(Received for publication, October 28, 1994; and in revised form, December 15, 1994)
From the
Salt-washed (0.6 M NaCl) zymogen granule membranes
(ZGM) of rat pancreatic acinar cells were utilized to identify and
characterize membrane protein(s) responsible for phospholipase and
lysophospholipase activities. Five major bands were identified in
salt-washed ZGM by Coomassie Brilliant Blue. A 70-kDa protein with
enzymatic activity was retained in significant quantities after several
washes with 0.6 M NaCl but could be displaced from ZGM by 2 M NaCl or by 100 mg/ml heparin. By contrast, GP2, an integral
membrane protein, was not displaced under these conditions. These
findings suggest that the enzyme is a peripheral membrane protein of
ZGM. Renaturation of ZGM proteins following electrophoresis revealed
that the 70-kDa protein possessed phospholipase activity.
Identification of the 70-kDa protein as a membrane-associated carboxyl
ester hydrolase was based upon: (a) the use of a specific
polyclonal antiserum, (b) N-terminal sequence, (c)
twodimensional gel analysis, (d) enzymatic characterization,
and (e) co-localization to an area of a non-reducing gel
containing significant phospholipase activity. Other ZGM proteins,
namely GP2 and GP3, could not be demonstrated to possess phospholipase
activity under the experimental conditions employed. Our finding that
carboxyl ester hydrolase from ZGM exhibits PLA and
lysophospholipase activities represents the first identification and
characterization of a protein responsible for phospholipase activity in
secretory granule membranes.
Phospholipases play a key role in diverse cellular processes
such as secretion by catalyzing the hydrolysis of sn-1 and sn-2 fatty acid acyl bonds of phospholipids to liberate free
fatty acids and lysophospholipids (Dennis et al., 1991).
Despite the fact that phospholipase activities reside on GM derived
from neuronal, endocrine, and exocrine cells (Moskowitz et
al., 1982; Husebye and Flatmark, 1987; Rubin et al.,
1990, 1991; Hildebrandt and Albanesi, 1991; Mizuno et al.,
1991), this activity has not been attributed to a specific granule
membrane protein. The zymogen granule membrane (ZGM) ()of
rat exocrine pancreas provides a particularly useful model system to
investigate which specific proteins might be responsible for
phospholipase activities of secretory granule membranes. Examination of
SDS-PAGE protein profiles of ZGM from Sprague-Dawley rats from several
laboratories has revealed the presence of five major protein bands that
are detectable with Coomassie dye (MacDonald and Ronzio, 1972; Ronzio et al., 1978; Paquet et al., 1982; LeBel and Beattie,
1984). Two of the major proteins are glycoproteins with molecular
masses (depending upon the gel separation system) between 74 and 92 kDa
(GP2) and between 54 and 58 kDa (GP3). The three other major proteins
have molecular masses of approximately 70, 50, and 30 kDa. Although the
functions of these ZGM proteins are unknown, GP2 and GP3 are related to
proteins that exhibit lipase activities. We have recently shown that
GP2 shares immunological and sequence similarity with PLA
,
which suggests its possible involvement in phospholipid hydrolysis
and/or binding (Withiam-Leitch et al., 1993). GP3 has
recently been cloned, and its deduced sequence is highly homologous to
triacylglycerol lipases (Wishart et al., 1993).
Carboxyl
ester hydrolase (CEH) is a 70-kDa secretory enzyme of exocrine pancreas
that catalyzes the digestion of dietary fats in the intestine (Wang and
Hartsuck, 1993). Although CEH has a recognized role in the hydrolysis
of cholesterol esters and neutral lipids (Rudd and Brockman, 1984), it
is also capable of hydrolyzing lysophospholipids and phospholipids,
including phosphatidylcholine (PC) (Lombardo et al., 1980;
Rudd and Brockman, 1984; Wang and Hartsuck, 1993). In this report, we
demonstrate that ZGM exhibits two Ca-independent
phospholipase activities of the PLA
and lysophospholipase
types. Both of these activities were found to be specifically
associated with the major protein band of 70 kDa. Based upon enzymatic,
immunological, and N-terminal sequence data, as well as two-dimensional
gel analysis, we conclude that this protein is a membrane-associated
form of carboxyl ester hydrolase.
The following buffers were used in the
standard assay to determine the pH dependence of ZGM phospholipase
activity: 50 mM sodium acetate-acetic acid (pH 3.5-5.5),
MES (pH 6.5), MOPS (pH 7.0), and Tris-HCl (pH 7.5-10.0).
Ca dependence was determined by adjusting the free
Ca
concentration in the standard assay between 0 and
5 mM by the use of Ca
-EGTA buffers as
described by Fabiato(1988).
For experiments to determine the
relative contribution of the PLA and PLA
pathways, the following substrates were used:
1-[1-
C]palmitoyl-2-[1-
C]palmitoyl
phosphatidylcholine (1*2*PC, where asterisk indicates point of
C labeling) (112 µCi/µmol) and
1-palmitoyl-2-[1-
C]palmitoyl phosphatidylcholine
(1,2*PC) (55 µCi/µmol). The generation of labeled lysoPC from
1*2*PC could be due either to PLA
(i.e. 2*lysoPC)
or PLA
(i.e. 1*lysoPC). The generation of labeled
lysoPC from 1,2*PC can only result from PLA
activity (i.e. 2*PC). Subtracting the amount of lysoPC derived from
1,2*PC (PLA
) from the lysoPC produced from 1*2*PC
(PLA
+ PLA
) yields the amount of lysoPC
produced by PLA
activity. Experiments to detect
lysophospholipase activity were performed using
1-[1-
C]-stearoyl lysoPC (56 µCi/µmol) in
the standard assay.
Phospholipase assays were performed on samples
renatured from SDS-PAGE by adding
1-stearoyl-2-[1-C]arachidonyl PC in 5 mM MOPS (pH 7.0) to 0.75-1.0 ml of protein solution to give a
final PC concentration of 20 µM. The assays were carried
out for 3 h at 37 °C. The extended incubation time was necessitated
by the fact that CHAPS, which was required in the renaturation buffer
to recover measurable enzyme activity, reduced specific phospholipase
activity by 80-90%. Recovery of ZGM phospholipase activity after
elution from gels ranged from 19-28% (23.2 ± 2.6%, n = 3).
Figure 1:
Membranes derived
from zymogen granules exhibit five major protein bands. Zymogen
granules were isolated from 10 adult rat pancreata as described under
``Experimental Procedures.'' A portion of this preparation
(approximately 100 µg of protein) of intact zymogen granules was
loaded on lane1. This represents 1% of the total
yield of zymogen granules. A soluble fraction containing secretory
contents (lane2) and a membrane pellet (lane3) were generated by lysis of the granules in 25 mM Tris-HCl (pH 8.2) followed by centrifugation at 100,000
g. The pellet, composed of lysed membranes, was washed
in 0.6 M NaCl to remove weakly bound material and pelleted
again (lane4). In each of the lanes, approximately
1% of the total corresponding fraction (intact granules, soluble
content, unwashed, and salt-washed membranes), was subjected to
electrophoretic separation to compare the pattern of protein bands
relative to the starting material in lane1.
Preparation of ZGM in this manner consistently produced protein
patterns consisting of five major protein bands that were readily
identified by Coomassie Brilliant Blue stain after separation by
SDS-PAGE under reducing conditions (Fig. 1). Molecular weight
estimates of these five protein bands differed slightly depending on
whether the acrylamide concentration used in the gel was 7.5 or 10%. In
experiments conducted using 10% acrylamide gels, the estimates (n = 3) of molecular weight obtained by regression analysis
were: 91.7 ± 4.3, 69.8 ± 2.6, 58.3 ± 3.1, 50.2
± 0.6, and 30.3 ± 0.9 kDa. For convenience, these
proteins will be referred to as 92, 70, 58, 50, and 30 kDa. The 92-kDa
protein has been identified by Western blotting as GP2 (Withiam-Leitch et al., 1993; also see below). The observation that washing of
ZGM with 0.6 M NaCl removed sPLA, amylase, and
other secretory proteins from ZGM pH 8.2 is important for two reasons.
First, washing with high salt produced a protein profile consistent
with previous studies (MacDonald and Ronzio, 1972; Ronzio et
al., 1978; Paquet et al., 1982; LeBel and Beattie, 1984).
Second, our ZGM preparation is of sufficient purity to study ZGM
phospholipase activity specifically associated with the membranes.
Figure 2:
Analysis of radiolabeled lipid products
produced by ZGM phospholipase activity. The standard assay (pH =
7, 1 mM EGTA; see ``Experimental Procedures'' for
additional details) was performed in the absence (lane1) and presence (lane2) of 0.6 M NaCl-washed ZGM using
1-stearoyl-2-[1-C]arachidonyl PC as substrate.
The lipids were then separated using thin layer chromatography, and the
labeled lipid products identified by autoradiography. In panelA, lipids were separated using absolute ether/petroleum
ether/acetic acid (50:50:1) to optimize the separation of neutral
lipids. In panelB, lipids were separated using
chloroform/methanol/water (92:49:8) to optimize the separation of
phospholipids. The position of the lipid standards, which were run in
adjacent lanes, were identified by iodine staining. The various lipid
products are abbreviated as follows: DAG, diacylglycerol; FFA, free fatty acids; LysoPC,
lysophosphatidylcholine; MAG, monoacylglycerol; NL,
neutral lipids; PA, phosphatidic acid; PC,
phosphatidylcholine; SM, sphingomyelin. This figure is
representative of three independent
experiments.
ZGM-mediated phospholipase activity showed a sharp pH profile, with
a pH optimum of 7.0 (Fig. 3) and no requirement for
Ca over the concentration range of 0-1 mM (data not shown). In fact, the presence of Ca
slightly inhibited (
20-25%) phospholipase activity at
concentrations ranging from 1 µM-1 mM. The
generation of lysoPC from the PC substrate (labeled on the fatty acid
in the sn-2 position) must be a consequence of a
PLA
-type activity. The labeled arachidonic acid identified
was not generated by phospholipase C ((PLC)/diacylglycerol lipase
activity) or phospholipase D (phospholipase D/phosphatidic acid
phosphohydrolase activity) because of the absence of labeled
diacylglycerol and phosphatidic acid, respectively (Fig. 2).
Figure 3: pH profile of ZGM phospholipase activity. Buffer systems employed were as follows: sodium acetate-acetic acid (pH 3.5-5.5), MES (pH 6.5), MOPS (pH 7.0), and Tris-HCl (pH 7.5-10.0). All buffers contained 1 mM EGTA. Salt-washed ZGM (0.6 M NaCl) were used to measure phospholipase activity. Enzyme activity was quantified as the total amount of PC breakdown, which is the sum of the amount of arachidonic acid and lysophopholipid produced. Data are expressed as the percent of maximal PC hydrolysis. Values are means (± S.E.) for three independent experiments.
To evaluate the roles of PLA or
PLA
/lysophospholipase in the liberation of free
arachidonate, three sets of experiments were performed. First, direct
measurements of phospholipase activity associated with salt-washed ZGM
via differentially labeled PC substrates (see ``Experimental
Procedures'') demonstrated that greater than 90% of the initial
breakdown of PC is via a PLA
mechanism, with only a minor
contribution from a PLA
pathway (Table 1). In
addition, an analysis of the time course of ZGM phospholipase activity (Fig. 4) revealed that the initial rate (5-20 min) of
2*lysoPC accumulation (0.98 nmol
min
mg
) was higher than the initial rate of AA* release
(0.30 nmol
min
mg
). At later time points (30-45 min), the
rate of AA* production increased (1.10 nmol
min
mg
), after which the rate of lysoPC
accumulation reached a plateau. This finding is consistent with free
AA* being formed from PC indirectly by a
PLA
/lysophospholipase pathway. That is, the rate of free
AA* formed by lysophospholipase activity is increased as the pool of
2*lysoPC is augmented by PLA
activity. Labeled lysoPC
levels reached a steady state due to an equilibrium between its
production from PC breakdown and its degradation due to
lysophospholipase activity. Finally, the additional finding that
salt-washed ZGM were also capable of hydrolyzing labeled lysoPC with an
initial rate (0.83 nmol
min
mg
) comparable to the rate of appearance of AA*
generated from PC after 30-45 min (Fig. 4) substantiated
the presence of a lysophospholipase. These collective findings
demonstrate that the major phospholipase activities of ZGM involve
Ca
-independent PLA
and lysophospholipase
activities, with only a minor contribution from a PLA
pathway.
Figure 4:
Time course of ZGM phospholipase activity. Salt-washed (0.6 M NaCl) ZGM were added to the
standard calcium-free assay mixture containing exogenous substrate for
the indicated time intervals, and the lipid products separated by thin
layer chromatagraphy using chloroform/methanol/water (92:49:8). The
amount of PC breakdown products formed is expressed as nmol
mg
protein. This figure represents the mean of three
independent experiments. Each experiment yielded qualitatively similar
curves. For clarity the standard errors are not shown, but in no case
were they more than 15% of the mean. AA, arachidonic acid; LPC, lysophosphatidylcholine.
Figure 5: Increasing concentrations of NaCl identify a membrane-associated CEH. ZGM isolated by pH 8.2 lysis of ZG were incubated with various concentrations of NaCl for 30 min at 4 °C. The salt-washed ZGM were then pelleted by ultracentrifugation. The pellets were solubilized in equal amounts of SDS sample buffer and prepared for immunoblotting with anti-CEH (A) and anti-GP2 (B). Lane1, no NaCl; lane2, 0.15 M NaCl; lane3, 0.30 M NaCl; lane4, 0.45 M NaCl; lane5, 0.60 NaCl; lane6, 0.80 M NaCl; lane7, 1.0 M NaCl; lane8, 2.0 M NaCl; lane 9, ZGM that had been washed twice in 0.6 M NaCl; lane 10, ZGM that had been washed three times in 0.6 M NaCl. This result is representative of three independent experiments. The mean values (± S.E.) for the three experiments obtained by laser densitometry (LKB Ultrascan) illustrated in C are represented by filledcircles and unfilledcircles for mCEH and GP2, respectively.
The inability of high salt to remove additional CEH was not due to insufficient washing, as repeated washings in 0.6 M NaCl did not produce an additional decrease in the amount of CEH associated with ZGM (Fig. 5A, lanes 9 and 10). Increasing the NaCl concentration to 2.0 M produced an additional decrease in the amount of CEH remaining on ZGM (<5%; Fig. 5A, lane8). Immunoblots of these same fractions with GP2 antiserum (GP2 is an integral membrane protein) show that the recovery of ZGM in each of these fractions was essentially equal (Fig. 5B). Thus, the changes in CEH associated with ZGM observed after high salt washes were not due to variability in the recovery of ZGM during the experimental procedures.
These data indicate that the CEH which associates with ZGM isolated without salt washing has two components. One can be removed by low concentrations of salt (0.15-0.45 M NaCl) and probably represents nonspecific adsorption of secretory CEH (sCEH). A significant proportion of the total CEH, however, remains tightly associated with the ZGM in salt-concentrations up to 1.0 M. This latter form of the enzyme, which will heretofore be referred to as mCEH, is probably a peripheral membrane protein.
The molecular mass of ZGM-associated CEH (mCEH), as determined by immunoblotting, coincides precisely with one of the five major ZGM proteins with an apparent molecular mass of 70 kDa (cf.Fig. 1and Fig. 5A). To determine further whether the 70-kDa protein of ZGM is composed of mCEH, ZGM proteins were blotted onto PVDF membrane, and the 70-kDa band cut out and subjected to amino acid sequencing. The N-terminal sequence of mCEH was determined to be A-K-L-G-A-V-Y-T-E-G, which exhibits 100% identity to the N-terminal sequence of the secretory form of CEH (Kissel et al., 1989).
To assess whether other proteins are present within the 70-kDa
protein band resolved on one-dimensional gels, the 70-kDa band derived
from 0.6 M NaCl-washed ZGM was also analyzed by
two-dimensional IEF SDS-PAGE (Fig. 6). Silver stain
revealed a string of three densely stained protein spots and an
additional minor species at the acidic end of the string. The molecular
mass of all four spots was equal to 70 kDa (Fig. 6A). A
similar two-dimensional gel was processed for Western blotting using
the anti-CEH specific polyclonal antiserum. In this case, a series of
five immunoreactive spots was detected at molecular mass = 70
kDa (Fig. 6B). A sixth spot exhibited slightly lower
electrophoretic mobility (72 kDa). Four of these spots overlapped
precisely with the silver-stained protein spots. These results indicate
that the 70-kDa protein band of salt-washed ZGM is composed entirely of
several different isoforms of mCEH. The inability of the silver-stain
method to detect an equivalent number of spots by immunoreactivity is
likely to be due to the greater sensitivity of the antiserum and
possibly to the differential sensitivity of the stain to variations in
the carbohydrate content of the isoforms (see Deh et al.,
1985).
Figure 6: Two-dimensional IEF/SDS-PAGE analysis of the 70-kDa protein band derived from 0.6 M NaCl-washed ZGM. Zymogen granules were collected, lysed, exposed to 0.6 M NaCl and the proteins separated by SDS-PAGE as described under ``Experimental Procedures.'' The 70-kDa protein band was processed further for two-dimensional gel electrophoresis. Resulting two-dimensional gels were either silver-stained (A) or immediately transferred to nitrocellulose for reaction with anti-CEH antiserum (B). The orientation of the IEF gel is acidic to basic, left to right. Molecular mass standards (kDa) are noted to the left of each panel. The positions of the four silver-stained spots (measured by their relative migration from the acidic end of the gel) overlap precisely (>92%) with four of the anti-CEH immunoreactive spots.
Figure 7:
Phospholipase activity and mCEH
immunoreactivity co-localize following SDS-PAGE. In A, 0.6 M NaCl-washed ZGM proteins (10 µg) were separated using a
10% gel in the absence of reducing agents and stained with Coomassie
Blue. This gel was then cut into five equal slices. Phospholipase
assays were then performed on proteins that were eluted and renatured
from each gel slice (see ``Experimental Procedures''). Three
independent experiments localized nearly all of the phospholipase
activity to R = 0.2-0.4 (see Table 2). In B, the R
= 0.2-0.4 region of the gel was further
subdivided into eight gel slices of decreasing molecular weight.
Fractions 1 and 8 contain the highest and lowest molecular weight
proteins, respectively. The proteins from each gel slice were eluted,
renatured, and then assayed for phospholipase activity as described
under ``Experimental Procedures.'' Phospholipase activity is
expressed as the counts/min for both AA* and 2*lysoPC. Aliquots of the
renatured proteins from these gel slices were also prepared for
immunoblotting with the mCEH and GP2 antisera. The results depicted in B were reproduced in a separate independent
experiment.
To determine the relative contribution of mCEH and GP2 to the
phospholipase activity, the region of the gel that harbored mCEH and
GP2 was cut into eight slices (R =
0.2-0.4, Fig. 7). Virtually all of the 2*lysoPC and AA*
breakdown products was localized to gel slice 5 (Fig. 7).
Western blot analysis showed that mCEH was found only in gel slice 5 (Fig. 7). The greatest amount of GP2 was also present in gel
slice 5, although there were significant amounts of GP2 in gel slices
1-4 and 6, which possessed minimal phospholipase activity.
To assess
further the relative contributions of mCEH and GP2 to ZGM phospholipase
activity, the glycosylphosphatidylinositol anchor property of GP2
(LeBel and Beattie, 1988) was used to selectively remove GP2 from ZGM.
Following treatment of ZGM with phosphatidylinositol-specific PLC, over
80% of GP2 was found in the soluble phase, whereas CEH was not
detectable in the soluble phase (data not shown). The premise of this
experiment was that phospholipase activity due to GP2 would be removed
from ZGM by PLC treatment, whereas the distribution of mCEH
phospholipase activity would be unaffected. Over 95% of the 2*lysoPC
produced (which is due to PLA) in control membranes was
retained after PLC treatment (Table 3). Since the GP2/mCEH region
of the gel contained all of the enzyme activity (cf. Fig. 7A), this finding clearly demonstrates that mCEH
is a ZGM protein with PLA
-type activity.
The possible
involvement of GP2 in the deacylation of PC was, nevertheless,
suggested by a 37% reduction in the amount of radiolabeled AA* produced
by ZGM pretreated with phosphatidylinositol-specific PLC (Table 3). The possibility that GP2 may be directly releasing AA*
from 1,2*PC by PLA activity seems unlikely since no rise in
AA* production was observed in the PLC supernatant, which contains most
of the GP2 originally found on the ZGM. Alternatively, the supposition
that GP2 functions as a lysophospholipase would explain the lack of AA*
accumulation in the PLC supernatant. The absence of mCEH in this
fraction implies that no 2*lysoPC would be enzymatically produced to
serve as a substrate for GP2. However, when the redistribution
experiments were performed using labeled lysoPC rather than PC as a
substrate, a translocation of lysophospholipase activity from ZGM to
the soluble phase could not be demonstrated (data not shown).
Collectively, these results suggest that mCEH is responsible for the
PLA
activity and at least 60-65% of the
lysophospholipase activity. Although there is no compelling explanation
for the loss of some phospholipase activity on PLC-treated ZGM, it may
be that detachment of GP2 from ZGM results in a loss of its enzymatic
activity.
Figure 8: Displacement of CEH from 0.6 M NaCl-washed ZGM by heparin. ZGM washed in 0.6 M NaCl were incubated for 30 min at 30 °C with various concentrations of heparin sulfate or protein and then the ZGM were retrieved by ultracentrifugation. In A, the resulting pellets were solubilized in equal amounts of SDS sample buffer and prepared for immunoblotting using both CEH and GP2 antisera. Lane 1, 1 mg/ml heparin; lane2, 10 mg/ml heparin; lane3, 100 mg/ml heparin; lane4, 100 mg/ml soybean trypsin inhibitor; lane5, 10 mg/ml ovalbumin. This experiment is representative of two other experiments. In B, previously washed ZGM were processed as in A for immunoblotting with anti-CEH. Lane1, control ZGM; lane2, 10 mg/ml chondroitin sulfate; lane3, 100 mg/ml chondroitin sulfate; lane4, 100 mg/ml heparin sulfate. Data shown are representative of two additional experiments.
This study has identified a 70-kDa membrane-associated carboxyl ester hydrolase (mCEH) as one of the five major ZGM proteins. This identification is based upon: (a) immunological cross-reactivity with a specific, high affinity polyclonal antiserum against rat CEH; (b) 100% sequence identity of the N terminus of the 70-kDa protein with the known sequence of CEH; (c) two-dimensional gel analysis; and (d) direct measurement of a characteristic enzymatic profile from the 70-kDa protein isolated from non-reducing gels.
The ZGM-associated mCEH shares similar
physicochemical properties with the secretory form of CEH (sCEH), as
evidenced by their similar mobility on SDS-PAGE, and the complete
conservation of the N-terminal sequence. The mCEH and sCEH also have
similar enzymatic properties since both enzymes are
Ca-independent phospholipase/lysophospholipases,
possess a pH optimum of approximately 7.0, and are inhibited by the
serine-reactive compound DFP (Murthy and Ganguly, 1962; Hyun et
al., 1969; Mizuno and Brockman, 1990; Wang and Hartsuck, 1993). On
the other hand, mCEH can be distinguished from sCEH based on its
association with ZGM even in the presence of high concentrations of
salt. These findings suggest that mCEH is a ZGM-associated form of
secretory CEH.
Retention of mCEH on ZGM, even at salt concentrations
as high as 1 M, is incompatible with binding via weak
electrostatic interactions. Furthermore, Scheele et al.(1978)
found that basic secretory proteins of exocrine pancreas are
nonspecifically adsorbed in larger quantities and with higher affinity
to membranes as compared to acidic proteins. Under high salt
conditions, therefore, pancreatic CEH, which is an acidic protein
(Erlanson, 1975; Ghosh and Grogan, 1991), would be expected to be
removed from ZGM more readily than the basic protein amylase (pI
= 8.4; Scheele, 1981). Our findings, however, demonstrate that
although amylase, sPLA, and other secretory proteins can be
removed by a single wash with 0.6 M NaCl, 30-40% of ZGM
pH 8.2-associated CEH was retained even after several washes with 0.6 M NaCl. This salt-stable association indicates that the
relationship of the enzyme to the membrane is based upon a more
specific interaction than simple adsorption. Increasing the salt
concentration to 2 M removes nearly all of the CEH from the
membrane, suggesting further that mCEH is a peripheral rather than an
integral membrane protein.
Our results using two-dimensional IEF/SDS-PAGE demonstrate that multiple isoelectric variants of mCEH can be resolved. Further studies are needed to determine whether the representation of these isoforms in the granule contents is, in any way, different from that found on the ZGM. Although mCEH of ZGM and sCEH may be composed of different post-translational variants, we cannot presently exclude the additional possibility that a limited number of high affinity binding sites is responsible for a specific interaction of sCEH with ZGM. A heparin-sensitive attachment site may mediate the binding of mCEH to ZGM. This supposition derives from our finding that heparin was capable of dissociating mCEH from ZGM. The possibility that a heparin-like moiety provides a specific binding site for mCEH gains support from the fact that ZGM contain heparin proteoglycans and other sulfated compounds (Kronquist et al., 1977; Reggio and Palade, 1978). The fact that a relatively high concentration of heparin was required for displacement of mCEH from ZGM implies that the putative heparin-binding sites may exhibit high affinity and low turnover. A charge effect to account for the displacement of CEH by heparin appears unlikely since a comparable concentration of chondroitin sulfate, which is more highly charged than heparin, was much less effective in displacing mCEH.
Our analysis of
ZGM phospholipase activity showed that over 90% of PC breakdown
involved a Ca-independent
PLA
/lysophospholipase pathway. These phospholipase
activities were all recovered from a 2-mm section of gel, which
contained mCEH and no other detectable protein, as determined by
immunoblotting and silver stain of salt-washed ZGM analyzed by
two-dimensional IEF/SDS-PAGE. Thus, the 70-kDa protein, which is one of
the five major proteins of purified ZGM, is composed of CEH exhibiting
measurable phospholipase activity. Our previous study demonstrated the
sequence similarity and immunological relatedness of GP2 to secretory
PLA
(Withiam-Leitch et al., 1993). This led us to
hypothesize that GP2 may be involved in phospholipid binding and/or
hydrolysis. Although phosphatidylinositol-specific PLC treatment of ZGM
slightly decreased phospholipase activity (cf. Table 3),
direct measurement of GP2-mediated phospholipid hydrolysis could not be
demonstrated. However, we cannot exclude the possibility that different
pH, phospholipid substrate, or detergent conditions than those utilized
for characterizing mCEH are required to detect the phospholipid
hydrolyzing activity of GP2.
Although phospholipase activities have
been described on secretory granule membranes of other secretory cells,
most studies attribute the release of sn-2 fatty acids from
phospholipids to PLA activity (Moskowitz et al.,
1982; Hildebrandt and Albanesi, 1991; Mizuno et al., 1991).
However, as considered in this and other studies (Waite, 1985; Dennis et al., 1991), sn-2 fatty acids can be released by
PLA
, PLA
-, PLC-, and PLD-mediated pathways.
Granule membranes of parotid acinar cells appear to contain primarily a
Ca
-independent PLA
activity, but at least
20% of PC breakdown is probably mediated via a PLA
pathway
(Mizuno et al., 1991). Adrenal chromaffin granule membranes
contain Ca
-independent PLA
,
PLA
, and lysophospholipase activities (Husebye and
Flatmark, 1987; Hildebrandt and Albanesi, 1991). The existence of
multiple Ca
-independent phospholipase activities in
other granule membranes coincides with the enzymatic properties of
mCEH. Studies to clarify both the enzymatic activity and the molecular
species responsible in each of these preparations would be extremely
useful in exploring the general applicability of the present findings.
In conclusion, our demonstration that the 70-kDa mCEH from ZGM exhibits enzymatic activity represents the first identification of a protein responsible for granule membrane phospholipase activity. The finding that ZGM mCEH catalyzes the release of unsaturated fatty acids and lysophospholipids from PC has important implications with regard to membrane fusion. Unsaturated fatty acids have been repeatedly implicated in the exocytotic process because of their potential to promote membrane fusion (Creutz, 1981; Burgoyne, 1991). LysoPC induces membrane destabilization, and could therefore also promote membrane fusion (Poole et al., 1970). The identification of mCEH as a phospholipase on ZGM should fuel further studies on the role of this enzyme in the secretory cycle.