From the Department of Molecular Physiological Chemistry, Osaka
University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan
A brush border membrane-associated phospholipase
B/lipase was solubilized from the distal two-thirds of rat small
intestine by autolysis during storage at
35 °C over 1 month, and
then the enzyme was purified to homogeneity and characterized
enzymatically and structurally. The purified enzyme exhibited broad
substrate specificity including esterase, phospholipase
A2, lysophospholipase, and lipase activities. SDS-gel
electrophoretic and reverse-phase high performance liquid
chromatographic analyses demonstrated that a single enzyme catalyzes
these activities. It preferred hydrolysis at the sn-2
position of diacylphospholipid and diacylglycerol without strict
stereoselectivity, whereas it apparently exhibited no positional
specificity toward triacylglycerol. Diisopropyl fluorophosphate, an
irreversible inhibitor of serine esterases and lipases, inhibited
purified enzyme. When the position of enzyme on SDS-gel electrophoresis
under the non-reducing conditions was determined by assaying the
activity eluted from sliced gels, brush border membrane-associated
enzyme corresponded to a ~150-kDa protein; autolysis gave a 35-kDa
product, in agreement with the results of immunoblot analysis. The
purified 35-kDa enzyme consisted of a 14-kDa peptide and a glycosylated
21-kDa peptide. Their NH2-terminal amino acid sequences
were determined and found in the second repeat of 161-kDa phospholipase
B/lipase with 4-fold tandem repeats of ~38 kDa each, which we cloned
and sequenced in the accompanying paper (Takemori, H., Zolotaryov, F.,
Ting, L., Urbain, T., Komatsubara, T., Hatano, O., Okamoto, M., and
Tojo, H. (1998) J. Biol. Chem. 273, 2222-2231). These
results indicate that the purified enzyme is the catalytic domain
derived from the second repeat of brush border membrane-associated
phospholipase B/lipase.
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INTRODUCTION |
Phospholipase A2
(PLA2)1 catalyzes
the hydrolysis of fatty-acyl ester bond at the sn-2 position
of glycerophospholipids and plays major roles in a variety of
biological processes such as digestion, membrane phospholipid
metabolism, inflammatory reactions, and eicosanoid synthesis.
PLA2s represent a diverse family of enzymes and can be
classified into broad two classes according to requirement of
Ca2+ ions for activity,
Ca2+-dependent and Ca2+-independent
PLA2s. Ca2+-dependent
PLA2s have been extensively studied so far in connection with important roles of Ca2+ ions in regulation of
intracellular signaling (1). To date, at least three isoenzymes are
known, i.e. group I and II secretory PLA2s and
85-kDa cytosol PLA2 (cPLA2). In the secretory
PLA2s Ca2+ ions involve in both catalysis and
substrate binding, whereas in cPLA2 calcium does not
function in the catalytic machinery, but enhances the enzyme's
affinity for membrane phospholipids (2, 3). On the other hand,
Ca2+-independent PLA2s, especially those of
mammalian origins, have not been so extensively studied. They were
recently purified from several mammalian sources, and their involvement
in phospholipids metabolism is now becoming evident; for example, a
40-kDa myocardial PLA2 may participate in alkyl and alkenyl
phospholipid metabolism (4), a 80-kDa macrophage PLA2 in
membrane phospholipid remodeling (5), and a 15-kDa lung acidic
PLA2 in surfactant metabolism (6). Very recently, some
information on primary structures has become available for these
PLA2s (7-9).
During the course of a study on the tissue distribution of rat group I
and II PLA2s, we found a high Ca2+-independent
PLA2 activity in rat distal intestine (10), and we decided
to purify and characterize this enzyme to better understand the
detailed structural and enzymatic properties of one of
Ca2+-independent PLA2s. This enzyme was similar
to those reported in rat and guinea pig brush border membranes, which
were partially purified after solubilized from those membranes by
detergent or papain treatment and characterized (11, 12). They had the characteristics of a digestive ectoenzyme and exhibited
lysophospholipase and lipase activities as well as PLA2
activity (13), therefore being hereafter named phospholipase B/lipase
(PLB/LIP). Unfortunately, there was no structural information available
on PLB/LIP, because it has not yet been purified to homogeneity in a
sufficient quantity.
In this study, we purified to homogeneity a truncated form of PLB/LIP
solubilized from rat intestine by autolysis during its storage at
35 °C for over 1 month, and characterized it both enzymatically
and structurally. The purified enzyme exhibited PLA2,
lysophospholipase, and lipase activities as a single 35-kDa molecule,
which consisted of a 14-kDa and a glycosylated 21-kDa peptides.
Molecular size analyses by SDS-gel electrophoresis suggested that the
purified enzyme was a catalytic domain derived from a ~150-kDa
membrane-associated enzyme. The accompanying paper (14) reports
cDNA cloning of PLB/LIP and expression of its entire protein, a
protein lacking COOH-terminal membrane anchoring domain, and the
catalytic domain in COS-7 cells. The results demonstrate that the
enzyme purified here is indeed the catalytic domain of membrane-bound PLB/LIP.
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EXPERIMENTAL PROCEDURES |
Materials--
The following glycerolipids were obtained from
Avanti Polar Lipids, Inc.:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
(POPG), 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine
(1-O-hexadecyl-OPC), and
1,2-dioleoyl-sn-glycerol.
2,3-Dioleoyl-sn-glycerol was purchased from Serdary Research
Laboratories, Inc. Monoolein was obtained from Nu-Check Prep, Inc.
1-Stearoyl-2-linoleoyl-sn-glycerol (SLDG) was purchased from
Biomol Research Laboratories, Inc.
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
2,3-dipalmitoyl-sn-glycero-1-phosphocholine, 1-palmitoyl-sn-glycero-3-phosphocholine (GPC),
1-myristoyl-GPC, 1,2-dioleoyl-rac-glycerol,
1,3-dipalmitoyl-2-oleoyl-rac-glycerol (POP),
1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol (POS),
1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC),
diisopropyl fluorophosphate (DFP), and Rhizopus
arrhizus lipase were obtained from Sigma. Triolein,
hydroxyapatite, trifluoroacetic acid, and octaethylene glycol dodecyl
ether (C12E8) were purchased from Nacalei
Tesque, Ltd. (Kyoto, Japan). Manoalide and p-bromophenacyl bromide (BPB) were obtained from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). HPLC-grade acetonitrile was purchased from Katayama
Chemical Industries Co., Ltd. (Osaka, Japan). Vectastain ABC kit was
obtained from Vector Laboratories, Inc. ConA-Sepharose was obtained
from Pharmacia Biotech Inc.
Assay for Lipolytic Activities--
PLA2,
lysophospholipase, and lipase activities were determined by a
non-radiometric HPLC method based on precolumn derivatization with
9-anthryldiazomethane (ADAM) as described previously (10). Individual
fatty acids released from mixed-acyl glycerophospholipids and tri- and
diacylglycerols were simultaneously determined by this method.
Substrate stock solutions used were as follows: mixed micelles of 5 mM diradyl-phospholipid and various concentrations of a
bile salt, cholate, or deoxycholate (DOC); 1-acyl-GPC (5 mM) micelles; and emulsions of 5 mM
triacylglycerol and 5% gum arabic. In a typical experiment, the
assay mixtures contained 10 mM EDTA, substrate micelles or
emulsion (10-µl stock solution), 0.1 M NaCl, 0.1 M Tris-HCl (pH 8.5), and the enzyme sample in a final
volume of 50 µl.
Cholesterol esterase activity was also determined by the ADAM method.
Cholesterol oleate (5 mM) was emulsified with 3.6 mg/ml mineral oil (15), 7.5 mM DHPC, or 5% arabic gum. DHPC was
used in place of phosphatidylcholine (16) because PLB/LIP has a high PLA2 activity. The assay mixtures contained cholesterol
ester emulsion (10 µl), either 10 mM DOC or 10 mM sodium taurocholate, 0.1 M NaCl, either 0.1 M MES (pH 6.5) or 0.1 M Tris-HCl (pH 8.5), and
the enzyme sample.
Solubilization of PLB/LIP from Brush Border Membranes--
Rats
anesthetized with pentobarbital were sacrificed by drawing blood from
the abdominal aorta, then the proximal half of the ileum was removed
and its mucosa was scraped off with a glass plate. Brush border
membrane fractions were prepared according to Hauser et al.
(17). The brush border membrane fraction was incubated with an equal
volume of either 2 M KCl, 2 M KBr, 1% DOC, or
2% Triton X-100 on ice for 1 h, and then 2 ml of the mixture was
centrifuged at 100,000 × g for 60 min. The pellets
were resuspended in 2 ml of 20 mM Tris-HCl containing 1 mM EDTA and 0.15 M NaCl (pH 7.4), and
PLA2 activity of the supernatant and pellet fractions was
determined with POPE/DOC mixed micelles as substrate. For autolysis
experiments, the brush border membrane fraction was kept at
35 °C,
and at the indicated times aliquots were taken, and handled in the same
manner as described above.
Purification of PLB/LIP--
Rat small intestines (lower 2/3
portions) were kept at
35 °C for at least 1 months, and then used
for the purification. The frozen tissues (80 g) were incubated in 800 ml of 10 mM Tris-HCl (pH 7.4), containing 2 mM
EDTA with stirring for 1 h at room temperature to remove the
mucosal cells and then the extract was sonicated for 5 min on ice.
After removing the residual intestine tissues, the extract was
centrifuged at 23,000 × g for 1 h. The pH of the supernatant was adjusted to 8.5, and then the solution was applied to a
QAE-Toyopearl column (3 × 20 cm) pre-equilibrated with 10 mM Tris-HCl (pH 8.5), containing 0.1% Triton X-100. The
PLA2 activity was eluted with the same buffer containing
0.5 M NaCl. Since the affinity of PLB/LIP for the column
was rather weak, the pass-through fraction was, if necessary, applied
again to the same QAE-Toyopearl column pre-equilibrated in the same
manner as described above. To the pooled PLA2 fraction was
added 1 M lithium sulfate, and then the solution was
applied to a phenyl-Sepharose column preequilibrated with 20 mM Tris-HCl (pH 7.4), containing 1 M lithium
sulfate. The PLA2 activity bound to this column under the
conditions, and was eluted with 10 mM Tris-HCl containing
10% ethylene glycol and 0.1% Triton X-100. The pooled
PLA2 fraction was dialyzed against 10 mM
Tris-HCl containing 50 mM NaCl, and then applied to a
QAE-Toyopearl column preequilibrated with 10 mM Tris-HCl.
The PLA2 activity was recovered in the flow-through
fractions, whereas a large amount of contaminants bound to the column.
The resultant PLA2-active fractions were dialyzed against
10 mM MES (pH 6.8), and then subjected to hydroxyapatite
column chromatography. The column was developed with a linear gradient
of potassium phosphate from 10 to 500 mM (pH 7.4), and the
PLA2 activity was eluted as a broad peak in the phosphate
concentration range of 30-150 mM. The pooled
PLA2 fraction was applied to a ConA-Sepharose affinity
column (1.5 × 7 cm) pre-equilibrated with 10 mM
Tris-HCl (pH 7.4). A major part (51%) of the PLA2 activity
bound to the column under these conditions, while a small part (34%)
did not. The column was washed with 10 mM Tris-HCl (pH
7.4), containing 0.1% C12E8 to remove strongly
uv-absorbing Triton X-100. The major bound enzyme activity was eluted
in a stepwise manner with the same buffer containing 0.5 M
methylmannoside, and the resultant solution was dialyzed against 20 mM Tris-HCl (pH 8.5), and further purified by HPLC on a
Cosmogel QA column (0.7 × 10 cm, Nacalei Tesque) preequilibrated with 20 mM Tris-HCl (pH 8.5), containing 0.1%
C12E8. The column was developed with a linear
concentration gradient of NaCl from 0 to 0.5 M in 60 min.
The PLA2 activity was eluted as a single peak at the
retention time of 18 min. The pooled PLA2 fraction was
rechromatographed on the same column; the pH of the eluent was
decreased to 8.0, and a shallower gradient of NaCl from 0 to 0.1 M in 60 min was used. The enzyme activity was eluted at the
retention time of 31 min, coinciding well with a protein peak detected
at 280 nm.
HPLC--
The HPLC system consisted of two Gilson model 302 liquid delivery modules, and a Gilson model 811 dynamic mixer. For a
Cosmogel-QA column of 7 × 100 mm (Nacalai Tesque), the system
equipped with a 1.8-ml mixing chamber was operated at 0.5 ml/min. When
a Cosmosil 5C8-300 column of 2.1 × 100 mm or 2.1 × 30 mm was used, the system equipped with a 65-µl mixing chamber
was operated in the microflow mode at 0.1 ml/min. Cosmosil
5C8-300 (Nacalai Tesque) was slurry-packed into a column
(2.1 × 100 mm or 2.1 × 30 mm) in our laboratory.
Extraction of Enzyme Activities from SDS-Polyacrylamide Gel
Electrophoresis--
SDS-PAGE was performed according to Laemmli
et al. (18) under the nonreducing conditions The gels were
cut into slices of 3-mm length, which were immersed in 200 µl of
buffer containing 0.25 M Tris-HCl and 0.25 M
NaCl (pH 8.5), overnight at 4 °C to extract PLB/LIP.
PLA2 and lysophospholipase activities in the extracts were
determined as described in the standard assay. The recovery of enzyme
activity was 52%.
Determination of the Positional Specificity and Stereospecificity
of PLB/LIP--
The positional specificity of PLB/LIP toward
glycerophospholipids was determined using the mixed micelles of 6 mM DOC and 1 mM DOPC, a symmetric acyl
phospholipid, as substrate. The reaction was followed under the
standard conditions, and the released lysophospholipids, 1-oleoyl- and
2-oleoyl-GPC, were separated by reverse-phase HPLC on a LiChrosorb
RP-18 column (4 × 150 mm, Merck) at the flow rate of 1 ml/min and
room temperature as reported (19). The eluent used was
methanol/acetonitrile/water (57:20:23) containing 20 mM
choline chloride, and the effluent was monitored at 205 nm. The
regiospecificity toward di- and triacylglycerols was determined with
mixed acyl glycerides, SLDG, POP, and POS, as substrates. The
stereospecificity was also assessed with enantiomeric pairs of DPPC and
dioleoylglycerol. The released fatty acids were derivatized, separated,
and quantitated by the same method as in the ADAM assay.
Protein Sequencing--
The purified PLB/LIP was reduced,
S-carboxymethylated, and purified as described in the legend
of Fig. 4. The amino acid sequences were analyzed with an Applied
Biosystems 477A sequencer and a 120A PTH analyzer. To identify a
phenylthiohydantoin S-carboxymethylated Cys precisely, both
S-carboxymethylated and unmodified proteins were analyzed
and the resultant data were compared with each other.
Other Analytical Methods--
Phospholipid phosphorus was
determined according to the method of Vaskovsky et al. (20).
ConA-peroxidase staining was performed as recommended by the
manufacturer's instructions (Honen, Tokyo, Japan). Immunoblotting was
performed as reported (21) and immunoreacted bands were visualized with
a Konica immunostain kit (Konica). Protein concentrations of crude
samples during purification were measured with a bicinchoninic acid
(BCA) protein assay kit (Pierce), and those of purified enzyme were
estimated by microbore reverse phase HPLC based on protein
concentrations determined by amino acid analysis with
-alanine
as an internal standard (22).
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RESULTS |
Solubilization of PLB/LIP from Brush Border Membranes--
We
determined the distribution of Ca2+-independent
PLA2 activity, which was immunochemically distinct from
Ca2+-dependent group I and group II
PLA2s, along rat intestine's length, and along the
villus-crypt units according to the method of Pinkus (23), and its
subcellular distribution. The results confirmed that
Ca2+-independent PLA2 is located in brush
border membranes of the absorptive cells of the lower intestine,
especially the proximal ileum (24). Hereafter, we will use the words
"PLA2 activity" and "Ca2+-independent
PLA2 activity" as the same meaning unless it is essential to distinguish between Ca2+-dependent and
Ca2+-independent PLA2 activities.
A high concentration of KCl or KBr and phosphatidylinositol-specific
phospholipase C treatment (0.57-570 milliunits/ml) did not solubilize
PLA2 activity at all, but treatment with 1% Triton X-100
solubilized 88% of PLA2 activity in the membrane
fractions. Limited proteolysis by endogenous and exogenous proteases
has been used to solubilize proteins associated with brush border membranes successfully (25). We found that autolysis gave an active
fragment that accounted for 50% of the total PLA2 activity of brush border membrane fractions during storage at
35 °C for 1 month.
The molecular size of the solubilized enzymes was determined by
SDS-PAGE under the non-reducing conditions, where PLA2 and lysophospholipase activities being recoverable from gel slices allowed
us to estimate the RF values of enzyme (Fig.
1). The size of Triton X-100-solubilized
enzyme (corresponding to 150 kDa) was identical to that of the enzyme
in brush border membrane fractions. Autolysis produced an active 35-kDa
enzyme and active PLB/LIP species of another size were not detectable
under the conditions used. PLA2 and lysophospholipase
activities in all solubilized enzyme solutions co-migrated on SDS-PAGE
as those in untreated membrane fractions, suggesting that a single
enzyme catalyzes those activities and that the 35-kDa autolytic product
is derived from the enzyme associated with brush border membrane.

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Fig. 1.
Solubilization of
Ca2+-independent PLA2 and lysophospholipase
activities from rat intestinal brush border membrane fractions. The membrane fractions were prepared, extracted, and subjected to
SDS-PAGE on a 7.5% gel as described under "Experimental
Procedures." Enzyme activities were eluted from sliced gels, and then
assayed as described under "Experimental Procedures." The
substrates used were 1 mM POPE plus 6 mM DOC
mixed micelles and 1 mM 1-myristoyl-GPC micelles.
A, brush border membrane fraction; B, 1% Triton
X-100 extract; C, autolytic extract.
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Purification of PLB/LIP--
We purified to homogeneity PLB/LIP
solubilized by autolysis during storage of the intestines at
35 °C. The results of purification are summarized in Table
I. The enzyme activity was separated into
the flow-through and bound fractions by ConA affinity chromatography. We purified PLB/LIP from the latter fraction because of its higher specific activity. Among several chromatographic steps involved in this
purification strategy, Cosmogel QA HPLC is the most effective (Fig.
2), leading to the overall purification
and yield of 22,000-fold and 4.2%, respectively. Inclusion of a
non-ionic detergent such as C12E8 in the eluent
of the HPLC was essential for improving the recovery, otherwise the
yield of enzyme activity was less than 10% on a single chromatographic
run. The ratios of lysophospholipase to PLA2 activities at
each purification step were rather constant (0.36-0.53), except for
that of the supernatant of intestine extracts (0.13), suggesting a
single enzyme catalyzes those reactions.
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Table I
Purification of PLB/LIP from rat small intestine
The PLA2 and lysophospholipase activities were determined as
described under "Experimental Procedures" using 1 mM
POPE plus 6 mM DOC mixed micelles and 1 mM
1-palmitoyl-GPC as substrates. Enzyme concentrations of crude
preparations were estimated by the BCA method, and that of the purified
PLB/LIP was determined as described under "Experimental
Procedures."
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Fig. 2.
HPLC profile of protein and lipolytic
activities on a Cosmogel QA column at pH 8.0. PLA2 and
lysophospholipase activities were measured as described under
"Experimental Procedures" using 1 mM POPE plus 6 mM DOC mixed micelles ( ) and 1 mM
1-palmitoyl-GPC ( ) as substrates. The sample volume applied was 6 ml, and 0.5-ml fractions were collected. The column was developed at
the flow rate of 0.5 ml/min as described under "Experimental
Procedures," and the eluent contained 0.1%
C12E8.
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On SDS-PAGE analysis, purified PLB/LIP gave a single band corresponding
to 35 kDa, which is the same as that in the autolytic extracts on a
10% gel under the non-reducing conditions, whereas reduction with
2-mercaptoethanol caused its separation into 14-kDa and 21-kDa
proteins. To examine whether the 35-kDa protein indeed represented
PLB/LIP, the same enzyme samples were loaded to adjacent two lanes on a
10% gel. A gel strip of one lane was stained with Coomassie Brilliant
Blue, and the other strip was sliced into 3-mm pieces from which
PLA2 and lysophospholipase activities were eluted and
assayed as described under "Experimental Procedures." The enzyme
activities were observed only in fraction 12 corresponding to a 35-kDa
protein band under the non-reducing conditions (Fig. 3A), whereas no activity was
detectable in the reduced protein lane. Furthermore, reverse-phase HPLC
analysis of purified enzyme on a Cosmosil 5C8-300 column (2.1 × 30 mm) showed a single protein peak coeluted with PLA2,
lysophospholipase, and lipase activities (Fig.
4A). These results demonstrate
that a single enzyme catalyzes the three lipolytic activities.

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Fig. 3.
SDS-PAGE analyses of purified PLB/LIP.
A, co-migration of a single Coomassie staining band (0.2 µg)
with both PLA2 ( ) and lysophospholipase ( ) activities
under the non-reducing conditions (lane 1). Enzyme
activities were eluted from sliced gels and then assayed as in Fig. 1.
Lane 2, an aliquot of the same sample was reduced with 5%
2-mercaptoethanol at 80 °C for 1 min. B, ConA-peroxidase
staining of purified PLB/LIP (50 ng each). Lane 1,
non-reduced; lane 2, reduced. Large and
small arrowheads and an arrow indicate the 35-kDa
PLB/LIP, 14-kDa fragment, and 21-kDa fragment, respectively.
C, comparison of purified and intestine extract PLB/LIPs by
immunoblot analyses using a specific IgG raised against amino acids
450-1450 of PLB/LIP (14). Tissue extracts were prepared as described
under "Experimental Procedures." Lane 1, dye-labeled
markers, of which molecular masses are indicated by small
arrowheads, from top to bottom, 175, 83, 62, 47, 32, 25, and 16.5 kDa as reference of lanes 2-5; lane 3, purified
PLB/LIP (2.4 ng); lanes 2, 4, and 5,
homogenate, supernatant, and pellet fractions of stored whole
intestinal mucosa, respectively (8 µl each). A small arrow
indicates an inactive digested product of PLB/LIP, as confirmed by
assaying of PLA2 activity of materials eluted from sliced
gels in a separate SDS-PAGE experiment, and a large
arrowhead the 35-kDa active enzyme. Freshly prepared ileum tissues
(8 µl each) were applied to lanes 6-9. Lane 6,
brush border membrane fractions, reduced; lane 7, ileum
homogenate, reduced; lane 8, brush border membrane
fractions, non-reduced; lane 9, ileum homogenate,
non-reduced. The small arrowheads indicate the positions of
the same molecular mass markers as in lane 1.
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Fig. 4.
Reverse phase HPLC profiles of purified
PLB/LIP on a Cosmosil 5C8-300 column (2.1 × 30 mm). The eluents used were as follows: eluent A, 0.1%
trifluoroacetic acid in water; eluent B, 95% acetonitrile containing
0.1% trifluoroacetic acid. A, purified PLB/LIP (~2 µg)
was applied to the column, and the enzyme was eluted with a linear
concentration gradient of eluent B: from 0 to 25% in 5 min; from 25 to
60% in 45 min; from 60 to 100% in 10 min, at a flow rate of 0.1 ml/min. PLA2 ( ), lipase ( ), and lysophospholipase
( ) activities in the fractions collected with a Gilson 201 fraction
collector in a manual mode were measured as described under
"Experimental Procedures." B, reduced and
S-carboxymethylated enzyme. L and S
indicate the large and small fragments of PLB/LIP, respectively. The
enzyme (~5 µg) purified as in A was reduced with
tributylphosphine in 0.5 M ammonium bicarbonate (pH
8.5)/1-propanol (1:1, v/v) under nitrogen for 2 h, and then
reacted with 3-fold molar excess of sodium iodoacetate over the
reductant for 30 min at 37 °C. The reaction was stopped by adding 5 µl of 2-mercaptoethanol. To the reaction mixture was added 0.5 volume
of formic acid and 2 volumes of 0.1% trifluoroacetic acid to reduce
the concentration of organic solvent and improve the solubility of
denatured PLB/LIP. The resultant solution (0.5 ml) was directly
injected onto the column preequilibrated with eluent A at a flow rate
of 0.4 ml/min, and the column was washed with the same solvent until
the absorbance at 210 nm returned to the base-line level. Then it was
developed with a linear concentration gradient of eluent B: from 0 to
30% in 5 min; from 30 to 60% in 50 min; from 60 to 100% in 10 min, at a flow rate of 0.1 ml/min.
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The majority of PLA2 and lysophospholipase activities bound
to a ConA-Sepharose column, suggesting the purified enzyme is a
glycoprotein that contains ConA-recognizable sugar chain(s). Moreover,
a ConA-peroxidase stain of the purified enzyme transferred on a
polyvinylidene difluoride membrane after SDS-PAGE showed that its large
fragment was indeed glycosylated (Fig. 3B). The presence of
PLB/LIP that did not bind to the column indicated a minor fraction of
PLB/LIP is either unglycosylated or modified with ConA-unrecognizable
sugar chain(s).
Fig. 3C shows the results of immunoblot analysis using
specific antibody raised against amino acids 450-1450 of PLB/LIP
expressed in Escherichia coli (14). Under non-reducing
conditions a 35-kDa band corresponding to purified enzyme was clearly
observed together with other degraded products in the supernatant and
membrane fractions of intestine homogenate prepared from stored
tissues. On the other hand, brush border membrane fractions and
intestine homogenate prepared freshly contained a single
~200-kDa2 form of enzyme
under the same conditions. SDS-PAGE in combination with detection by
enzyme assay gave a molecular mass estimation of ~150 kDa (Fig. 1).
This difference was due to a lower resolution of the latter method.
Denaturation treatment led to its splitting into 90- and 130-kDa
proteins.
Enzymatic Properties of PLB/LIP--
Purified PLB/LIP did not
require calcium ions for activity; the addition of calcium up to 10 mM did not affect PLA2, lysophospholipase, and
lipase activities significantly. We examined the substrate specificity
of PLB/LIP in the presence and absence of DOC using diradylphospholipids (POPC, POPE, POPG, or
1-O-hexadecyl-OPC), 1-palmitoyl-GPC, glycerides (triolein,
diolein, or monoolein) and cholesterol oleate as substrates (Table
II). The enzyme exhibited a broad
substrate specificity, and the apparent order of activity under the
standard conditions in the presence of 6 mM DOC was as
follows: diolein > POPC ~ POPE > 1-O-hexadecyl-OPC ~ POPG > L-palmitoyl-GPC ~ triolein > monoolein. The
enzyme also had esterase activity toward p-nitrophenyl fatty
acid esters. Cholesterol oleate, which was emulsified by three
different methods as described under "Experimental Procedures," was
not a substrate for PLB/LIP. PLB/LIP required a bile salt for the
activities toward diradylphospholipids and triacylglycerols, but not
for those toward lysophospholipids and mono- and diacylglycerols (Table
II). Fig. 5 shows typical dependence of
the PLA2, lysophospholipase, and lipase activities on the
DOC/glycerolipid molar ratio. PLB/LIP exhibited virtually no activity
toward POPC and TOG in the absence of the bile salt, but its presence
dramatically enhanced the activities with increasing the bile
salt/glycerolipid molar ratio. The activity toward POPC reached a
maximum at the molar ratio of 6 and then gradually decreased, whereas
that toward TOG exhibited saturation and did not decrease at higher
ratios (up to 100). In contrast, the enzyme was active to
1-palmitoyl-GPC micelles and the bile salt maximally increased the
activity 1.8 times that in its absence at the molar ratio of 2, and
then the activity gradually decreased (Fig. 5). The optimal activity
toward POPC, 1-palmitoyl-GPC, or triolein was observed over the pH
range of 8-9, and activity was hardly detected below pH 4.
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Table II
Substrate specificity of PLB/LIP
Lipolytic activities were determined by the ADAM method in the presence
or absence of 6 mM DOC as described under "Experimental Procedures." Activity toward p-nitrophenyl pentanoate was
determined by continuously monitoring absorbance changes at 400 nm in
the presence or absence of 10 mM DOC at pH 8.0. The total
p-nitrophenol released was calculated with its pK
value of 6.98. , not detectable.
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Fig. 5.
Effects of DOC on lipolytic activities of
PLB/LIP. Substrates (1 mM each) used were POPC,
1-palmitoyl-GPC ( ), and TOG (×). POPC/DOC mixed micellar solutions
were prepared as described previously (52). DOC dissolved in water was
added to the reaction mixtures for assaying lysophospholipase and
lipase activities. Oleic acid-releasing ( ) and palmitic
acid-releasing ( ) activities from POPC were simultaneously monitored
as described under "Experimental Procedures."
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To determine the positional specificity of PLB/LIP toward
diacylphospholipids, diacylglycerols and triacylglycerols, the time courses for release of the individual fatty acids from mixed acyl phospholipid and glycerides, i.e. POPC, SLDG, POP, and POS,
by the enzyme's action were followed by the ADAM method as described under "Experimental Procedures" (Fig.
6). The results were compared with those
with an sn-2-specific rat pancreatic PLA2 and an
sn-1(3)-specific R. arrhizus lipase. PLB/LIP
produced oleic acid and linoleic acid at much higher rate than palmitic
acid and stearic acid from POPC and SLDG as substrate, respectively,
suggesting its preference to sn-2 hydrolysis of
diacylphospholipid and diacylglycerol. In contrast, the rates of fatty
acid release from each ester bonds of POP and POS were practically
identical, suggesting low regiospecificity as to triacylglycerol
hydrolysis of PLB/LIP. The stereospecificity of this enzyme was also
examined by the ADAM method with enantiomeric pairs of DPPC and
dioleoylglycerol as substrates. (Table
III). PLB/LIP hardly discriminated
between these enantiomeric pairs.

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Fig. 6.
Positional specificity of PLB/LIP toward
diacylphospholipid, diacylglycerol, and triacylglycerol. The
substrates used were as follows: A and D, 1 mM POPC, 6 mM DOC mixed micelles; B
and E, 1 mM SLDG emulsified with 1% arabic gum
and 6 mM DOC; C and F, 1 mM POP or POS emulsified with 1% arabic gum and 6 mM DOC. Enzymes used were as follows: A-C,
purified PLB/LIP (A and B, 7.1 ng/ml;
C, 23.5 ng/ml); D, purified rat pancreatic
PLA2 (0.22 µg/ml); E and F,
R. arrhizus lipase (0.4 µg/ml). Time courses were followed
as described under "Experimental Procedures." Fatty acids released
were as follows: , oleic acid from POPC (A and D) and from POP (C and F); , oleic
acid from POS (C and F); , palmitic acid from
POP (C and F); , palmitic acid from POPC
(A and D) and from POS (C and
F); , stearic acid from SLDG (B and E) and from POS (C and F); ,
linoleic acid from SLDG (B and E).
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Table III
Hydrolysis of diolein and DPPC enantiomers by PLB/LIP
Lipolytic activities were determined in the presence of 6 mM DOC for PLB/LIP and 6 mM cholate for rat
pancreatic PLA2 as described under "Experimental
Procedures." , not detectable; ND, not determined.
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We confirmed the sn-2 specificity for diacylphospholipid
hydrolysis by PLB/LIP by identifying which lysophospholipid
regioisomers, 1-oleoyl- or 2-oleoyl-GPC, the enzyme produces from DOPC.
These isomers were well separated by reverse-phase HPLC (Fig.
7A) as reported (19). PLB/LIP
time-dependently produced oleic acid and 1-oleoyl-GPC, but
not appreciably 2-oleoyl-GPC (Fig. 7, B and C); a
small amount of 2-oleoyl-GPC may be formed through base- and silica
gel-catalyzed 1,2-acyl migration (26, 27), because the slow production
of the isomer was also observed in the reaction with pancreatic
PLA2. The rate of oleic acid release was greater than that
of 1-oleoyl-GPC release; this is consistent with the fact that
1-oleoyl-GPC is an intermediate which the enzyme further converts to
oleic acid and GPC. On the other hand, the time courses for the
formation of two products were superimposable on hydrolysis by
sn-2-specific rat pancreatic PLA2 (Fig.
7D). These results unequivocally demonstrated that PLB/LIP
is specific for the hydrolysis at the sn-2 position of
diacylglycerophospholipids.

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Fig. 7.
Confirmation of the positional specificity of
PLB/LIP toward DOPC by reverse phase HPLC. A, separation of
authentic 1-oleoyl-GPC (peak 3) and 2-oleoyl-GPC (peak
2). 1-Oleoyl- and 2-oleoyl-GPC were prepared from DOPC by the
action of rat pancreatic PLA2 and R. arrhizus
lipase (53), respectively. B, HPLC profiles of the products
formed by the reaction of purified PLB/LIP (23.5 ng/ml) with 2 mM DOPC/12 mM cholate mixed micelles at
37 °C. Peak 1, oleic acid. C, time courses of
the formation of oleic acid ( ), 1-oleoyl-GPC ( ), and 2-oleoyl-GPC
( ). The assay conditions were the same as in B.
D, time courses of the formation of oleic acid ( ),
1-oleoyl-GPC ( ), and 2-oleoyl-GPC ( ) for the reaction of rat
pancreatic PLA2 and 2 mM DOPC/12 mM
cholate mixed micelles.
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Inhibition of PLB/LIP by DFP, Manoalide, and BPB--
Since
PLB/LIP exhibited PLA2, lysophospholipase, and lipase
activities as a single enzyme, we tested the ability of known potent
inhibitors for secretory 14-kDa PLA2 (manoalide and BPB) and lipases (DFP) to inhibit the lipolytic activities of PLB/LIP. When
the enzyme was preincubated with various concentrations of inhibitors
for 1 h at 37 °C, they dose-dependently inhibited
the lipolytic activities. The extent of inhibition was expressed as an
inhibitor concentration required for half-maximal inhibition (Table
IV). Notably, the dependence of activity
on inhibitor concentration was similar irrespective of substrates
tested, suggesting a single active site is involved in catalysis. DFP
was apparently most effective, and treatment with 0.5 mM
DFP completely inhibited activity in all cases.
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Table IV
Inhibition of PLB/LIP by DFP, BPB, and manoalide
PLB/LIP (23.5 ng/ml) was incubated with various concentrations of the
inhibitors (DFP, 1 µM to 2 mM; manoalide,
0.01-0.5 mM; BPB, 0.05-2 mM) for 1 h at
37° C and pH 8.5, and then enzyme activity was determined as
described under "Experimental Procedures" using the following
substrate/DOC combinations: 1 mM POPC plus 6 mM DOC, 1 mM 1-palmitoyl-GPC plus 6 mM DOC, and 1 mM TOG and 1% arabic gum plus 10 mM DOC. Oleic
acid-releasing and palmitic acid-releasing activities from POPC were
simultaneously monitored.
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NH2-terminal Amino Acid Sequences of PLB/LIP
Fragments--
The purified PLB/LIP was reduced and
S-carboxymethylated, and then subjected to reverse-phase
HPLC, by which the S-carboxymethylated enzyme was separated
into two peaks (Fig. 4B). SDS-PAGE confirmed that the
earlier and later peaks corresponded to the small and large fragments
of PLB/LIP, respectively. Automated Edman analysis of these fragments
revealed the NH2-terminal sequences of 13 residues of the
small fragment and of 31 residues of the large fragment. A data base
search showed that both sequences were similar to parts of the sequence
of AdRab-B protein that has been cloned from an adult rabbit
intestine-specific cDNA library (28). AdRab-B contained four
internal repeats (designated repeats 1 through 4) of about 350 amino
acids each, and the identity between the sequences of either small or
large fragment and those of each repeat of AdRab-B was as follows: 22, 67, 40, and 27% for the small fragment versus repeats 1, 2, 3, and 4, respectively; 39, 81, 52, and 45%, for the large fragment
versus repeats 1 through 4, respectively (Fig.
8). The sequences of both fragments
showed the highest similarity to repeat 2 of AdRab-B.

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Fig. 8.
Comparisons of NH2-terminal amino
acid sequences of the small and large fragments of PLB/LIP with the
sequences of each repeats of AdRab-B. The numbers on both sides of
sequences indicate those of amino acids of rat PLB/LIP (14) and rabbit AdRab-B (28) deduced from their cDNA sequences. The residues conserved among all five sequences are boxed. The
asterisks indicate the conserved amino acid residues of the
fragments and repeat 2 of Ad-RabB.
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DISCUSSION |
Purified Enzyme Is a Catalytic Domain of PLB/LIP--
We purified
to homogeneity a phospholipase B/lipase solubilized from rat intestinal
brush border membranes by autolytic limited proteolysis. This truncated
form of PLB/LIP (35 kDa) has a considerably smaller size than the brush
border membrane form, but actively catalyzes PLA2,
lysophospholipase, and lipase reactions (Table II). Co-migration of
these three activities on reverse phase HPLC and SDS-PAGE of the
purified enzyme confirmed that a single enzyme is responsible for such
broad specificity (Figs. 3A and 4A). The NH2-terminal amino acid sequences of two peptides derived
from the purified enzyme were most similar to the sequence of the
second repeat of AdRab-B that contains 4-fold internal repeats with
about 38 kDa molecular mass each. In the following paper (14), we deduced the amino acid sequence of PLB/LIP by its cDNA sequencing, and found that it contains the same structural organization as AdRab-B.
The sequence of the small fragment was found in the
NH2-terminal part of repeat 2 domain and that of the large
fragment in its mid-portion, confirming that the purified enzyme is
indeed derived from the second repeat; hence, repeat 2 is a catalytic
domain of PLB/LIP.
We used mild autolysis conditions, that is, the storage of intact
intestine tissues at a low temperature of
35 °C over 1 month, and
tissue extracts were prepared without homogenization. This gave an
enzymatically active autolytic product, which consisted of the 14-kDa
and 21-kDa peptides, as revealed by reverse phase HPLC and SDS-PAGE
(Figs. 3 and 4). This raises a question whether these peptides are
autolytic products or genetically distinct subunits. The amino acid
sequence deduced from cDNAs of rat PLB/LIP showed that the nascent
PLB/LIP is a single polypeptide with a molecular weight of 161,070, and
that it contains Lys367 and Arg528 just before
the NH2 termini of the small and large fragments, respectively (14). This confirmed the two peptides are indeed proteolytic products, but not distinct gene products, and suggested that trypsin or trypsin-like protease rather specifically cleaved PLB/LIP to give the solubilized homogeneous enzyme as judged by NH2-terminal amino acid, SDS-PAGE, and reverse-phase HPLC
analyses. Guinea pig phospholipase B similar to rat PLB/LIP was
effectively solubilized from brush border membrane fractions with
papain treatment, and the solubilized 97-kDa enzyme was partially
purified (12).
Immunoblot analysis under non-reducing conditions showed that the
autolytically solubilized form was hardly detected in fresh intestine
homogenate, which instead contained a single ~200 kDa form;
denaturation gave two smaller species (90 and 130 kDa). This suggested
that limited proteolysis split the 200-kDa form into a few fragments,
which remain associated, like the purified enzyme composed of two
peptides. Smaller-sized fragments dissociating on denaturation could
not be detected by the antibody used. The formation of nick by
proteolysis might modulate enzyme's function; for example, the
substrate specificity of rat hepatic lipase was modified by limited
proteolysis by collagenase (29). Although the full-length PLB/LIP
expressed in COS-7 cells exhibited the substrate specificity similar to
the enzyme purified here (14), it is necessary to examine in more
detail whether the structural changes caused by proteolytic cleavage
modulate PLB/LIP's function.
Catalytic Properties of PLB/LIP--
Purified PLB/LIP exhibited
high PLA2 and triacylglycerol lipase activities. Its
specific activity toward the mixed micelles of 1 mM POPC
and 6 mM cholate (713 µmol/min/mg) was 6.7 times that of
rat pancreatic PLA2 (107 µmol/min/mg) under the same
conditions (22). The specific activity toward triolein (Fig. 5) was
lower than that of rat pancreatic lipase (5330 µmol/min/mg; Ref. 30), but was comparable with those of other rat lipases, such as lingual lipase (230 µmol/min/mg; Ref. 31), hepatic lipase (750 µmol/min/mg; Ref. 32), and adipose tissue lipoprotein lipase (258 µmol/min/mg; Ref. 33). This unique substrate specificity was also found in partially
purified guinea pig intestinal phospholipase B (13); the reported
specific activities for PLA2 (22.8 µmol/min/mg) and triacylglycerol lipase (19.9 µmol/min/mg) were significantly lower than those obtained in this study under similar conditions (Table II).
Since the structural information on the guinea pig enzyme has not been
available, it is unknown at present whether it is a homolog of rat
PLB/LIP. On the other hand, rabbit AdRab-B homologous to PLB/LIP
exhibited virtually no triacylglycerol lipase activity and much lower
diacylglycerol lipase activity than PLA2 activity (28),
when expressed in COS-7 cells. An AdRab-B preparation purified recently
from rabbit intestine by immunoaffinity chromatography was a mixture of
several forms of its proteolytic digests, and the specific activity
values of purified enzyme have not yet been reported (34). Since the
physical states of substrate significantly influence lipolytic
activities, the specificities of different enzymes should be compared
side by side under the same assay conditions. The positional
specificity of AdRab-B toward diacylphospholipids has not yet been
firmly established. In this study, we demonstrated the A2
specificity by separating two positional isomers by reverse-phase HPLC
with DOPC, a symmetrical acyl phospholipid, as substrate. This avoids
the problem on positional impurity of substrate that may be encountered
in experiments with a mixed acyl phospholipid or doubly-radiolabeled
substrates. Following the time course of the formation of each isomers
unequivocally determined the positional specificity of PLB/LIP.
It was also unique that PLB/LIP preferred the sn-2
hydrolysis of diacylphospholipids and diacylglycerols without clear
chiral selectivity, whereas it hydrolyzed triacylglycerols without
regiospecificity (Fig. 6). Comparable inhibition of these lipolytic
activities by DFP, BPB, and manoalide suggested that a single active
site is involved in catalysis and that the binding mode of
triacylglycerol to the enzyme differs from that of the former
substrates in the same active site. Further extensive structural and
functional studies on its active site should be required to clarify the
mechanism underlying the complex, at a glance, positional specificity
of PLB/LIP.
Recent studies revealed that members of the lipase superfamily (35)
exhibit a wide range of ratios of phospholipase A1 to lipase activities; classical pancreatic lipases have no significant phospholipase activity, the guinea pig and coypu lipases belonging to
the type 2 pancreatic lipase-related proteins, a pancreatic lipase
subfamily, exhibit high phospholipase A1 and lipase
activities (36), and new members of the lipase superfamily, hornet
venom phospholipase A1 (37) and platelet serine
phospholipid-specific phospholipase A1 (38), are devoid of
lipase activity. Recent development of x-ray crystallographic studies
on natural and mutated lipases and PLA2s provided important
clues to unravel how lipolytic enzymes regulate their substrate
selectivities (39, 40). However, the studies along this line are still
on the starting line. The catalytic domain of PLB/LIP with the unique
substrate and positional specificity and the primary structure entirely
unrelated to those of the enzymes belonging to the known lipase
superfamily provides a good model for studying the mechanism of
controlling substrate specificity. Its comparison with recently cloned
and sequenced PLA2s with an active site serine,
including cPLA2 (41, 42) and PAF acetylhydrolases (43,
44) will provide further insights into the structure-function
relationship of lipases and phospholipases A.
DFP, an irreversible inhibitor for serine esterases and lipases (45),
completely inhibited the three lipolytic activities of PLB/LIP,
suggesting that a serine nucleophile is involved in its catalytic
mechanism. In the best-established lipase family (35), the serine is
present within the conserved pentapeptide sequence
G-X-S-X-G. Very recently, Upton and Buckley
proposed another class of lipase family, of which enzymes, such as
Aeromonas hydrophila lipase/acyltransferase (46) and
E. coli thioesterase I (47), contain the active serine in
another conserved G-D-S-L sequence (48). The repeat 2 domain of PLB/LIP
(14) contains two serines, Ser414 and Ser459,
in the former conserved sequence, and a Ser404 in the
latter conserved sequence. We recently studied by chemical modification
and site-directed mutagenesis which serine residues were responsible
for catalysis, and obtained evidence that Ser404, but not
Ser414 and Ser459, was essential for
catalysis.3
In contrast to protein and polysaccharide digestion involving
coordinated luminal and membrane digestion (49), glycerolipid digestion
was believed to proceed exclusively in the lumen by the action of the
major secretory enzymes, preduodenal acid lipase, pancreatic lipase,
and pancreatic phospholipase A2 until recently (50). The
presence of PLB/LIP in ileal brush border membranes suggests that it is
the first lipolytic enzyme participating in membrane digestion and
indicates a need to reassess the individual roles of these enzymes in
lipid digestion and absorption. Recently, a quantitative study on the
relative contributions of gastric and pancreatic lipases to lipolysis
in the upper gastrointestinal tracts was reported in normal subjects
(51). Furthermore, it is interesting to determine its
pathophysiological significance in the intestine under diseased
conditions, such as pancreatic insufficiency and inflammatory bowel
disease. In addition to its digestive roles on the luminal side, the
physiological roles of PLB/LIP should be further examined in
spermatids, and Paneth cells as described in the following paper
(14).
During preparation of these manuscripts, we noted a
report that described evidence that DFP reacted with a
Ser400 in the G-D-S-L sequence of AdRab-B purified from
rabbit intestine by immunoaffinity chromatography (34).