Further characterization of a novel phospholipase B (phospholipase A 2 ...

Further characterization of a novel phospholipase B (phospholipase A2 - lysophospholipase) from intestinal brush-border membranes

STEVEN PIND' AND ARNIS KUKSIS~

Department of Biochemistry, University of Toronto, Toronto, Ont., Canada M5S IA8

and

Department of Medical Research, University of Toronto, C.H. Best Institute, 112 College St., Toronto, Ont., Canada M5G IL6

Received December 21, 1990

PIND, S., and KUKSIS, A. 1991. Further characterization of a novel phospholipase B (phospholipase A, - lysophospholipase) from intestinal brush-border membranes. Biochem. Cell Biol. 69: 346-357.

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as a 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.

Key words: phospholipase, brush-border membrane, intestine, phospholipid hydrolysis, antibodies.

PIND, S., et KUKSIS, A. 1991. Further characterization of a novel phospholipase B (phospholipase A, - lysophospholipase) from intestinal brush-border membranes. Biochem. Cell Biol. 69 : 346-357.

Les membranes intestinales de la bordure en brosse des rats et des cobayes renferment une phospholipase B (activitts phospholipase A2 - lysophospholipase) de poids mol&ulaire tlevk, indtpendante du calcium et douk des caracttistiques d'une ectoenzyme digestive. Dans le but de purifier la forme compltte, quoique dtnaturk, de cette enzyme chez le rat, nous avons utilist une combinaison des techniques suivantes : fractionnement subcellulaire, stparation de phases avec le Triton X-114, chromatofocalisation et Clectrophortse prtparative en gel de polyacrylamide avec dodtcyl sulfate de sodium. La renaturation de la fraction purifite sur gel confirme que les dew activitts enzymatiques sont assocites a cette prottine. Des tranches de gel renfermant la phospholipase B purifite sont utilistes pour gtntrer chez les lapins un antistrum polyclonal qui peut servir a I'immunotransfert. La mobilitt relative de la phospholipase B durant l'tlec- trophortse en gels avec dodtcyl sulfate de sodium est dramatiquement affectte par le pourcentage d'acrylamide et la presence ou I'absence d'agents rkducteurs dans les gels. Cela est vrai pour la prottine purifite visualiske par coloration a I'argent et, aprts Bectrophortse des prottines totales, avec la phospholipase observk par immunotransfert. Les valeurs de la masse molkulaire de l'enzyme varient de 130-170 kDa dans les gels 7 3 % et de 120-130 kDa dans les gels en gradient de 5-10% (la meilleure valeur est de 120 kDa). Aprks solubilisation de la membrane de la bordure en brosse par digestion avec la papahe, la principale bande immunortactive se dtplace avec une masse apparente de 80 kDa dans les dew gels (73% et gradient de 5-10%). Une bande reactive importante est detect& a 90 kDa a p r b immunotransfert des prottines des membranes de la bordure en brosse du cobaye solubilistes par la papaine (cela concorde avec les dimensions du fragment purifit rapport6 dans la littrature) et a 140 kDa a p r b immunotransfert des protkines intactes. Le mCme immunotransfert produit une reaction avec une prottine de 135 kDa provenant de la membrane de la bordure en brosse du lapin de mCme qu'avec une prottine de 95 kDa aprts solubilisation avec la papaine. Ces rtsultats suggtrent que malgrt I'existence de poids moltculaires sptcifiques des esptces, la phospholipase B des membranes de la bordure en brosse intestinale est conservte entre les esptces.

Mots clks : phospholipase, membranes de la bordure en brosse, intestin, hydrolyse des phospholipides, anticorps. [Traduit par la rtdaction]

'present address: Department of Molecular Biology, Imm-11, Research Institute of the Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.

'~u thor to whom correspondence and reprint requests should be addressed at the Banting and Best Department of Medical Research. Prinled in Canada / Imprime au Canada

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PIND AND KUKSlS 347

Introduction The intestinal brush-border membrane is characterized by

the presence of several hydrolytic enzymes that project from the membrane surface into the intestinal lumen, where they function in the terminal digestion of dietary nutrients (for reviews, see Kenny and Maroux, 1982; Semenza, 1986; Noren et al. 1986; Maroux, 1987; and a monograph edited by Kenny and Turner, 1987b). Common structural features among these proteins include a small, hydrophobic, membrane-anchoring domain, and a large, glycosylated, globular domain containing the active site(s). These two regions are connected via a polypeptide stalk, which in many cases is susceptible to proteolytic cleavage. As a reflection of the fact that their catalytic sites are completely external to the cell, these hydrolases have also been classified as "ectoenzymes" (Kenny and Turner 1987a). In addition to the well-characterized peptidases and glycosidases, recent studies have identified a novel phospholipase B in brush- border membranes purified from rat (Pind and Kuksis 1988, 1989) and guinea pig (Gassama-Diagne et al. 1989) small intestines. Initially detected as a calcium-independent phospholipase A2 activity (Subbaiah and Ganguly 1970; Pind and Kuksis 1987; Diagne et al. 1987), it was subse- quently shown that the enzyme also displayed lysophospholipase activity towards sn-1 acyl lysophospholipids, hence its designation as a phospholipase B (Pind and Kuksis 1988, 1989; Gassama-Diagne et al. 1989). Enzyme activity was not released from the membranes by high salt or EDTA treatments, but was efficiently solubilized by a variety of detergents (Pind and Kuksis 1988), or more diagnostically, by a proteolytic digestion with papain (Pind and Kuksis 1989; Gassarna-Diagne et al. 1989). Based on these results, it was concluded that phospholipase B may be another digestive ectoenzyme of the intestinal brush-border membrane.

Several lines of evidence suggest that phospholipase B from rat and guinea pig are very similar enzymes or isozyrnes of the same enzyme. Besides having similar enzymatic properties and solubilization characteristics, the enzymatic activity of both can be renatured following nonreducing SDS- PAGE. While both phospholipases B also appear to be large enzymes, a direct comparison between the enzymes of the two species has not yet been made. Following renaturation from SDS-polyacrylamide gels, we found that the full-length phospholipase B migrates as an apparent 170-kDa protein in 7.5% nonreducing gels (Pind and Kuksis 1989). In contrast, the papain-solubilized form of phospholipase B, purified by Gassama-Diagne et al. (1989), has an apparent molecular weight at 97 kDa. We have now attempted to reconcile these differences by a further characterization of the enzyme from the rat small intestine. The preparation of a polyclonal antiserum in the present study has allowed for the first time a demonstration of the immunological similarity among the phospholipase B from the rat, guinea pig, and rabbit brush border membranes, as well as between the intact enzymes and the corresponding papain-solubilized preparations.

Materials and methods Materials

Polybuffer 74 and the Polybuffer exchanger PBE 94 were purchased from Pharmacia Canada, Ltd. (Dorval, Que.). Lubrol-PX

(Surfact-Amps grade) was from Pierce (Rockford, IL). Nitrocellulose membranes (0.45 pm pore size) were from Schleicher and Schuell (Keene, NH). The polyvinylidine membranes (Immobilon, 0.45 pm pore size) were obtained from Millipore (Toronto, Ont.). Reagents for irnmunoblotting were obtained in the protoblotR Western Blot AP system from Promega (purchased through Bio/Can Scientific, Inc., Mississauga, Ont.). All other materials were as previously described (Pind and Kuksis 1987, 1988, 1989), or were reagent grade or better quality.

Animals Male Wistar rats (250-300 g) and male Hartley guinea pigs

(350 g) were purchased from Charles River Canada Inc. (LaSalle, Que.). Female New Zealand rabbits (10-12 weeks of age) were obtained from Rieman's Fur Ranch (St. Agatha, Ont.).

Electrophoresis SDS-PAGE was performed according to Laemmli (1970) in slab

gels (16 x 12 x 1.5 cm) containing 7.5% polyacrylamide or in gels containing a 5-10% linear gradient of polyacrylamide (Hames 1981). Samples were usually run under nonreducing conditions, although in some instances samples were reduced with fl-mercaptoethanol. Gels to be silver stained were fixed and stained as previously described (Pind and Kuksis 1989) or as described by Rabilloud et al. (1988). Other gels were stained with Coomassie blue R-250 (Hames 1981).

Phospholipase assays Phospholipase A, and lysophospholipase activities were assayed

using egg yolk phos hatidylcholine mixed with tracer amounts of !' 1-palmitoyl-2-[1-1 C]oleoyl-sn-glycerol-3-phosphorylcholine (- 150 cpm/nmol), and using sn-1-acyl-lysophosphatidylcholine (produced from eg yolk phosphatidylcholine) mixed with tracer E quantities of 1-[I- Clpalmitoyl-sn-glycerol-3-phosphorylcholine ( - 150 cpm/nmol), respectively (Pind and Kuksis 1988, 1989). AU assays were done at pH 8.0 in the presence of 1% CHAPS detergent and 300-340 nrnol substrate. The amount of protein was varied in such a way that less than 20% of substrate was hydrolyzed in a 15-min incubation. In some instances assays were also done in the presence of 5 mM EDTA. Phospholipase B activity refers to samples having both phospholipase A, and lysophospholipase activities.

Phase partitioning of brush-border membrane proteins Brush-border membranes were purified (20- to 25-fold, 50%

yield) from the proximal (early studies) or distal (later studies) two- thirds of the small intestines from 3 rats (Pind and Kuksis 1987). The purified membranes (7-10 mg protein) were suspended in 8 mL of 10 mM Tris-HC1 (pH 7.4), 150 mM NaCI, and solubilized by adding 1 mL of 10% (w/v) precondensed Triton X-114 (Bordier 1981) and mixing well. Following 10 min at 4OC with occasional stirring, the insoluble material was pelleted by centrifugation at 27 000 x g x 40 min (Sorvall RC 5B; SS-34 rotor). From the resulting supernatant, 4 mL (in duplicate) was subjected to phase partitioning using a 10-fold scaled-up version of the method intro- duced by Bordier (1981), with the exception that the detergent and aqueous phases were separated by centrifugation at 1000 x g for 5 min. The final, detergent-rich pellets were diluted to 8 mL with 25 mM imidazole (pH 7.4), 0.05% (w/v) Lubrol-PX (buffer I).

Chromatofocusing Proteins associated with the detergent phase following Triton

X-114 partitioning, diluted as described above with buffer I, were applied to a column of PBE 94 (0.7 x 13 cm, 5 mL bed volume) which had been previously equilibrated with 20-30 volumes of the same buffer. Sample application and elution were carried out at 4"C, using gravity flow at approximately 10 mL/h. Following loading, the column was washed with 2 mL of buffer I and then eluted with 16 volumes of 1:8 diluted Polybuffer 74-HC1 (pH 4.0) containing 0.05% Lubrol-PX. The pH and A,, of the fractions were measured and 20 to 25-pL aliquots of alternate fractions were

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348 BIOCHEM. CELL BIOL. VOL. 69, 1991

used in assays of phospholipase activity. Each chromatofocusing column was used only once. Fractions from the column that contained measurable phospholipase B activity were also monitored for purity by SDS-PAGE and silver-staining. For this purpose, 10-pL aliquots of the active fractions were resolved under nonreducing conditions on gels containing a 5-10% gradient of acrylamide. A specific, silver-stained band that correlated with the phospholipase activity was identified (see Results). Generally, this band was well resolved from the other proteins in the first 5 to 7 fractions containing phospholipase activity; in subsequent fractions another protein migrated to a similar position on the gel.

Identification of the phospholipase band in 5-10% gradient gels Fractions from the chromatofocusing column in which the

putative phospholipase band was free of the closely migrating contaminant were pooled, dialyzed for 2 days against 3 changes of 100 volumes of 10 mM ammonium acetate (pH 7.0), and then lyophilized. This pooled fraction was reconstituted with Laemmli sample buffer (nonreducing ) and separated in a 5-10% gradient gel. After staining with 0.3 M CuClz (Lee et al. 1987), the putative phospholipase band was excised, destained with EDTA (Lee et al. 1987), crushed in 2 mL of 50 mM Hepes (pH 8.0), 2 mM EDTA, 150 mM NaCl, 0.1% (w/v) SDS, and allowed to elute overnight at room temperature. Following a brief centrifugation to pellet the gel material, aliquots of the resulting supernatant were then resolved by SDS-PAGE in a 7.5% gel. BSA (200 pg) was added to the rest of the supernatant and the proteins were precipitated with acetone, denatured in guanidine-HC1, and then renatured by dilution as previously described (Pind and Kuksis 1989; modified from Hager and Burgess, 1980). Aliquots (0.2 mL) of the resulting solution were assayed for phospholipase A, and lysophospholipase activities.

Preparation of anti-phospholipase B sera For the initial immunization of two rabbits, the contaminant-

free fractions from six chromatofocusing columns were pooled before dialysis and subsequent lyophilization. The lyophilized sample was reconstituted with 1 mL of Laemmli sample buffer (nonreducing) and separated by SDS-PAGE in two 5-10% gradient gels. Protein bands were visualized with Coomassie blue R-250, and the phospholipase bands were excised with a clean razor blade, minced into small pieces, suspended into Freund's complete adjuvant by repeated passage from one syringe to another via a double-hub connector, and injected subcutaneously at several sites along the backs of the rabbits (Hurn and Chantler 1980; Harlow and Lane 1988). It was estimated by Coomassie blue staining intensity that each rabbit received 30-60 pg of protein during this initial injection. Booster injections were given 8, 16. and 30 weeks later, using approximately one-half of the initial amount of gel-purified antigen, prepared in the same manner as above but emulsified in Freund's incomplete adjuvant. The preparations used for booster injections were qualitatively identical to those used for immunization.

Immunoblotting Proteins were transferred electrophoretically from 7.5% or

5-10% gradient gels onto nitrocellulose membranes in ice-cold transfer buffer (20 mM Tris-HC1 (pH 8.0), 150 mM glycine, 20% methanol; Winston et al. 1987) at 80 V for 16 h (overnight) in a Transblot apparatus (Bio-Rad). The nitrocellulose membranes were blocked by incubation in 10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% (w/v) Tween 20. 1% (w/v) BSA. at room temverature for 1 h, and then incubated for 1 h with a-1/1000 dilution of rabbit serum in the same buffer. The bound antibody was detected using an alkaline phosphatase conjugated goat anti-rabbit IgG (FC) secondary antibody following methods described by the supplier (Promega).

Amino acid analysis Fractions from the chromatofocusing columns were prepared

for electrophoresis as described above. The lyophilized sample was

separated under nonreducing conditions on a 5-10% gradient gel in which both the separating and stacking gels had been allowed to polymerize for 24 h prior to use. Following electrophoresis the gel was soaked in transfer buffer (10 mM Hepes (pH KO), 0.5 mM dithiothreitol, 10% methanol; Matsudaira 1987; Moos et al. 1988) for 5 min to reduce the amount of Tris and glycine. Proteins were then transferred electrophoretically onto a polyvinylidene difluoride membrane (Immobilon) in ice-cold transfer buffer at 80 V for 16 h in the Transblot apparatus. The membrane was then removed, soaked in 10 mM iodoacetamide for 5 min, rinsed in H,O for 10 min, and then stained with Coomassie blue R-250 (Matsudaira 1987). A portion of the Coomassie-stained phospholipase B band was hydrolyzed in vacuo in 6 M HCI at llO°C for 24 h and the resulting amino acids were derivatized with phenylisothiocyanate and analyzed using the Waters Pico Tag system (Biotechnology Service Centre, The Hospital for Sick Children, Toronto, Ont.). Amino sugars were similarly analyzed on a second portion following a 4-h digestion at llO°C.

Other methods Brush-border membranes were digested with papain as described

earlier (Pind and Kuksis 1989). Aliquots of the papain-solubilized proteins and the residual membrane fraction were concentrated prior to electrophoresis by precipitation with deoxycholate and trichloroacetic acid (Peterson 1977) containing 1% phosphotungstic acid (Hoffman and Kuksis 1979). Precipitates were washed with acetone and adjusted to pH 7.0 in the sample buffer prior to electrophoresis. Protein concentrations were determined using the bicinchoninic acid protein assay reagent (Pierce). Brush-border membranes were purified from guinea pig and rabbit small intestines as described by Kessler et al. (1978).

Results

Purification of phospholipase B The goal of the present experiments was to purify

phospholipase B from the small intestinal brush-border membranes of rats, in either its native, active form, or in a form amenable for use as an antigen. Preliminary attempts involving chromatography of the detergent-solubilized membrane proteins on a wide variety of column matrices, which fractionated proteins by size, charge, carbohydrate content, or affinity, failed to separate the phospholipase activity from the bulk of the solubilized proteins. Some promising (2-fold) purifications were obtained on hydrophobic matrices, including phenyl-Sepharose and phospholipid affinity columns, but were difficult t o reproduce as binding was dependent upon removal of the detergent. A more reproducible and cleaner separation of the proteins based on hydrophobicity was obtained by Triton X-114 phase partitioning. We previously reported (Pind and Kuksis 1989) that the intact phospholipase partitioned into the detergent phase during this procedure. Using a 10-fold scaled-up version of this method, more than 85% of the phospholipase activity was solubilized by Triton X-114 extraction and associated with the detergent phase following partitioning. Approx- imately 35% of the total proteins solubilized with Triton X-114 was also associated with this phase, resulting in a 2.2-fold purification of the phospholipase activity. Figure 1 shows the electrophoretic profiles of the Triton X-114 solubilized proteins in the 7.5% (lanes 1-3) and 5-10% gradient (lanes 4-6) gels following phase partitioning. There is a striking difference in the distribution of the proteins, with some bands confined t o the detergent phase (lanes 2 and 5 ) , while others are found only in the aqueous phase (lanes 3 and 6). It is interesting to note that the intensely staining sucrase- and isomaltase-rich bands are found in the

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PIND AND KUKSlS 349

FIG. 1 . SDS-PAGE of the brush-border membrane proteins following Triton X-114 phase partitioning. The total membrane proteins solubilized with Triton X-114 (lanes 1 and 4) and equal aliquots of those found in the detergent (lanes 2 and 5), or aqueous (lanes 3 and 6) phases following partitioning were resolved under nonreducing conditions on 7.5% (lanes 1-3) and 5-10% gradient (lanes 4-6) gels. Bands were visualized by silver staining. The mobilities of molecular mass standards (Bio-Rad high molecular mass standards) are shown along the side margins. I, isomaltase-rich bands; S, sucrase-rich bands.

aqueous phase, even though the sucrase-isomaltase complex is a transmembrane protein (Hunziker et al. 1986). Similar protein profiles have been published by Tiruppathi et al. (1986). These authors also confirmed by enzyme assay that more than 90% of the disaccharidases, including sucrase- isomaltase, remain in the aqueous phase following Triton X-114 phase partitioning. A considerable portion of the ca. 170-kDa band in the 7.5% gel (lanes 1-3) remained in the aqueous phase following partitioning. This is the region of the gel where phospholipase B activity was previously found after renaturation (Pind and Kuksis 1989). The detergent phase contained two bands in the 170-kDa region of the gel as possible candidates for the phospholipase (lane 2, not completely resolved in this photograph). While most of the major proteins migrate to similar positions relative to molecular weight standards on the 5-10% gradient gel (lanes 4-6), there are a number of mobility shifts in the 170-kDa region of the gel; the importance of this will become apparent below.

Phospholipase A2 and lysophospholipase activities were recovered in a single peak eluting between pH 5 and 6 following chromatofocusing of the proteins associated with the Triton X-114 detergent phase (Fig. 2). The large absorbance

at 280 nm at the beginning of this profile is due to Triton X-114 rather than to protein; eluting the column with pH 4.0 Polybuffer containing 0.05% Lubrol-PX resulted in a reproducible Azso nm profile from the proteins eluting in fractions 30-70. Figure 3 shows that this column resulted in a considerable fractionation of the proteins. Electrophoresis in the 7.5% gel (Fig. 3A) revealed that the lower one of the two bands in the 170-kDa region correlated with those fractions having phospholipase activity (band marked by the arrow). In the 5-10% gradient gel (Fig. 3B), this band was well resolved from the other proteins (arrow), but its mobility had increased such that it migrated in this gel with an apparent molecular mass of 130 kDa. By fraction 50 of the elution profile, a protein began to elute from the column that migrated slightly faster than the 130-kDa band in fractions 42-47. In all subsequent chromatofocusing column eluants, fractions were monitored for the presence of this contaminating band by SDS-PAGE and silver staining. Of the phospholipase B activity loaded onto the column in different preparations, 40 to 70% eluted before the contaminating band appeared and was pooled for subsequent use. This fractionation resulted in a further 2- to 4-fold enrichment of activity, or a combined purification of 100-

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FIG. 2. Chromatofocusing profile of the proteins associated with the Triton X-114 detergent phase. The detergent pellet arising from phase partitioning the Triton X-114 soluble proteins from the brush-border membranes of 3 rats was suspended in 8 mL of 25 mM irnidazole (pH 7.4), 0.05% Lubrol-PX, loaded onto a chromatofocusing column (0.7 x 13 an) and eluted with pH 4.0 Polybuffer 74-HC1, 0.05% Lubrol-PX. Aliquots (25 pL) of alternate fractions were used in single determinations of phospholipase A, and lysophospholipase activity. @-@, pH profile; ....., A,,, ,,,; o, phospholipase A,; e--o , lysophospholipase activity. The large A,,, ., in fractions 2-20 was due to Triton X-114.

FIG. 3. SDS - polyacrylamide gel analysis of fractions obtained following chromatofocusing of the Triton X-114 detergent phase associated brush-border membrane proteins. Aliquots (10 PL) of the fractions obtained from the elution profile (shown in Fig. 2) were resolved by nonreducing electrophoresis in (A) 7.5% and (B) 5-10% gradient gels. Bands were visualized by silver staining. The mobilities of molecular mass standards are shown along the margins. The arrows mark the band whose staining intensity correlates with the phospholipase activity profile.

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PIND AND KUKSIS 351

to 150-fold with a 20-25% yield from the initial mucosal homogenate.

Although the staining intensity of the putative phospholipase band identified in fractions 42-47 (Fig. 3) correlated with the phospholipase activity profile in Fig. 2, further analysis was necessary to ascertain whether this was indeed the phospholipase. Figure 4 (lanes 2 and 3) shows that when the 130-kDa band was excised from a 5-10% gradient gel, eluted, electrophoresed under nonreducing conditions in a 7.5% polyacrylamide gel, and silver stained, a single band migrating at 160-165 kDa was observed. A single band was also observed when the eluted protein was electrophoresed under reducing conditions (lane 5), but interestingly, it now migrated with an apparent molecular mass of 130 kDa. Fur- ther evidence that this band was the phospholipase B was obtained by renaturation of the 130-kDa band from nonreducing 5-10% gels as described in Materials and methods. The renatured protein had both phospholipase A2 and lysophospholipase activities, whereas material recovered from gel slices both above and below the 130-kDa band had neither activity. Although accurate specific activity measurements could not be made on the renatured material, it was estimated from the relative silver staining inten- sities that the phospholipase band accounted for 5-10% of the protein in the pooled chromatofocusing fraction. With the caveat that silver staining may not be representative of the relative protein concentrations, a 1000- to 2000-fold purification of the phospholipase was achieved following the preparative electrophoresis step, an estimate similar to the 1700-fold enrichment that was obtained during purification of the papain-solubilized enzyme from guinea pig (Gassama-Diagne et al. 1989).

Immunoblot analysis of phospholipase B A rabbit polyclonal antiserum against rat brush-border

membrane phospholipase B was generated by subcutaneous injection of SDS-polyacrylamide gel pieces containing purified phospholipase B. Figure 5 shows by immunoblot analysis that the antiserum recognized a single band when total membrane proteins were resolved in 7.5% (lanes 1-4) or in 5-10% gradient gels (lanes 5-8) under both nonreducing (lanes 2 and 6) and reducing (lanes 3 and 7) conditions. There was no reaction with preimmune serum in either of these gels (lanes 4 and 8). It is also obvious from this immunoblotting analysis that reducing agents dramatically influence migration of the immunoreactive band in both of these gels. In the 7.5% gel, 0-mercaptoethanol caused an increase in the mobility of the immunoreactive band from 170 to 130 kDa (lanes 2 vs. 3), similar to the effect of this reducing agent on the purified protein (Fig. 4). In the 5-10% gradient gel the mobility of the immunoreactive band increased from 130 to approximately 120 kDa (Fig. 5, lanes 6 vs. 7) following reduction. To further verify the specificity of the antiserum, Fig. 6 shows that reactivity on an immunoblot paralleled the activity profile from a chromatofocusing column. Immunoreactive bands were only present in those fractions having assayable phospholipase activity; those fractions with the greatest activity also had the most intensely staining bands. In addition, irnmunoblots with 50 pg of the total brush-border membrane protein, or 25 pg of the detergent phase-associated proteins, or approximately 1 pg from the peak fractions of the chromatofocusing column, gave immunopositive bands of similar inten-

FIG. 4. Electrophoresis in a 7.5% polyacrylamide gel of the proteins recovered from the 130-kDa band of a 5-10% gradient gel. The 130-kDa band from a 5-10% gradient gel of the chromatofocusing-purified phospholipase B was identified and eluted as described in Materials and methods. The eluted proteins were electrophoresed in a 7.5% gel under nonreducing (lanes 1-3) or reducing (lanes 4 and 5) conditions and visualized by silver staining (Rabilloud et al. 1988). Lanes 1 and 4, total brush-border membranes from the distal two-thirds of rat small intestine; lanes 2 and 5, 25 pL of proteins eluted from the 130-kDa band; lane 3, 50 pL of eluted proteins. The mobility of molecular weight standard (M, x are shown along the left-hand margin.

sities (not shown), indicating that the antigen was being purified during phospholipase fractionation. Taken together, these results support the conclusion that the antiserum was specific for phospholipase B.

The preparation of a polyclonal antiserum allowed for the first time a direct comparison between the intact and papain-solubilized phospholipase B and a demonstration of cross-reactivity among the different species. Papain digestion of rat brush-border membranes solubilized phospholipase B activity and resulted in a decrease in size from 120 (Fig. 7, lane 1) to 80 kDa (lane 4) for the major immunoreactive band in an immunoblot (5-10% gel, reducing conditions). This band also migrated as an 80-kDa band in 7.5% gels (under reducing or nonreducing conditions, not shown). A less intensely staining band(s) was also apparent at approximately 60 kDa in the solubilized fraction (lane 4), possibly reflecting the size heterogeneity of the papain-digested prod- ucts observed previously (Pind and Kuksis 1989). Figure 7 further shows that the antiserum identified a major

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FIG. 5. Immunoblot analysis of the anti-phospholipase B serum with brush-border membrane proteins. Lanes 1-4, results from 7.5% gels; lanes 5-8, results from 5-10% gradient gels. Lanes 1 and 5, 2 pg of total brush-border membrane proteins visualized by silver staining. In lanes 2-4 and 6-8, 25 pg of total brush-border membrane proteins from the distal two-thirds of the small intestine were electrophoresed, transferred to nitrocellulose, and immunoblotted, as described in Materials and methods. Lanes 3 and 7 were run under reducing conditions; all others were run under nonreducing conditions. Lanes 2, 3, 6, and 7 were incubated with a 1/1000 dilution of antiphospholipase B serum; lanes 4 and 8 were incubated with a 1/1000 dilution of preimmune serum. The antigen-antibody com- plexes were detected using alkaline phosphatase conjugated secondary antibodies and bromochloroindoyl phosphate - nitro blue tetrazolium as chromogenic substrates.

immunoreactive band at approximately 140 kDa and minor bands at 125 kDa and lower molecular weights when the proteins from guinea pig brush-border membranes were immunoblotted (lane 2). The possibility that one of these bands was indeed the phospholipase B was strengthened following immunoblotting of the proteins solubilized by papain digestion of the membranes. Lane 5 shows that the most intensely staining immunoreactive band in the solubilized fraction migrated as a 97-kDa protein, in exact agreement with the reported size of the purified, papain-solubilized protein from guinea pig (Gassama-Diagne et al. 1989). Further- more, Fig. 7 indicates that this enzyme is also present in the rabbit intestine as the antiserum reacted with a protein band of approximately 135 kDa in the rabbit brush-border membranes (lane 3) that decreased in size to approximately 95 kDa following papain solubilization (lane 6). These results provide strong evidence that similar phospholipases B occurring in the three species contain epitopes that are recognized by the same antiserum.

Intestinal distribution of the rat phospholipase B Figure 8 shows that the phospholipase A2 and

lysophospholipase activities were restricted to the distal two- thirds of the small rat intestine when assayed in total mucosal

homogenates. Phospholipase B activity was apparent in regions corresponding to the distal jejunum and distal ileum, but was highest in the region corresponding to the proximal ileum. These assays were done in the presence of 5 mM EDTA to inhibit any adherent pancreatic or Paneth cell phospholipase A2 present in the scrapings (Volwerk and De Haas 1982; Mansbach et al. 1982), and in 1 % CHAPS detergent, to inhibit any intracellular lysophospholipases present (Brockerhoff and Jensen 1974; Charbonnier et al. 1988). That the phospholipase activities measured were actually those of the brush-border membrane phospholipase B was further verified by membrane purification studies and by immunoblotting. The specific activities of phospholipase A2 and lysophospholipase in the total mucosal homogenates from the distal two-thirds of rat small intestine (each approximately 16 nrnol/(mg protein/min)) were increased more than 20-fold by brush-border membrane purification (each to approximately 330 nmol/(mg proteidmin)). These values are 2.5- to 3-fold higher than the specific activities reported earlier (Pind and Kuksis 1988) from studies involving the proximal two-thirds of the small intestine, but are similar to the values reported for homogenates and purified membranes from guinea pig (Gassama-Diagne et al. 1989). In addition, positive reaction

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PIND AND KUKSIS 353

0.00 0 0 10 20 3 0 40 5 0 60 70

Fraction (1 mL)

FIG. 6. Immunoblot analysis of fractions obtained following chromatofocusing of the Triton X-114 detergent phase associated brush- border membrane proteins. (A) Chromatofocusing profile showing A,,, ., (broken line) and phospholipase A2 activity (solid line). Chromatofocusing was performed as described in the legend to Fig. 2 except that a single 10-mL fraction was collected before the 1-mL fraction collections began. (B) Aliquots (40 pL) of the indicated fractions were electrophoresed in a 5-10% gradient gel, transferred to nitrocellulose, and immunoblotted as described in the legend to Fig. 5. Only a portion of the total immunoblot is shown. The mobility of two prestained molecular mass markers (Amersham Rainbow markers) are shown on the left margin.

on an immunoblot paralleled the activity profile along the length of the small intestine (not shown).

Composition of rat phospholipase B Amino acid and amino sugar analyses were performed on

gel-purified samples of the phospholipase B that had been electroblotted onto an inert support (Immobilon). This sample had 40% nonpolar residues, with Asx and Glx predominating over the positively charged acids (Table 1). Amino sugar analysis on another portion of the membrane showed a 70:30 ratio of N-acetylglucosamine to N-acetyl- galactosamine. Amino terminal sequence analysis was performed using approximately 20 pmol of protein, but was inconclusive, as an unambiguous amino acid assignment could not be made in some cycles.

purified protein confirmed the presence of both phospholipase A2 and lysophospholipase activities, reinforcing the identification of this enzyme as a phospholipase B. Although this purification method is impractical for enzyme activity studies, the availability of this purified fraction allowed the generation of a polyclonal antiserum and a further characterization of this protein by immunoblotting.

Mobility of the phospholipase B in SDS-polyacrylamide gels was dependent upon the percentage of acrylamide in the gels and upon whether samples were reduced or not prior to electrophoresis. Estimations of the molecular mass of the protein varied from 120 to 170 kDa under the selected exper- imental conditions. Anomalous migration of proteins during electrophoresis is known to be caused by both carbohydrate constituents and disulphide bonds (Hames 1981). All of the previously studied ectoenzymes in the microvillus

Discussion membrane are glycoproteins containing considerable quan- A combination of Triton X-114 phase partitioning, tities of carbohydrate (10-35% by weight; Kenny and

chromatofocusing, and SDS-PAGE resulted in the purifica- Maroux 1982). The phospholipase B binds to Conconavalin tion of a full-length, albeit denatured, phospholipase from A-Sepharose (S. Pind and A. Kuksis, unpublished data) and rat intestinal brush-border membranes. Renaturation of this contains amino sugars, indicating that this enzyme is also

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FIG. 7. Cross-reactivity of the anti-phospholipase B serum with intact and papain-solubilized proteins from rat, guinea pig, and rabbit brush-border membranes. Proteins of the brush-border membrane from rat (lanes 1 and 7, 25 pg), guinea pig (lanes 2 and 8, 50 pg), and rabbit (lanes 3 and 9, 50 pg) were separated by electrophoresis (5-10% gel, reducing conditions) and immunoblotted as described in the legend to Fig. 5. In lanes 4-6, 75 pg of the proteins solubilized by a 60-min papain digestion of rat (lane 4), guinea pig (lane 5), or rabbit (lane 6) brush-border membranes were immunoblotted. Lanes 1-6 show the reaction with a 1/750 dilution of the anti-phospholipase B serum; lanes 7-9 show the reaction with a 1/750 dilution of the preimmune serum.

glycosylated. We previously observed (Pind and Kuksis 1989) that phospholipase activity could be renatured from the denatured enzyme only if reducing agents were not used, suggesting that disulphide bonds keep the denatured protein in a renaturation-competent conformation. During electrophoresis, glycoproteins probably bind SDS only to their protein portions, while intact disulphide bonds oppose complete denaturation and prevent saturation of the protein with SDS (Hames 1981). Both of these circumstances result in a reduced net charge of the molecule during electrophoresis, thereby lowering the mobility and yielding artificially high molecular weight estimates. It has been suggested that electrophoresis under reducing conditions in gradient gels is the method of choice for glycoprotein molecular weight deter- mination (Hames 1981). This being the case, the best estimate for the mass of the full-length rat phospholipase B would be 120 kDa, and for the major papain-solubilized fragment would be 80 kDa. The structural basis for such a dramatic size difference between the two forms is not presently known. Papain solubilization of other intestinal ectoenzymes generally results in a smaller difference, as the membrane-anchoring domains comprise less than 5% of the total mass of the proteins (Kenny and Maroux 1982; Semenza 1986; Maroux 1987). The present results suggest

that the phospholipase B may have a larger membrane anchor or cytoplasmic domain than other ectoenzymes, or that papain cleavage was not restricted to the stalk region in the ectodomain of this enzyme. In any event, the present results also show that there are correspondingly large size differences between the intact and papain-solubilized forms of the protein(s) from guinea pig and rabbit brush-border membranes that cross-react with the rabbit antiserum to rat phospholipase B. In the papain-solubilized fraction from guinea pig membranes, the antiserum reacts most intensely with a 97-kDa band suggesting that the cross-reacting bands originated from phospholipase B. We conclude that the phospholipases B are immunologically related and that the previously reported discrepancies in the estimation of the size resulted from a comparison between the full-length protein and a papain-solubilized fragment and from the effect of the selected electrophoretic conditions.

In addition to having a greater activity of the phospha- tidylglycerol-specific phospholipase A2 of Paneth cell origin (Mansbach et al. 1982; Senegas-Balas el al. 1984), mucosal homogenates of the distal (ileum) region of the rat small intestine have been demonstrated to have higher specific activities than homogenates from the proximal region of other, undefined phospholipases A (Shakir et al. 1982;

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PIND AND KUKSIS

Intestinal segment

FIG. 8. Distribution of phospholipase A, and lysophospholipase activities in homogenates of the intestinal mucosa. The entire small intestine was removed from rats and divided into 6 equal-sized segments (18-20 cm each) from the pyloric valve (segment 1) to the ileocecal junction (segment 6). Mucosal scrapings from each segment were homogenized in 2 mM Hepes (pH 7.0), 300 mM mannitol, 5 mM EDTA, using five 30-s pulses (power setting, 4) of a Polytron (Brinkmann). Aliquots were assayed for phospholipase A, (hatched bars) and lysophospholipase (open bars) activities and for protein content (in duplicate), as described in Materials and methods. Phospholipase assays were done in 5 mM EDTA. Results are expressed as the amount of radioactive fatty acid released per 100 pg protein in a 16-min assay, and are the average * range/2 for assays done on two separate rats.

Walters et al. 1986). The present results have shown that the brush-border membrane phospholipase B activity has a similar distribution gradient. The phospholipase A2 and lysophospholipase activities were absent in the duodenum and proximal jejunum and were highest in the proximal part of the ileum. In contrast, the guinea pig enzyme was previously shown to be present in all regions of the small intestine, although the middle third had the highest specific activity (Diagne et al. 1987). It is tempting to speculate that the difference in the intestinal distribution between the rat and guinea pig has functional implications, especially since the guinea pig is known to lack pancreatic phospholipase A2 (Fauvel et al. 1981). As a contributor to the digestion of phospholipids, it has been proposed that phospholipase B acts to hydrolyze diacyl or monoacyl phospholipids solubilized in bile-salt micelles that reach the luminal surface of the brush-border membrane (Pind and Kuksis 1989; Gassama-Diagne et al. 1989). The concentration of the enzyme in the ileum of rats suggests that phospholipase B has a role secondary to that of pancreatic phospholipase A,, and acts to digest phospholipids that evade the pancreatic enzyme in the proximal region of the small intestine. Such a "clean-up" function may be important to scavenge luminal phospholipids arising from the bile in the absence of pancreatic secretions, or from the membranes of sloughed intestinal cells. The presence of phospholipase B in all regions of the small intestine in the guinea pig suggests that this enzyme plays a primary role in phospholipid digestion in this species.

The importance of brush-border membrane ectoenzymes for the intestinal digestion of dietary carbohydrates and proteins has been well established (Kenny and Turner 1987b). In contrast, the extent of this membrane's involve-

TABLE 1. Amino acid composition of the purified phospholipase B

Amino Acid Residues/100 residues

Asx Glx Ser G ~ Y His Arg Thr Ala Pro T Y ~ Val Met Ile Leu Phe L Y ~ T ~ P C Y ~

11.9 10.9 8.4 8.2 1.6 4.7 7.6 7.7 5.5 2.8 7.1 1.6 3.9 9.8 4.5 3.9 nd* nd

Nom: The values are from a single 24-h hydrolysate of approximately 8.3 pmol of protein blotted onto Immobilon.

*nd, not determined.

ment in lipid digestion is only now becoming evident. In addition to phospholipase B, earlier studies have shown the brush-border membrane to contain enzymes that hydrolyze glycosylcerarnides (Leese and Semenza 1973), sphingomyelin, and ceramides (Nilsson 1969). Recent studies have now demonstrated that two pancreatic enzymes, cholesterol esterase and triacylglycerol lipase, are localized to the brush-

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border membrane through a receptorlike binding t o KENNY, A.J., and TURNER, A.J. (Editors). 1987a. Mammalian membrane-associated heparin (Bosner et 01. 1988, 1989), ectoenzymes. Elsevier Science Publishers, B.V., Amsterdam.

suggesting that it may be necessary t o reevaluate the site of - 1987b- What are ectoenzJ'mes? In Wtmmalian ectoen-

intraluminal lipid digestion. zymes. Elsevier Science Publishers B.V., Amsterdam. pp. 1-13. KESSLER, M., ACUTO, O., STORELLI, C., MURER, H., MULLER,

Acknowledgements These studies were supported by grants from the Medical

Research Council of Canada, Ottawa, Ont., and the Heart and Stroke Foundation of Ontario, Toronto. The authors thank Dr. Sashi Joshi for helpful suggestions during the amino acid and N-terminal sequence analyses and Dr. H.W. Davidson for critically reading the manuscript.

BORDER, C. 1981. Phase separation of integral membrane proteins in Triton X-114. J. Biol. Chem. 256: 1604-1607.

BOSNER, M.S., GULICK, T., RILEY, D. J.S., SPILBURG, C.A., and LANGE, L.G. 1988. Receptor-like function of heparin in the binding and uptake of neutral lipids. Proc. Natl. Acad. Sci. U.S.A. 85: 7438-7442. - 1989. Heparin-modulated binding of pancreatic lipase and

uptake of hydrolyzed triglycerides in the intestine. J. Biol. Chem. 264: 20 261 - 20 264.

BROCKERHOEE, H., and JENSEN, R.G. 1974. Phospholipases: carboxyl esterases. In Lipolytic enzymes. Academic Press Inc., New York. pp. 194-266.

CHARBONNIER, M., GRATAROLI, R., PORTUGAL, H., LAFONT, H., and NALBONE, G. 1988. Subcellular distribution of lysophospholipase of rat intestinal mucosa. Biochem. Cell Biol. 66: 813-820.

DIAGNE, A., MITJAVILA, S., FAUVEL, J., CHAP, H., and DOUSTE- BLAZY, L. 1987. Intestinal absorption of ester and ether glycerophospholipids in guinea-pig. Role of a phospholipase A, from brush-border membrane. Lipids, 22: 33-40.

FAUVEL, J., BONNEFIS, M.J., CHAP, H., THOUVENOT, J.P., and DOUSTE-BLAZY, L. 1981. Evidence for the lack of classical secretory phospholipase A, in guinea-pig pancreas. Biochim. Biophys. Acta, 666: 72-79.

GASSAMA-DIAGNE, A., FAUVEL, J., and CHAP, H. 1989. Purifica- tion of a new, calcium-independent, high molecular weight phospholipase A,/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane. J. Biol. Chem. 264: 9470-9475.

HAGER, D.A., and BURGESS, R.R. 1980. Elution of proteins from sodium dodecyl sulfate - polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase and other enzymes. Anal. Biochem. 109: 76-86.

HAMES, B.D. 1981. An introduction to polyacrylamide gel electrophoresis. In Gel electrophoresis of proteins: a practical approach. Edited by B.D. Hames and D. Rickwood. IRL Press Ltd., Oxford. pp. 7-91.

HARLOW, E., and LANE, D. 1988. Immunization. In Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. pp. 53-137.

HOFFMAN, A.G.D., and KUKSIS, A. 1979. Improved isolation of villus and crypt cells from rat small intestinal mucosa. Can. J. Physiol. Pharmacol. 57: 832-842.

HUNZIKER, W., SPIESS, M., SEMENZA, G., and LODISH, H.F. 1986. The sucrase-isomaltase complex: primary structure, membrane-orientation, and evolution of a stalked, intrinsic brush border protein. Cell, 46: 227-234.

HURN, B.A.L., and CHANTLER, S.M. 1980. Production of reagent antibodies. Methods Enzyrnol. 70: 104-142.

KENNY, A.J., and MAROUX, S. 1982. Topology of microvillar membrane hydrolases of kidney and intestine. Physiol. Rev. 62: 91-128.

M., and SEMENZA, G. 1978. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim. Biophys. Acta, 506: 136-154.

LAEMMLI, U.K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (London), 227: 680-685.

LEE, C., LEVIN, A., and BRANTON, D. 1987. Copper staining: a five minute protein stain for sodium dodecyl sulfate - polyacrylamide gels. Anal. Biochem. 166: 308-3 12.

LEESE, H.J., and SEMENZA, G. 1973. On the identity between the small intestinal enzymes phlorizin hydrolase and glyco- sylceramidase. J. Biol. Chem. 248: 8170-8173.

MANSBACH, C.M., 11, PIERONI, G., and VERGER, R. 1982. Intestinal phospholipase, a novel enzyme. J. Clin. Invest. 69: 368-376.

MAROUX, S. 1987. Structural and topological aspects. In Mam- malian ectoenzymes. Edited by A.J. Kenny and A.J. Turner. Elsevier Science Publishers, B.V., Amsterdam. pp. 15-45.

MATSUDAIRA, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262: 10 035 - 10 038.

Moos, M., JR., NGUYEN, N.Y., and LIU, T.-Y. 1988. Reproduc- ible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem. 263: 6005-6008.

NILSSON, A. 1969. The presence of sphingomyelin- and ceramide- cleaving enzymes in the small intestinal tract. Biochim. Biophys. Acta, 176: 339-347.

NOREN, 0.. SJOSTROM, H., DANIELSEN, E.M., COWELL, G.M., and SKOVBJERG, H. 1986. The enzymes of the enterocyte plasma membrane. In Molecular and cellular basis of digestion. Edited by P. Desnuelle, H. Sjostrom, and 0. Noren. Elsevier Science Publishers, B.V., Amsterdam. pp. 335-365.

PETERSON, G.L. 1977. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83: 346-356.

PIND, S., and KUKSIS, A. 1987. Isolation of purified brush-border membranes from rat jejunum containing a ca2+-independent phospholipase A, activity. Biochim. Biophys. Acta, 901: 78-87.

1988. Solubilization and assay of phospholipase A, activity from rat jejunal brush-border membranes. Biochim. Biophys. Acta, 938: 21 1-221.

1989. Association of the intestinal brush-border membrane phospholipase A, and lysophospholipase activities (phospholipase B) with a stalked membrane protein. Lipids, 24: 357-362.

RABILLOUD, T., CARPENTIER, G., and TARROUX, P. 1988. Improvement and simplification of low-background silver staining of proteins using sodium dithionite. Electrophoresis, 9: 288-291.

SEMENZA, G. 1986. Anchoring and biosynthesis of stalked brush border membrane proteins: glycosidases and peptidases of enterocytes and renal tubuli. Annu. Rev. Cell Biol. 2: 255-3 13.

SENEGAS-BALAS, F., BALAS, D., VERGER, R., DE CARO, A., FIGARELLA, C., FERRATO, F., LECHENE, P., BERTRAND, C., and RIBET, A. 1984. Imrnunohistochemical localization of intestinal phospholipase A, in rat Paneth cells. Histochemistry, 81: 581-584.

SHAKIR, K.M.M., GABRIEL, L., SUNDARAM, G., and MARGOLIS, S. 1982. Intestinal phospholipase A and triglyceride lipase: localization and effect of fasting. Am. J. Physiol. 242: G168-G176.

Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

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A&

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11/1

4/14

For

pers

onal

use

onl

y.

PIND AND KUKSIS 357

SUBBALAH, P.V., and GANGULY, J. 1970. Studies on the phospholipases of rat intestinal mucosa. Biochem. J. 118: 233-239.

TIRUPPATHI, C., ALPERS, D.H., and SEETHARAM, B. 1986. Phase separation of rat intestinal brush border membrane proteins using Triton X-114. Anal. Biochem. 153: 330-335.

VOLWERK, J. J., and DE HAAS, G.H. 1982. Pancreatic phospholipase A,: a model for membrane-bound enzymes. In Lipid- protein interactions. Vol. 1. Edited by P.C. Jost and O.H. Griffith. John Wiley & Sons, New York. pp. 69-141.

WALTERS, J.R.F., HORVATH, P.J., and WEISER, M.W. 1986. Preparation of subcellular membranes from rat intestinal scrap- i n g ~ or isolated cells. Different ca2+ binding, non-esterified fatty acid levels, and lipolytic activity. Gastroenterology, 91: 34-40.

WINSTON, S.E., FULLER, S.A., and HURRELL, J.G.R. 1987. Western blotting. In Current protocols in molecular biology. Unit 10.8. Edited by F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. Green Pub- lishing Associates and Wiley-Interscience, New York.

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Further characterization of a novel phospholipase B (phospholipase A 2 ...

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