©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of Carboxyl Ester Hydrolase as a Phospholipid Hydrolyzing Enzyme of Zymogen Granule Membranes from Rat Exocrine Pancreas (*)

(Received for publication, October 28, 1994; and in revised form, December 15, 1994)

Matthew Withiam-Leitch Ronald P. Rubin Svetlana E. Koshlukova John M. Aletta (§)

From the Department of Pharmacology and Toxicology, State University of New York, School of Medicine and Biomedical Sciences, Buffalo, New York 14214

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(1) and lysophospholipase activities represents the first identification and characterization of a protein responsible for phospholipase activity in secretory granule membranes.


INTRODUCTION

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) (^1)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(2), 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(1) 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.


EXPERIMENTAL PROCEDURES

Materials

Antiserum to porcine secretory PLA(2) (sPLA(2)) was obtained from Upstate Biotechnology. Purified porcine sPLA(2) was obtained from Boehringer Mannheim. GP2 antiserum was a kind gift of Dr. Adrien Beaudoin of the University of Sherbrooke, Quebec, Canada. Antiserum to carboxyl ester hydrolase was kindly provided by Dr. Howard Brockman of the Hormel Institute, University of Minnesota. All electrophoretic reagents were purchased from Bio-Rad.

Isolation of Zymogen Granules and Zymogen Granule Membranes

Zymogen granules (ZG) were isolated from male Sprague-Dawley rats as described previously (Rubin et al., 1990). ZG were lysed for 15 min in 25 ml of lysis buffer containing 25 mM Tris-HCl (pH 8.2) at 4 °C. ZGM were isolated from the ZG lysate by ultracentrifugation at 100,000 times g. The resulting pellet was resuspended in 500 µl of 5 mM MOPS (pH 7.0) and is referred to as ZGM pH 8.2, or unwashed ZGM. ZGM pH 8.2 were processed further in lysis buffer that contained various concentrations of NaCl. For most experiments, the entire yield of ZGM pH 8.2 was washed once in 25 ml of lysis buffer containing 0.6 M NaCl. For experiments designed to determine the affinity of proteins to ZGM, a 20-µl aliquot of unwashed ZGM was placed in 2 ml of lysis buffer containing 0.0-2.0 M NaCl. ZGM were washed for 30 min at 4 °C with mixing, and the ZGM then collected by ultracentrifugation at 100,000 times g for 60 min. The resulting pellets were brought up in 100 µl of Laemmli sample buffer in preparation for SDS-PAGE and immunoblotting.

Purity of Zymogen Granules and Zymogen Granule Membranes

Amylase activity (zymogen granule marker) was assayed as described by Bernfeld(1955). Lactate dehydrogenase (cytosolic marker) and fumarase (mitochondrial marker) activities were assayed as described previously by Whittaker and Barker(1972). Acid phosphatase (lysosomal marker) and alkaline phosphatase (plasma membrane marker) were assayed using Sigma Diagnostic kits as per the manufacturer's specifications. Rotenone-insensitive NADH-cytochrome c reductase (endoplasmic reticulum marker) was assayed as described by Sottocasa et al.(1967). All marker enzyme activities were readily detected in the homogenate. Amylase, a marker enzyme for ZG, was enriched 6.6-fold compared to the homogenate. Enzyme markers for plasma membrane (alkaline phosphatase) and cytosol (lactate dehydrogenase) were not detectable in the ZG fraction. The relative specific activity of amylase in the ZG fraction was enriched 738-fold relative to NADH-cytochrome c reductase activity (endoplasmic reticulum marker), 300-fold compared to fumarase activity (mitochondrial marker), and 33-fold compared to acid phosphatase activity (lysosomal marker).

Electrophoretic Separation of ZGM Proteins and Immunoblotting

Samples were prepared for SDS-PAGE as described by Laemmli(1970) using a Bio-Rad Minigel system. After transfer of the proteins to nitrocellulose or PVDF, the membrane was blocked by a 2-h exposure to 5% fatty acid free bovine serum albumin (BSA) in Tris-buffered saline (TBS; pH 7.4). The blots were then incubated for 2 h at room temperature with the primary antibody diluted in 5% BSA/TBS. Antisera dilutions were as follows: anti-CEH, 1:5000; anti-GP2, 1:10,000; and anti-PLA(2), 1:1000. After being washed in TBS, 0.5% Tween 20, the blots were incubated with 167 nCi/ml I-labeled anti-rabbit IgG in 5% BSA/TBS for 1 h at room temperature, followed by four 20-min washes. The blots were exposed for 2-72 h to Kodak XAR film for autoradiography.

Protein Elution from Polyacrylamide Gels and Renaturation of Proteins

Proteins were eluted from SDS-PAGE gels and renatured using modifications of the procedures developed by Hager and Burgess (1980) and Pind and Kuksis(1987). ZGM protein samples (400 µg) were solubilized in Laemmli sample buffer devoid of reducing agents. The samples were not boiled. Following SDS-PAGE, one lane of the resolved proteins stained with Coomassie Blue was used as a reference for identifying proteins. The gel fraction of interest was then cut away from the rest of the gel and homogenized in a Dounce homogenizer containing 5 ml of elution buffer consisting of 0.1% SDS, 50 mM MOPS (pH 7.0), 0.1 mM EGTA, 0.1 mg/ml BSA, and 150 mM NaCl. The gel homogenate was rotated overnight at room temperature and then centrifuged at 4000 times g to pellet the gel fragments. The supernatant containing the eluted proteins was precipitated using 25 ml of ice-cold acetone, followed by centrifugation at 10,000 times g. The protein precipitate was then solubilized in 40 µl of 6 M guanidine-HCl in 5 mM MOPS (pH 7.0). After incubating the mixture for 20 min at room temperature, 2 ml of renaturation buffer consisting of 50 mM MOPS (pH 7.0), 20% glycerol, 0.5% CHAPS, and 1 mM EGTA was added. The renatured proteins were then used for phospholipase assays and/or solubilized in sample buffer for SDS-PAGE and Western blotting.

Phospholipase Assays

Phospholipase activity was assayed using a sonicated dispersion of 1-stearoyl-2-[1-^14C]arachidonyl-phosphatidylcholine (PC). The reaction mixture contained: 10 µg of ZGM protein, 1 mM EGTA, 50 mM MOPS (pH 7.0), and 20 µM of the substrate (55 µCi/ml) in 100 µl of assay buffer. Unless otherwise noted, the reaction was allowed to proceed for 15 min at 37 °C and was terminated by the addition of 3 ml of chloroform/methanol (2:1) containing 0.06 N HCl. After extraction (Bligh and Dyer, 1959), the lipids were separated by thin layer chromatagraphy using chloroform/methanol/water (92:49:8) as a solvent to separate phospholipids, or petroleum ether/absolute ether/acetic acid (50:50:1) to separate neutral lipids. Radioactive spots were then scraped from the TLC plates and quantified by scintillation spectrometry.

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(1) and PLA(2) pathways, the following substrates were used: 1-[1-^14C]palmitoyl-2-[1-^14C]palmitoyl phosphatidylcholine (1*2*PC, where asterisk indicates point of ^14C labeling) (112 µCi/µmol) and 1-palmitoyl-2-[1-^14C]palmitoyl phosphatidylcholine (1,2*PC) (55 µCi/µmol). The generation of labeled lysoPC from 1*2*PC could be due either to PLA(1) (i.e. 2*lysoPC) or PLA(2) (i.e. 1*lysoPC). The generation of labeled lysoPC from 1,2*PC can only result from PLA(1) activity (i.e. 2*PC). Subtracting the amount of lysoPC derived from 1,2*PC (PLA(1)) from the lysoPC produced from 1*2*PC (PLA(1) + PLA(2)) yields the amount of lysoPC produced by PLA(2) activity. Experiments to detect lysophospholipase activity were performed using 1-[1-^14C]-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-^14C]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).

Release of GP2 from ZGM

Membranous GP2 was solubilized by incubating 400 µg of ZGM for 2 h at 37 °C with 0.3 units of phosphatidylinositol-specific PLC (Bacillus thuringiensis) contained in 300 µl of 20 mM MOPS (pH 7.0). This mixture was then centrifuged at 100,000 times g to obtain soluble and particulate fractions. This procedure results in the translocation of more than 80% of ZGM-associated GP2 to the soluble phase (Withiam-Leitch et al., 1993).

N-terminal Sequence Analysis

ZGM proteins washed with 0.6 M NaCl were separated by SDS-PAGE under reducing conditions, followed by Western blotting to PVDF membranes. Approximately 100 pmol of 70-kDa membrane protein was subjected to automated Edman degradation using a Porton sequencer (Beckman) equipped with an on-line detection unit to identify phenylthiohydantoin-amino acid derivatives. The analysis was provided by the Protein Sequencing Facility of the State University of New York, Buffalo, NY.

Two-dimensional Gel Electrophoresis

Salt-washed (0.6 M NaCl) zymogen granules (400 µg) were separated by SDS-PAGE under reducing conditions. The 70-kDa protein was excised, electroeluted from the gel, and precipitated with 9 volumes of ice-cold methanol. After centrifugation, the protein pellet was solubilized for isoelectric focusing (IEF) as described by O'Farrell(1975) and adapted for the Bio-Rad Mini-Protean II gel system. Protein samples prepared in an equivalent manner for IEF/SDS-PAGE were visualized either by silver staining (Bio-Rad) or by Western blot analysis with CEH-specific antiserum as described above.


RESULTS

ZGM Washed in 0.6 M NaCl Are Free of Major Secretory Proteins from the Granule Content

ZGM isolated by pH 8.2 lysis contained a substantial amount of the digestive enzymes amylase and secretory PLA(2) (sPLA(2)). The relative specific activity of amylase was elevated 1.25-fold (± 0.71) on unwashed ZGM relative to the homogenate, indicating substantial amylase contamination of these membranes. Washing these membranes in 0.6 M NaCl reduced the relative specific activity of amylase on ZGM to 0.004 (± 0.002) relative to the homogenate, indicating that a single high salt wash is capable of removing 99.6% of the amylase from ZGM pH 8.2. Similarly, immunoblot analysis of ZGM pH 8.2 revealed significant contamination of sPLA(2); however, a single 0.6 M NaCl wash rendered sPLA(2) undetectable by Western blotting (data not shown). In addition, Coomassie Blue stains comparing the protein composition of ZGM pH 8.2 and 0.6 M NaCl-washed ZGM revealed that many other secretory proteins were also removed from pH 8.2 membranes by a single high salt wash (Fig. 1).


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 times 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(2), 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.

PLA(1) and Lysophospholipase Activities Associated with ZGM Are Calcium-independent

ZGM-mediated breakdown of PC, the major phospholipid component of ZGM, in the pH range of 3.5-10.0 and in the Ca concentration range of 0-5 mM resulted in the production of two reaction products (Fig. 2). The sole neutral lipid product of PC hydrolysis was labeled free arachidonic acid (AA*), whereas the sole phospholipid product of PC breakdown was lysophospholipid.


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-^14C]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(1)-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(2) or PLA(1)/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(1) mechanism, with only a minor contribution from a PLA(2) 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 times min times mg) was higher than the initial rate of AA* release (0.30 nmol times min times mg). At later time points (30-45 min), the rate of AA* production increased (1.10 nmol times min times 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(1)/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(1) 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 times min times 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(1) and lysophospholipase activities, with only a minor contribution from a PLA(2) 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 times 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.



Carboxyl Ester Hydrolase Is a ZGM Protein

Rat exocrine pancreas expresses CEH, which has both lysophospholipase and PLA(1) activities that are known to be calcium-independent. Therefore, we next tested whether a CEH-specific polyclonal antiserum cross-reacts with any of the proteins of the ZGM. As illustrated in Fig. 5A, the antiserum recognizes a protein of 70 kDa on these membranes. To examine the potential of the soluble form of CEH to adsorb nonspecifically to ZGM during isolation, ZGM were washed with increasing concentrations of NaCl to determine the strength and specificity of CEH binding to ZGM. Washing with NaCl concentrations up to 0.45 M removed a significant proportion (65-70%) of the CEH (Fig. 5C). These results are consistent with the data presented in Fig. 1regarding the removal of the secretory content from ZGM. There was, however, a significant amount of CEH that remained associated with ZGM (30% of the starting material associated with lysed membranes) even when the salt concentration was increased to 1.0 M NaCl (Fig. 5C). By contrast, there was no change in GP2 associated with ZGM at any of the salt concentrations (Fig. 5C).


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 times 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.



mCEH Exhibits Phospholipase Activity

The first approach to determining which ZGM proteins were associated with phospholipase activity involved an assessment of the locus of enzyme activity in a non-reducing polyacrylamide gel. The gel was divided into five equal slices of R(F) = 0.0-0.2, 0.2-0.4, and so on (Fig. 7A). Proteins in these slices were eluted and prepared for phospholipase assay as described under ``Experimental Procedures.'' Virtually all of the ZGM phospholipase activity was localized with ZGM proteins having a relative mobility of R(F) = 0.2-0.4 (mass = 60-100 kDa; Table 2). Both 2*lysoPC and AA* were produced by the enzyme(s) in this fraction, which is consistent with the products formed from PC in native ZGM. Coomassie Blue staining (Fig. 7A) and Western blot analysis (Fig. 7B) showed that mCEH and GP2 were the only major proteins in this gel slice, and that the non-reducing conditions employed produced an overlapping distribution of these two proteins. The phospholipase activity of ZGM, therefore, was due to either mCEH or GP2.


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(F) = 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(1)) 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(1)-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(2) 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(1) 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.

Displacement of mCEH by Heparin

The secretory form of CEH binds specifically and saturably to heparin localized to epithelial cell membranes in the small intestine (Bosner et al., 1988, 1989). To investigate whether a similar mode of binding might account for the attachment of mCEH, ZGMs were washed with various concentrations of heparin sulfate. Indeed, heparin differentially displaced CEH, without affecting GP2 membrane association (Fig. 8A). The relative specificity of the displacement by heparin was revealed by the finding that equivalent concentrations of chondroitin sulfate, another glycosaminoglycan, did not dissociate mCEH as effectively as heparin (Fig. 8B). In addition, ovalbumin and soybean trypsin inhibitor, which might compete for putative nonspecific protein binding sites, were ineffective in displacing mCEH (Fig. 8A).


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.



Inhibition of ZGM Phospholipase Activity by Diisopropyl Fluorophosphate

Previous studies have shown that the soluble form of CEH is completely inhibited by the serine-reactive compound diisopropyl fluorophosphate (DFP)(Van Den Bosch et al., 1973; Mizuno and Brockman, 1990). To examine whether the phospholipase activity associated with ZGM has a serine-reactive site like that of soluble CEH, ZGM were washed with 0.6 M NaCl and then pretreated with 1 mM DFP for 20 min prior to the phospholipase assay. PC breakdown was inhibited 97.1 ± 3.2% (n = 3) in DFP-treated ZGM compared to control ZGM. This result provides additional evidence that mCEH is responsible for measurable phospholipase activity.


DISCUSSION

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(2), 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(1)/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(2) (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(2) 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(1), PLA(2)-, PLC-, and PLD-mediated pathways. Granule membranes of parotid acinar cells appear to contain primarily a Ca-independent PLA(2) activity, but at least 20% of PC breakdown is probably mediated via a PLA(1) pathway (Mizuno et al., 1991). Adrenal chromaffin granule membranes contain Ca-independent PLA(2), PLA(1), 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AM-28029, DE-05764, and Pharmacology Training Grant GM-07145. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, State University of New York, School of Medicine and Biomedical Sciences, 102 Farber Hall, Buffalo, NY 14214-3000. Tel.: 716-829-3237; Fax: 716-829-2801.

(^1)
The abbreviations used are: ZGM, zymogen granule membrane(s); ZG, zymogen granule; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; CEH, carboxyl ester hydrolase; mCEH, membranous carboxyl ester hydrolase; PLC, phospholipase C; PLA, phospholipase A; sPLA, secretory PLA; PVDF, polyvinylidene difluoride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; AA*, free arachidonic acid; PC, phosphatidylcholine; BSA, bovine serum albumin; TBS, Tris-buffered saline; sCEH, secretory CEH; DFP, diisopropyl fluorophosphate.


ACKNOWLEDGEMENTS

Margaret Haldeman in the laboratory of Dr. Cecile Pickart graciously provided technical advice and assistance in the preparation of the IEF gels. We thank Dr. Adrien Beaudoin for the GP2 antibody and for helpful discussions during the course of this study. Dr. Howard Brockman generously provided the CEH antibody. We also appreciate the comments of Drs. Mulchand Patel and Jerome Roth on the manuscript.


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