(Received for publication, September 5, 1996, and in revised form, January 29, 1997)
From the A human liver microsomal Bile acid glucosides have been shown to be formed in human liver
microsomes by a glucosyltransferase that is sugar
nucleotide-independent and utilizes dolichyl phosphoglucose as natural
cosubstrate (Ref. 1, for recent reviews on glycosidic conjugation of
bile acids, see Refs. 2 and 3). The position of the glucose moiety in these bile acid glucosides has been determined from enzymatically synthesized bile acid glucosides to be the 3-position (4) with a
Bile acid 3-O-glucosides have been recently shown to be
hydrolyzed in vitro by a human liver microsomal
Preliminary studies gave evidence (8) that hydrolytic activity toward
bile acid 3-O-glucosides differs from the two known prominent Sources of chemicals (1, 8, 13) and of human liver (13) were the
same as described in previous papers. The following materials were
obtained from Sigma, Munich: various 4-methylumbelliferyl glycosides,
phospholipids, and sheep anti-rabbit IgG agarose. The following
compounds were synthesized as described:
CBE1 and Br-CBE (14), carboxynonyl-dNM and
dodecyl-dNM (15). The following bile acid glucosides were synthesized
enzymatically with human liver microsomes as previously published:
[24-14C]chenodeoxycholic acid glucoside (8 µCi/µmol)
(5) and hyodeoxycholic acid [U-14C]glucoside (1.6 µCi/µmol) from UDP-[U-14C]glucose and hyodeoxycholic
acid (7). The following samples were generous gifts: lithocholic acid
[U-14C]glucoside (17 µCi/µmol) from Prof. Dr. F. Dallacker, Department of Organic Chemistry, Aachen University of
Technology, Germany; dNM from Dr. D. Schmidt, Bayer AG, Wuppertal,
Germany, and rabbit immune serum prepared against human placental
glucocerebrosidase from Dr. C. Incerti, Genzyme Therapeutics, Modena,
Italy.
Analytical Methods
Glucocerebrosidase was assayed fluorimetrically with a mixture of
glucosylceramide and the synthetic analog NBD-glucosylceramide as
described (17). Hydrolytic activity toward various glycosides of
4-methylumbelliferone was estimated fluorimetrically according to a
published procedure (18) with the following modifications. The assay
mixtures contained 0.1 M sodium acetate, pH 5.5, as buffer,
5 mM MgCl2, and a glycoside of
4-methylumbelliferone, each 1 mM.
For reconstitution of enzyme activities prior to assays aliquots of
enzyme samples from column fractions were preincubated in the presence
of 0.1 mg/ml L- Protein was assayed by the dye-binding method of Bio-Rad using bovine
serum albumin as standard. After the final purification step the
concentration of protein was too low to be measured by a common method
(about 0.05 µg of total protein). Therefore, the amount of protein in
the eluate after affinity chromatography was estimated from the density
of the silver-stained protein band that was visible in the final enzyme
preparation after SDS-polyacrylamide gel electrophoresis (see below).
Quantitation of density was performed by comparison to the density of
bands from a bovine serum albumin standard using a laser densitometer
(Ultroscan XL, LKB, Bromma, Sweden).
SDS-polyacrylamide gel electrophoresis was performed in 7.5% (w/v)
polyacrylamide gels under reducing conditions as described (19).
Immunoinhibition and immunoprecipitation studies were performed using
microsomes prepared as described below and a mitochondrial-lysosomal fraction from human liver prepared by differential centrifugation as
described in a previous report (10). These subcellular fractions were
solubilized in 10 mM Bis-Tris propane HCl, pH 7.2, containing 20% (v/v) glycerol, 1 mM dithioerythritol, 15 mM NaCl, and 0.5% (w/v) CHAPS and centrifuged at
100,000 × g for 1 h. For immunoinhibition studies
aliquots of the supernatants (about 15 µg of protein) were incubated
for 30 min at 37 °C in the buffer mixture used for solubilization
with a final concentration of 0.17% (w/v) CHAPS, containing 15 µl of
various dilutions of immune rabbit serum prepared against human
placental glucocerebrosidase diluted into nonimmune rabbit serum or 15 µl of nonimmune rabbit serum as control (total volume, 45 µl).
Aliquots of the incubation mixtures with solubilized microsomes or
mitochondria lysosomes were then assayed for Purification of Bile Acid If not stated otherwise, all steps were
carried out at 4 °C. Standard buffer was 20 mM Bis-Tris
propane HCl, pH 6.5, containing 1 mM dithioerythritol and
20% (v/v) glycerol except for step 4 and chromatofocusing where 10%
(v/v) glycerol was used. The following protease inhibitors were added
to buffers where indicated: antipain, chymostatin, leupeptin, and
pepstatin, 0.1 µg/ml each, and phenylmethylsulfonyl fluoride, 10 µM final concentration.
Microsomes were prepared from 40 g of human liver
and washed with 0.15 M KCl as described previously (20).
The washed microsomes were suspended in 60 ml of 0.25 M
sucrose containing 5 mM Tris/HCl, pH 7.4, and protease
inhibitors. Aliquots of this suspension could be stored at Fractionation
with polyethylene glycol 6000 was carried out as described previously
(20) except that the protein precipitating between 5 and 7% (w/v) of
polyethylene glycol was used for subsequent purification steps. The
precipitate was dissolved in standard buffer containing 10% (v/v)
acetonitrile, 0.2% (w/v) SB12, 0.2% (w/v) octyl The protein fraction
of step 2 was centrifuged for 35 min at 100,000 × g.
The clear supernatant was applied to a DEAE-Trisacryl column (1.6 × 5 cm; flow rate, 50 ml/h) equilibrated with standard buffer
containing 10% (v/v) acetonitrile, 0.2% (w/v) SB12, and 0.2% (w/v)
octyl The active fractions of step
3 were diluted by an equal volume of standard buffer containing 0.2%
(w/v) SB12 and 0.2% (w/v) octyl To the active fractions of
step 4 MgCl2 and NaCl were added to a final concentration
of 5 mM and 0.3 M, respectively. This protein
solution was treated batchwise with 150 µl of affinity gel which was
prepared by coupling carboxynonyl-dNM to AH-Sepharose 4B according to
the instructions given by Pharmacia (ligand concentration, 2 µmol/ml
of settled gel). The suspension was shaken for 4 h to allow enzyme
binding. The gel was then washed by repeated suspension and
centrifugation (2 min, 2000 × g) with the following
buffers: 5 times with 1 ml of standard buffer, containing 0.05% (w/v)
SB12, 0.1% (w/v) CHAPS, 0.3 M NaCl, and 5 mM
MgCl2; 10 times with 1 ml of the same buffer containing 3 M NaCl; 2 times with 1 ml of this buffer containing 0.3 M NaCl; 10 times with 0.25 ml of 10 mM Bis-Tris
propane HCl, pH 6.5, containing 90% (v/v) ethylene glycol and 1 mM dithioerythritol. After these successive washings bile
acid For enzyme characterization an enzyme
preparation after Mono Q chromatography (step 4 of the purification
procedure) was further purified by chromatofocusing on a Mono P column
after dilution with 2 volumes of standard buffer (see above) containing
0.2% (w/v) octyl The purification of human
liver bile acid
Purification of bile acid Data are given for enzyme activity toward lithoholic acid glucoside.
Enzyme preparations from steps 2 and 3 containing octyl Department of Internal Medicine III,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-glucosidase has been
purified to apparent homogeneity in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis where a single protein band
of Mr 100,000 was obtained under reducing
conditions. The enzyme was enriched about 73,000-fold over starting
microsomal membranes by polyethylene glycol fractionation, anion
exchange chromatographies on DEAE-Trisacryl, and Mono Q followed by
affinity chromatography on
N-(9-carboxynonyl)-1-deoxynojirimycin-AH-Sepharose 4B. The
purified enzyme had a pH optimum between 5.0 and 6.4, was activated by
divalent metal ions, and required phospholipids for exhibition of
activity. The enzyme catalyzed the hydrolysis of
3
-D-glucosido-lithocholic and
3
-D-glucosido-chenodeoxycholic acids with high affinity
(Km, 1.7 and 6.2 µM, respectively) and of the
-D-glucoside (Km, 210 µM) and the
-D-galactoside of
4-methylumbelliferone. The ratio of relative reaction rates for these
substrates was about 6:3:11:1. No activity was detectable toward
6
-D-glucosido-hyodeoxycholic acid, glucocerebroside, and the following glycosides of 4-methylumbelliferone:
-D-glucoside,
-L-arabinoside,
-D-fucoside or
-D-xyloside.
Immunoinhibition and immunoprecipitation studies using antibodies
prepared against lysosomal glucocerebrosidase showed no
cross-reactivity with microsomal
-glucosidase suggesting that these
two enzymes are antigenically unrelated.
-glucosidic linkage (1). The physiological significance of bile acid
3-O-glucosidation is suggested by the identification of
glucosides of unconjugated as well as glycine- and taurine-conjugated bile acids in urine from healthy humans (5) and patients with extrahepatic cholestasis (6). In addition to bile acid
3-O-glucosidation, the synthesis of bile acid
6-O-glucosides was recently described in human liver
microsomes by a UDP-sugar-dependent mechanism (7).
-glucosidase (8). This
-glucosidase is localized predominantly in
the microsomal fraction of human liver (8) where it is confined to the
smooth endoplasmic reticulum (9). Hydrolytic activity toward bile acid
3-O-glucosides thus occurs in the same subcellular
compartment as synthesis of these bile acid conjugates, which has been
shown to be localized in the smooth endoplasmic reticulum of human
liver (10).
-glucosidases in human tissues, the lysosomal
membrane-bound enzyme glucocerebrosidase
(N-acylsphingosyl-1-O-
-D-glucoside:glucohydrolase) (11) and a cytosolic broad specificity
-glucosidase (12). The
following study will show that
-glucosidase activity toward bile
acid 3-O-glucosides is a novel microsomal
-glucosidase
that is not identical to the known
-glucosidases described before. The present report describes properties and a method for the
purification of human liver microsomal
-glucosidase activity toward
bile acid 3-O-glucosides to apparent homogeneity in
SDS-polyacrylamide gel electrophoresis. According to its natural
substrates the isolated enzyme will be named bile acid
-glucosidase
(3
-D-glucosido-bile acid:glucohydrolase).
-Glucosidase activity toward bile acid glucosides was
estimated as described for [14C]chenodeoxycholic acid
glucoside as substrate (8) with the following modifications: hydrolysis
of hyodeoxycholic acid [14C]glucoside was estimated with
a substrate concentration of 106 µM; the assay mixture
with lithocholic acid [14C]glucoside (9 µM), containing maximally 4 µg of protein in 30 µl,
was stopped with 100 µl of 0.5 M glycine HCl, pH 2.0. The water phase was extracted for 5 min with 300 µl of
chloroform/n-butyl alcohol (9:1, v/v) resulting in transfer
of >99% of unreacted lithocholic acid [14C]glucoside
into the organic phase. The labeled reaction product ([14C]glucose) remained in the water phase with a yield
of about 97%. A 100-µl aliquot of the upper aqueous phase was
counted for radioactivity in 5 ml of Rotiszint Eco-Plus (Roth,
Karlsruhe) scintillation mixture. For identification of
[14C]glucose as reaction product, thin layer
chromatography was performed as described (16) with
[U-14C]glucose (DuPont NEN) as standard.
-phosphatidylinositol from bovine liver
and 8.3 mg/ml human serum albumin for 30 min on ice. Aliquots of these
preincubation mixtures were added to assay mixtures which additionally
contained 0.7 mg/ml CHAPS. Phospholipids were freshly prepared as
dispersions in the respective sample buffers as described (13).
-glucosidase activities
toward lithocholic acid glucoside or glucocerebroside, respectively.
For immunoprecipitation studies, the mixtures with solubilized enzyme
preparations and antiserum or nonimmune serum as described above were
incubated for 16 h at 4 °C. A second antibody (sheep
anti-rabbit IgG) bound to agarose beads, equilibrated in 10 mM Bis-Tris propane HCl, pH 7.2, containing 20% (v/v)
glycerol, 1 mM dithioerythritol, and 15 mM NaCl
in a volume of 100 µl of gel suspension (about 75 µl of settled
gel) was then added to precipitate the immune complexes. After 30 min
with shaking at 37 °C and 2 h at 4 °C, the incubation
mixtures were centrifuged for 15 min at 2000 × g.
Hydrolytic activities toward lithocholic acid glucoside or
glucocerebroside were measured in the respective supernatants as
described above.
-Glucosidase
70 °C
for at least 3 months with no apparent loss of enzyme activity. For
solubilization an equal volume of 40 mM Bis-Tris propane
HCl, pH 7.2, containing 40% (v/v) glycerol, 20% (v/v) acetonitrile, 2 mM dithioerythritol, 0.4% (w/v) SB12, 0.44% (w/v) octyl
-glucoside, and protease inhibitors was added to the microsomal
suspension. After 30 min the mixture was centrifuged at 100,000 × g for 60 min.
-glucoside, and
protease inhibitors.
-glucoside. After the column was washed with 30 ml of
equilibration buffer followed by washing with 30 ml of equilibration
buffer without the addition of acetonitrile, containing protease
inhibitors, a linear gradient from 0 to 0.16 M NaCl in the
same buffer was applied (total volume, 90 ml; flow rate, 20 ml/h). Bile
acid
-glucosidase activity emerged at approximately 0.06 M NaCl.
-glucoside and were subjected to
anion-exchange chromatography on a Mono Q column using a Pharmacia FPLC
system (flow rate, 1 ml/min; pressure, 2.5 megapascal). After
application of the sample the column, previously equilibrated with
standard buffer containing 0.2% (w/v) SB12 and 0.1% (w/v) CHAPS, was
washed with 5 ml of the same buffer and then subjected to elution with
two linear NaCl gradients in equilibration buffer, first from 0 to 0.07 M NaCl (total volume, 7 ml) and then from 0.07 to 0.2 M NaCl (total volume, 48 ml). Enzyme activity was eluted at
about 0.1 M NaCl.
-glucosidase was eluted from the gel by repeated suspension and
centrifugation of the gel in 0.25 ml of the last washing buffer with
90% ethylene glycol saturated with NaCl (15 times). For enzyme assays
aliquots of these fractions were diluted with 10 mM
Bis-Tris propane HCl, pH 6.5, containing 1 mM
dithioerythritol to reduce the ethylene glycol to a final concentration
of 30%; 0.05% (w/v) SB12 and 0.1% (w/v) CHAPS were added (final
concentrations), and samples were reconstituted with
L-
-phosphatidylinositol and human serum albumin (see
above).
-glucoside using a method previously described (1) with the following modifications. The buffer used for column
equilibration was standard buffer (see above), containing 0.05% (w/v)
SB12 and 0.2% (w/v) octyl
-glucoside. A pH gradient was developed
on the column from 6.5 to 4.0 with 10% (v/v) Polybuffer 74 adjusted
with HCl to pH 4.0, containing the additions used in the equilibration buffer (eluent volume, 40 ml).
Purification of -Glucosidase
-glucosidase was achieved from a solubilized
microsomal preparation by polyethylene glycol fractionation, followed
by anion-exchange chromatographies on DEAE-Trisacryl and on Mono Q and
affinity chromatography on carboxynonyl-dNM-AH-Sepharose (Table
I). The purification was about 73,000-fold with respect
to the solubilized microsomal preparation; the yield was 1.4%.
-glucosidase from human liver
-glucoside
were corrected for octyl
-glucoside inhibition. Enzyme preparations
from step 4 and 5 were assayed after reconstitution as described under
"Experimental Procedures."
Step
Volume
Total
protein
Specific activity
ml
µg
nmol × min
1 × mg
1
1. Solubilized
microsomes
110
260,000
0.34
2. Polyethylene glycol
fractionation
54
16,600
2.74
3.
DEAE-Trisacryl
12.5
1,300
16.7
4. Mono
Q
8.7
64
210
5. Affinity
purification
3.5
0.05
25,000
The supernatant of the solubilized microsomal preparation contained
39% of the microsomal protein and 56% of the bile acid -glucosidase activity toward lithocholic acid glucoside present in
microsomes before solubilization. The loss of activity of about 40%
can be attributed to the addition of SB12 and acetonitrile to
microsomes, whereas octyl
-glucoside exerted a stabilizing effect on
enzyme activity and prevented a greater loss of active enzyme during
solubilization. Other detergents, e.g. CHAPS (0.3%, w/v) or
Triton X-100 (0.2%, v/v), were also suitable for extraction of bile
acid
-glucosidase from the microsomes with yields of 74 and 51%,
respectively. However, after solubilization of microsomal protein with
these detergents, the formation of aggregates of variable composition
with protein, detergent, and phospholipids appeared to be favored, so
that separation of bile acid
-glucosidase from contaminating
proteins could not be achieved in subsequent chromatographic
procedures.
Following solubilization of microsomes an initial polyethylene glycol
fractionation proved to be favorable for further purification of the
-glucosidase. When polyethylene glycol fractionation was omitted,
purification of bile acid
-glucosidase was only about 23-fold after
DEAE-Trisacryl and Mono Q chromatographies in contrast to about
600-fold purification with polyethylene glycol fractionation as the
initial purification step. In anion exchange chromatographies bile acid
-glucosidase showed the characteristics of an acidic protein being
bound at pH 6.5 to the column materials. The acidic nature of the
protein was further confirmed by chromatofocusing. With this method the
apparent pI of the
-glucosidase was estimated to be about 5.0 from
the pH of the buffer which led to elution of the enzyme.
Affinity purification of the -glucosidase was carried out as the
final step of the purification procedure. Under the conditions described, the enzyme was nearly quantitatively bound to
carboxynonyl-dNM as ligand of the affinity gel. In the presence of the
-glucosidase inhibitor dNM (1 M), no binding of the
enzyme to the affinity gel was observed suggesting that interaction of
the
-glucosidase with the gel was of specific nature. Elution of the
enzyme from the affinity gel was possible with 90% ethylene glycol
saturated with NaCl. Under this condition about 10% of bile acid
-glucosidase activity applied to the affinity gel could be
recovered, whereas with 3 M NaCl or 90% ethylene glycol
the recovery of enzyme activity from the affinity gel was less than
0.1%. The enzyme was present in the eluate in an inactive form which
could be reconstituted with phospholipids (see below). However, the
enzyme preparation rapidly lost the potential for reconstitution of
activity when stored in the final elution buffer at 4 °C or
70 °C (half-life, <2 h). In the reconstituted form, the enzyme
could be stored for 40 h at 4 °C (half-life, 14 days) and for
at least 2 months at
70 °C with minimal loss of activity (<20%
of the initial value). The instability of the enzyme prior to
reconstitution may be the reason for the low recovery of enzyme
activity in the last step of the purification procedure.
As shown in Fig. 1, the final enzyme preparation
exhibited a single protein band of Mr 100,000 in
SDS-polyacrylamide gel electrophoresis under reducing conditions. A
protein band of Mr 100,000 was also obtained
after treatment of the affinity gel with 1 mM dodecyl-dNM and analysis of this eluate by SDS-polyacrylamide gel electrophoresis (not shown). However, no -glucosidase activity was detectable in the
eluate with dodecyl-dNM, which had been subjected to DEAE-Trisacryl chromatography for removal of this inhibitory dNM derivative. Therefore, bile acid
-glucosidase appears to have been eluted by
dodecyl-dNM but could not be reactivated due to either its lability or
its strong binding property to this inhibitor (Table III) which
remained bound to the enzyme during DEAE-Trisacryl chromatography. That
-glucosidase was specifically eluted from the affinity gel by
dodecyl-dNM is further supported by the fact that a successive elution
with 90% ethylene glycol saturated with NaCl resulted in less than
0.5% of the applied
-glucosidase activity being detected. In
comparison a yield of enzyme activity of about 10% was obtained in
this elution step without pretreatment of the affinity gel with
dodecyl-dNM. In contrast to dodecyl-dNM the parent compound dNM (1 M) was not able to elute the enzyme from the affinity gel
which may be explained by the weak inhibitory potency of dNM (Table
III).
|
Properties of -Glucosidase
As shown in Table II the
pure bile acid -glucosidase hydrolyzes 3
-glucosides of the bile
acids lithocholic and chenodeoxycholic acids, whereas the
6
-glucoside of the bile acid hyodeoxycholic acid is not a substrate
of the enzyme. Hydrolysis of
6
-D-glucosido-hyodeoxycholic acid was also not
detectable with a partially purified enzyme preparation after Mono Q
chromatography or with microsomes suggesting that bile acid
6-O-glucosides are not subject to enzymatic hydrolysis in
human liver in contrast to bile acid 3-O-glucosides. As may be seen from Table II glucocerebroside, which is the natural substrate of glucocerebrosidase, showed no reaction with bile acid
-glucosidase. Activity toward glucocerebroside was detectable in
human liver microsomes (8) but was separated from the activity toward
bile acid glucosides by DEAE-Trisacryl chromatography where
glucocerebrosidase activity appeared in the flow-through, whereas bile
acid
-glucosidase was eluted within a salt gradient (results not
shown). Of the glycosides of 4-methylumbelliferone tested as artificial
substrates of the pure
-glucosidase, the
-D-glucoside
supported a high activity, whereas the
-D-galactoside
was a poor substrate of the enzyme (Table II). No reaction was observed
with the following glycosides of 4-methylumbelliferone:
-D-glucoside,
-L-arabinopyranoside,
-D-fucoside, and
-D-xyloside (limit of
detection, 0.5 µmol per min per mg of pure enzyme protein).
|
The apparent Km values given in Table II were
obtained from double-reciprocal plots of initial rates of enzyme
activity as a function of varying substrate concentrations yielding
straight lines. From the ratio of the
kcat/Km values it may be seen
that the enzyme exhibited about 10-70-fold higher catalytic efficiency
with bile acid glucosides as compared with the -glucoside of
4-methylumbelliferone (Table II).
The purified enzyme showed a broad pH optimum for hydrolysis of lithocholic acid glucoside between pH 5.0 and 6.4 with half-maximal activities at pH 4.5 and 7.3 (not shown). A different pH dependence for hydrolysis of bile acid glucosides was observed in the same buffer system with the crude microsomal enzyme showing a sharp optimum close to pH 5.0 as described in a previous report (8).
As shown in Fig. 2 the purified -glucosidase could be
stimulated about 6-fold by the addition of divalent metal ions such as
Mn2+, Co2+, or Mg2+ compared with a
control without metal ions and with 1 mM EDTA. Ni2+, Ca2+, or Zn2+ were less
effective in activation of bile acid
-glucosidase. Ba2+
had no significant effect on enzyme activity (Fig. 2). In the absence
of metal ions EDTA (1 mM) produced a decrease in activity of the purified enzyme by about 20%. The purified enzyme showed a
similar metal ion dependence for hydrolysis of the
-glucoside of
4-methylumbelliferone as observed for bile acid glucoside hydrolysis (not shown).
Effect of Inhibitors
The microsomal -glucosidase had
already been shown in a previous report to be sensitive to inhibition
by various glucosidase inhibitors such as 1-deoxynojirimycin and
natural or synthetic glucosides (8). In addition to these compounds
N-alkyl derivatives of 1-deoxynojirimycin and the catalytic
site-directed covalent inhibitors conduritol B epoxide (CBE) and
bromoconduritol B epoxide (Br-CBE) (14) have been studied as effectors
of the purified bile acid
-glucosidase. As shown in Table
III, N-alkylation of 1-deoxynojirimycin
increased the inhibitory potential of the parent structure by several
orders of magnitude. Thus, dodecyl-dNM produced about 50% inhibition
of the enzyme with a concentration of 0.002 µM, whereas
for 1-deoxynojirimycin a concentration of 50 µM was necessary to achieve a similar inhibitory effect.
Whereas bromoconduritol A/B reacted as an inhibitor of the purified
-glucosidase as already described for the microsomal enzyme (8), CBE
and Br-CBE showed only a marked inhibitory effect on the enzyme in
microsomes (Table III). The purified
-glucosidase was not
significantly affected by these compounds in concentrations of 2 mM even after preincubation of the enzyme with the
inhibitors for 12 h at 4 °C with or without pretreatment of the
enzyme with phospholipids (see below). A marked loss in sensitivity of
the enzyme to CBE and Br-CBE was already observed when microsomes were
solubilized as in step 1 of the purification procedure. Whereas enzyme
activity in untreated microsomes was inhibited by about 50% in the
presence of 20 µM CBE or 1 µM Br-CBE (Table
III), a similar inhibitory effect on the enzyme from solubilized
microsomes required 2 mM concentrations of these epoxide
inhibitors (not shown). This decrease in sensitivity of the enzyme from
solubilized microsomes to CBE and Br-CBE was already observed before
insoluble microsomal membrane components were removed by high speed
centrifugation. The yield of bile acid
-glucosidase activity in
these solubilized microsomes using a small scale preparation was about
80% as compared with the activity in untreated microsomes. Therefore,
the possibility can be excluded that two forms of microsomal bile acid
-glucosidase may have been separated which differ in sensitivity to
CBE and Br-CBE. After polyethylene glycol fractionation (step 2 of the purification procedure) the enzyme was as resistant against CBE and
Br-CBE as the enzyme preparation after Mono Q chromatography (shown in
Table III). No separation of two different forms of bile acid
-glucosidase was observed during polyethylene glycol fractionation. All fractions obtained in this procedure exhibited only bile acid
-glucosidase activity with low sensitivity to CBE and Br-CBE.
All other compounds tested as inhibitors of microsomal bile acid
-glucosidase (8) retained their inhibitory potency for the purified
enzyme with the exception of the inhibitors castanospermine and octyl
-glucoside. These compounds affected only weakly the microsomal
enzyme (8) and showed no reaction with the purified enzyme in
concentrations of 3 and 5 mM, respectively.
The isolated bile acid
-glucosidase was dependent on the presence of phospholipids for
exhibition of enzyme activity. Without the addition of phospholipids
the pure enzyme was inactive suggesting that the
-glucosidase was
obtained in a delipidated form from the final column step and that
phospholipids are necessary to maintain the enzyme in an active
conformation. Whereas phospholipids did not increase enzyme activity in
the first three steps of the purification procedure, a first activatory
effect of about 2-fold could be observed after Mono Q chromatography.
With a more purified and therefore more delipidated enzyme preparation
after chromatofocusing (purification as compared with solubilized
microsomes, 6400-fold), the effect of naturally occurring phospholipids
on enzyme activity was studied. As shown in Fig. 3,
treatment of the enzyme with various concentrations of
phosphatidylinositol, phosphatidylserine, phosphatidylcholine, or
lysophosphatidylcholine resulted in a maximal activation about 3.7-, 2.9-, 2.7-, or 2.2-fold, respectively. Phosphatidylethanolamine and
sphingomyelin were less potent activators of the enzyme (Fig. 3). All
of these compounds were inhibitory in higher concentrations.
Since detergents may act as phospholipid substitute on the activity of
membrane bound enzymes, the effect of various detergents on a partially
purified preparation of bile acid -glucosidase activity was studied.
As shown in Fig. 4, addition of CHAPS, taurocholate, or
Triton X-100 led to a maximal activation of enzyme activity of about
2.5-, 2-, or 1.4-fold, respectively. Brij 58 was only inhibitory to the
enzyme. Whereas in microsomes, detergents such as taurocholate or
Triton X-100 had no effect or led to inhibition of enzyme activity in
higher concentrations (8), in the partially purified state,
e.g. after Mono Q chromatography, detergents could replace
phospholipids to produce a maximally activated form of the enzyme. Very
dilute preparations after chromatofocusing (protein, 0.7 µg/ml)
additionally required the presence of 0.5-1% (w/v) human serum
albumin for enzyme reconstitution. The pure enzyme, however, could not
be activated by the addition of detergents and albumin. Only after
reconstitution in the presence of phospholipids the pure enzyme
exhibited hydrolytic activity.
Immunochemical Cross-reactivity
In an effort to explore the
possible structural relationship between bile acid -glucosidase and
glucocerebrosidase solubilized liver preparations of microsomal bile
acid
-glucosidase and lysosomal glucocerebrosidase were incubated
with antibodies directed against human placental glucocerebrosidase. As
shown in Fig. 5, at the highest antibody concentration
used, glucocerebrosidase was inhibited by about 70% and could be
quantitatively immunoprecipitated, whereas under these conditions the
antibodies were incapable of precipitating or inactivating bile acid
-glucosidase (shown in Fig. 5 only for immunoprecipitation of the
enzyme). The same result was obtained with a partially purified
preparation of bile acid
-glucosidase after Mono Q chromatography.
These results suggest that glucocerebrosidase and bile acid
-glucosidase are antigenically unrelated, whereas human liver
lysosomal glucocerebrosidase is recognized by antibodies to the
placental form of the enzyme.
The present study describes the isolation of a microsomal human
liver bile acid -glucosidase to apparent electrophoretical homogeneity using carboxynonyl-dNM-AH-Sepharose for affinity
purification of the enzyme. N-
-Carboxyalkyl derivatives
of dNM and its D-manno analog have already been used for
the purification of various glycosidases (24, 25). Thus, the
carboxynonyl-dNM support used in the present study was also suitable
for the isolation of a cytosolic calf liver
-glucosidase (15) and of
lysosomal human placental glucocerebrosidase (26). Whereas, however,
the cytosolic and the lysosomal enzyme eluted under mild conditions from the affinity support, with 1 mM octyl
-glucoside
(15) or 40-60% ethylene glycol (26), respectively, bile acid
-glucosidase activity could not be recovered under these conditions
but emerged from the column with 90% ethylene glycol saturated with
NaCl.
In addition to conditions for elution from the affinity column, further
properties of bile acid -glucosidase show that the enzyme is
distinct from the previously described
-glucosidases, e.g. the effect of lipids on enzyme activities. Whereas bile
acid
-glucosidase was activated by taurocholate, acidic, and neutral phospholipids (Figs. 3 and 4), these compounds were inhibitory to the
cytosolic
-glucosidase (12). Glucocerebrosidase was only activated
by taurocholate and acidic phospholipids, e.g. phosphatidylserine or phosphatidylinositol (27), whereas the neutral
phospholipids phosphatidylcholine or phosphatidylethanolamine were
without effect on enzyme activity (28). In contrast to the pure form of
glucocerebrosidase, which was also active without the addition of
phospholipids (29), bile acid
-glucosidase showed an absolute
phospholipid requirement for exhibition of activity in the pure state.
Furthermore, bile acid
-glucosidase is the only glucosidase
described at present that is metal ion-dependent for
expression of full activity (Fig. 2). However, various
-mannosidases have been shown to require divalent metal ions for exhibition of
activity (25, 30).
In addition to the membrane-bound lysosomal glucocerebrosidase a second
membrane-bound -glucosidase has been described from a crude membrane
fraction of human spleen and has been termed "nonspecific
-glucosidase" since it was active toward the artificial substrate
4-methylumbelliferyl-
-D-glucoside (31, 32). The subcellular location of this enzyme has not been determined. To evaluate the relationship between the bile acid
-glucosidase and the
"nonspecific" membrane-bound
-glucosidase more information is
needed on the latter enzyme.
A comparison of the properties of the purified bile acid
-glucosidase with those of the corresponding microsomal-bound enzyme shows that some marked differences in characteristics between the
purified enzyme and its membrane-bound form are apparent. The purified,
soluble form of enzyme exhibited a broad pH optimum of activity between
pH 5.0 and 6.4, whereas the membrane-bound form showed a sharp optimum
of activity close to pH 5.0 (8). Furthermore, the membrane-bound enzyme
was highly sensitive to inhibition by the active site-directed
inhibitors CBE and Br-CBE, whereas these compounds did not
significantly affect the soluble purified enzyme even after
reconstitution with phospholipids (Table III). Changes in properties
between membrane-bound enzymes and the corresponding soluble forms have
been described for a variety of enzyme activities (33, 34). As a first
example, the membrane-bound form of mitochondrial ATPase has been
described to be sensitive to inhibition by oligomycin, whereas in its
soluble form the enzyme was oligomycin-resistant (34). This phenomenon
has been termed allotopy (34) and may be applied to bile acid
-glucosidase. Membrane-bound proteins that show the phenomenon of
allotopy are dependent on lipid-protein interactions for the exhibition
of activity. After removal of the natural membrane environment an artificial lipid support has to be provided which may lead to changes
in properties of the respective enzyme as observed in the present
report for bile acid
-glucosidase.
Even though the enzyme studied here resembled most other
-glucosidases in being strongly inhibited by the basic analog of D-glucose dNM and its N-alkyl derivatives, it
was not inhibited by castanospermine, a rigid, bicyclic analog of dNM
which inhibits other
-glucosidases with Ki values
in the micromolar range (see Ref. 35 for reviews). This feature and the
exceptional resistance of the enzyme against CBE and Br-CBE sets it
aside from most other
-glucosidases and might point to details of
the catalytic mechanism that differ from the generally accepted model (35).
The isolated enzyme appears to be highly specific for bile acid
3-O--D-glucosides since bile acid
6-O-
-D-glucosides were not hydrolyzed by the
enzyme. Furthermore, of the various glycosides of 4-methylumbelliferone
tested only the
-D-glucoside and the
-D-galactoside showed a reaction with the enzyme with a
ratio of relative reaction rates of 11:1. Thus, bile acid
-glucosidase cannot be classified as a broad specificity
-glucosidase in contrast to the cytosolic
-glucosidase. This
enzyme was shown to hydrolyze not only the
-D-glucoside
or the
-D-galactoside but also the
-L-arabinoside, the
-D-fucoside, or the
-D-xyloside of 4-methylumbelliferone with high activity
(12). Bile acid 3-O-glucosides are at present the only
natural compounds that could be identified as substrates of bile acid
-glucosidase. The physiological role of bile acid glucoside
hydrolysis is, however, unknown at present and has to be explored by
further studies.
Dedicated to Professor Dr. Helmut Holzer on the occasion of his 75th birthday.
We are greatly indebted to Melanie Flecken and Rita Gartzen for excellent technical assistance.