibility of
artificial membranes to sPLA2 pertain to biological
membranes. This important observation emphasizes that the detailed
biophysical investigations accomplished with model systems can be
applied to biological systems. Whether these principles are responsible for the differential susceptibility of normal healthy cells
versus those in apoptosis (51, 52) or with elevated
intracellular calcium (31, 53) remains to be investigated. A step
toward resolving that question is achieved in the accompanying article (54) addressing the relationship between physical changes that occur in
erythrocytes treated with calcium ionophore and hydrolysis of the cell
membrane by sPLA2.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of Scott Weed at the Department of Zoology, and Dr. John Gardner of the Electron Microscopy Laboratory at Brigham Young University. Two-photon scanning microscopy experiments were performed at the Laboratory for Fluorescence Dynamics, Urbana, IL; gratitude is expressed to Drs. Theodore Hazlett, Enrico Gratton, and Susana Sanchez for providing technical assistance and access to the facility for these experiments.
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FOOTNOTES |
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* This work was supported by Grant MCB-9904597 from the National Science Foundation (to J. D. B.) and by a graduate fellowship from the Office of Graduate Studies at Brigham Young University (to F. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 801-378-8160;
Fax: 801-378-7499; john_bell{at}byu.edu.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M010879200
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ABBREVIATIONS |
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The abbreviations used are: sPLA2, secretory phospholipase A2; ADIFAB, acrylodan-labeled fatty acid-binding protein; bis-pyrene, 1,3-bis-(1-pyrenyl)propane; laurdan, 6-dodecanoyl-2-dimethylaminonaphthalene; PBS, phosphate-buffered saline solution; MBSS, balanced salt solution; PS, phosphatidylserine; GP, generalized polarization.
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