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J. Biol. Chem., Vol. 277, Issue 30, 26858-26864, July 26, 2002
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From the
Received for publication, February 21, 2002, and in revised form, May 9, 2002
An obligatory step in the mammalian nutritional
utilization of pyridoxine-5'- A significant dietary source of vitamin B6 for
humans is provided by a glycosylated form of the vitamin,
pyridoxine-5'- This laboratory has examined the intracellular (i.e.
cytosolic) hydrolysis of PNG in mammalian small intestinal mucosa.
Intestinal cytosolic PNG hydrolysis was initially thought to be
catalyzed by a broad specificity A recent reassessment of mucosal PNG hydrolase activity in the rat
small intestine indicated that ~40% of the activity associated with
the total membrane fraction could be attributed to enzymatic activity
in the brush border
membrane.3 Hydrolysis of
glucosides and oligosaccharides by enzymes present in the intestinal
brush border often precedes the absorption of individual
monosaccharides or the aglycone of glycosylated molecules such as
flavonoid and isoflavone glucosides. Day et al. (18) found
that quercetin, genistein, and daidzein We report herein the kinetic characterization of LPH-catalyzed
hydrolysis of PNG and include lactose as a substrate for comparison of
catalytic properties. We also report the in vitro formation of a PN-disaccharide, which is evidence of a transferase activity for
mammalian LPH.
Materials--
Pyridoxine (PN) hydrochloride, lactose, glucose,
phloridizin (phlorizin), phloretin, Sephacryl S400, Sephacryl S200,
2',4'-dinitrophenyl-2-fluoro-2-deoxy- Purification of LPH--
LPH was purified according to the
Triton X-100 solubilization method of Wacker et al. (19)
with the exceptions of Sephacryl S200 substituted for Sephadex G-200
and omission of the final anti-lactase affinity purification column.
During this purification, all materials were kept on ice or at 4 °C.
Brush border membrane was isolated from rat intestine for the
purification of LPH according to the method of Kessler et
al. (22) with some modifications (23). Weanling male and female
rats (Hsd:Sprague-Dawley, Harlan, Indianapolis, IN)
(n = 45) were used for the purification of LPH. Small
intestine was harvested, flushed with 0.9% (w/v) NaCl, and cut
longitudinally, and mucosa was scraped with a glass slide, yielding
~20 g of total mucosa. Electrophoretic characterization of the
purified enzyme was performed under denaturing conditions using 8%
(w/v) polyacrylamide gels according to Laemmli (24) using a mini-gel
electrophoresis apparatus from Novex (Invitrogen). A commercially
available prestained protein molecular mass standard (Invitrogen) ranging from 10-173 kDa was used. The gel was stained with Simply Blue SafeStain for 1 h followed by destaining
overnight in distilled deionized water.
Antibody Production and Western Blot Analysis--
The peptide
CTLFHFDLPQALEDQG was synthesized with an additional cysteine at the
carboxyl terminus and verified by mass spectrometry amino acid analysis
by Research Genetics (Huntsville, AL). This peptide was conjugated at
the terminally added cysteine residue to maleimide-activated KLH
according to the manufacturer's instructions. KLH-conjugated peptide
was injected into New Zealand white rabbits (Cocalico Biologicals,
Reamstown, PA) for production of polyclonal antiserum. Immunoblotting
or Western analyses were done as described by Harlow and Lane (25).
Purified LPH protein was resolved on an 8% (w/v) SDS-PAGE gel at 130 V
for 2 h. The gel was electroblotted to polyvinyldifluoride
for 2.5 h at 12 V in a transfer buffer containing 192 mM glycine, 25 mM Tris, 0.05% SDS, 10%
methanol. The blot was stained with Amido Black and destained. For the
protein detection, the stained blot was cut into strips for different antiserum treatments. Primary antibody treatments included whole immune
serum, pre-immune serum, and peptide-competed serum. The peptide-competed serum was prepared by incubating the whole immune serum (0.5 ml) with 10 µg of peptide at room temperature for 1 h
prior to application to the blot. The strips were blocked in phosphate-buffered saline (PBST) (137 mM NaCl, 2.7 mM KCl, 5.4 mM Na2HPO4,
1.8 mM KH2PO4, 0.05% (v/v) Tween
20) containing 5% (w/v) nonfat dry milk (NFDM) for 1 h. Primary
antibody treatments were applied at 1:500 dilution (in PBST-NFDM)
followed by washing with PBST without NFDM and subsequent addition of
the secondary antibody (goat anti-rabbit IgG horseradish peroxidase
conjugate) at 1:5000 dilution. Unbound secondary antibody was removed
by washing with PBST (no NFDM). A chemiluminescent/fluorescent
substrate (ECL-Plus) was then applied and incubated at room temperature for 5 min. The excess substrate was blotted away, and protein bands
were visualized by fluorescence emission (Storm 840 fluorescent optical
scanner; Amersham Biosciences).
Enzyme Activity Assays--
The standard assay for PNG
hydrolytic activity was performed according to Nakano and Gregory (14).
Standard activity assays for PNG hydrolysis were performed in a
reaction mixture containing 10 µg of purified LPH and 0.25 mM PNG in 80 mM sodium phosphate, pH 6.0, that
was incubated at 37 °C for 1 h. Pyridoxine release was measured
using reverse-phase HPLC with fluorometric detection (21). Lactase
activity was measured according to the method of Dahlqvist (26) with a
modification of the 50 mM maleate buffer to a 50 mM sodium phosphate, pH 6.0, assay buffer. Standard
activity assays for the hydrolysis of lactose and other disaccharides
were performed using 25 mM disaccharide. Glucose release
was quantified using a reagent kit based on the glucose oxidase
reaction with colorimetric detection. Phlorizin hydrolase activity was
measured by reverse-phase HPLC with UV detection of the aglycone,
phloretin (27). Standard assay mixtures contained 100 mM
phlorizin in 50 mM sodium citrate, pH 5.9. All activity
assays were conducted under conditions that allowed measurement of
initial rate.
For kinetic studies, reactions involving LPH-catalyzed hydrolysis of
PNG and lactose were conducted at various concentrations as
specified in the text or the table. Experiments examining active site
inhibition, along with selective active site protection using 2F-DNPGlc
and a glycal (D-glucal), were conducted as described by Day
et al. (18) and Arribas et al. (20). Other
potential inhibitors as specified were added to standard reaction
mixtures without preincubation of enzyme. Kinetic constants
(Km, Vmax, and
Ki) were calculated by nonlinear regression using
SigmaPlot Enzyme Kinetics Module, v 1.0 (SPSS, Inc., San Rafael, CA)
software. Protein concentration was determined by a colorimetric method
(28) using bovine serum albumin as the standard.
Liquid Chromatography-Mass Spectrometry (LC-MS)--
To obtain
molecular mass and fragmentation information regarding a novel reaction
product formed during incubation of purified LPH with PNG and lactose,
LC-MS and LC-MS/MS analyses were performed in atmospheric pressure
chemical ionization mode (APCI) (LC: Hewlett Packard model 1100, Agilent, Palo Alto, CA; and MS: Thermo Finnigan model TSQ7000;
San Jose, CA). The HPLC separation was performed isocratically using 5 mM ammonium acetate with 0.1% (v/v) acetic acid as the
mobile phase at a flow rate of 0.6 ml/min with a reverse-phase column
(C18 Phenosphere, 50 × 4.6 mm, 5 µm column; Phenomenex, Torrance, CA). The analysis was conducted by monitoring ions from 75-600 m/z. For the MS-MS analysis, a collision
energy of 30 V was used to fragment the parent mass (M+1) of
m/e 494.
LPH Purification--
LPH from rat small intestinal mucosa was
purified 300-fold relative to the lactase activity measured in mucosal
crude homogenate. The enzyme was purified to near homogeneity, and the
purification was repeated with similar results. The approximate
molecular mass of purified LPH by SDS-polyacrylamide gel
electrophoresis was 135-140 kDa. In the published method used for
purification, an anti-lactase antibody affinity column was used to
further separate LPH from aminopeptidase N (molecular mass, 145 kDa)
(19). This investigation did not use an affinity chromatography
purification step to remove peptidase activity. Although aminopeptidase
N may not have been separated completely from the final purified LPH, the enrichment of lactase activity from the crude homogenate was consistent with that reported in the purification method (19). Staining
of the SDS-PAGE revealed two major bands of protein, one at ~135-140
kDa and another at ~200-220 kDa (Fig.
1A). The band of protein at
135-140 kDa corresponds to mature LPH. The band at 200-220 kDa is
consistent with the molecular mass of a precursor form of LPH (29).
Western analyses were performed to confirm further the relative purity
of the enzyme preparation (Fig. 1B). The peptide used for
antibody production had amino acid sequence homology with regions in
the precursor and mature forms of LPH but no other mammalian intestinal
enzymes cataloged in Swiss-Prot and GenBankTM databases. No
protein bands were detected in the blot strips incubated with PBST-NFDM
alone and the secondary antibody alone (Fig. 1B, lanes
1 and 2, respectively). The whole immune serum-treated strip revealed three immunogenic protein bands (lane 3),
which were not visible in the peptide-competed antibody treatment
(lane 4). The fourth visible band (~60 kDa) was found to
be an artifact of the antibody under the conditions of this analysis.
The band at ~80 kDa detected by the whole immune serum was also
detected by the pre-immune serum, indicating that the recognition of
the band by the whole immune serum was not sequence-specific
(lane 5). The protein bands at 135-140 and 220-220 kDa
that were recognized by this polyclonal antibody appear to be the
mature and precursor forms of LPH. However, upon closer inspection of
the Coomassie Blue-stained gel, trace amounts of additional high
molecular mass bands were observed. Further assays for disaccharidase
activity of the purified LPH preparation revealed residual
maltase-glucoamylase (EC 3.2.1.20 and 3) at a specific activity of 3.8 µmol/min·mg protein. This activity was controlled for in later
kinetic characterization studies.
Kinetic Characterization--
Purified LPH from rat small
intestinal mucosa catalyzed the hydrolysis of PNG and followed
Michaelis-Menten kinetics. Values for Km,
Vmax, and kcat were lower
when PNG was used as the substrate as compared with lactose. Values for
kcat and
Vmax/Km indicated that
lactose hydrolysis was catalyzed more efficiently than the hydrolysis
of PNG (Table I).
Inhibition Studies--
The substrate PNG exhibited substrate
inhibition at concentrations greater than 3 mM (Fig.
2). Because of limited availability of
PNG, only concentrations ranging from 0.01 to 5 Km were tested. The reaction products (glucose and pyridoxine), lactose, and other disaccharides also were tested for inhibitory properties. Pyridoxine (0.5-10 mM) did not inhibit LPH-catalyzed PNG
hydrolysis, but the monosaccharide glucose inhibited the reaction at
concentrations Identification of the Site of PNG Hydrolysis by LPH--
The
inhibitor 2F-DNPGlc binds to both the lactase and phlorizin hydrolase
active sites, inhibiting both hydrolytic activities (31, 32). The
lactase activity was preserved by preincubation with
D-glucal, which yields reversible blockage of the lactase active site (20). Subsequent addition of 2F-DNPGlc to the
glucal-modified LPH led to the selective inhibition of the phlorizin
hydrolase active site (20). This experimental protocol allowed the
contribution of each activity from the respective active sites to be
calculated (20). Hydrolysis of PNG and lactose was reduced 100-fold in the presence of 2F-DNPGlc without glucal as compared with the uninhibited enzyme. Phloretin release was reduced by 85-90% in the
presence of 2F-DNPGlc with or without glucal as compared with the
activity of the free enzyme. Incubation of purified LPH with glucal
prior to the addition of 2F-DNPGlc maintained the hydrolytic activities
toward lactose and PNG to 50-65% of the uninhibited enzyme (Fig.
4). These findings strongly suggest that
PNG was hydrolyzed at the lactase active site of LPH.
Formation of Other Pyridoxine Glycosides--
HPLC analyses of
enzyme reaction mixtures containing LPH, PNG (substrate), and lactose
(inhibitor) detected not only the product, PN, and the substrate, PNG,
but also another fluorescent compound with a retention time of 5.3 min
(Fig. 5). The amount of this compound
formed was dependent on the concentration of lactose. Initial LC-MS
(APCI positive ion mode) analysis of the enzyme reaction mixture showed
the unidentified compound to have M+1 ion of 494 (m/z), corresponding to a disaccharide of
pyridoxine. This was further supported by an MS/MS experiment on the
M+1 ion using a collision energy of 30 V (Fig. 5). In addition to the residual M+1 ion of the disaccharide, major fragments were
observed at m/z 332 and 170, corresponding to the
M+1 ions of pyridoxine glucoside and pyridoxine (Fig.
6). The LC-MS and MS/MS experiments produced the same results when the HPLC-purified disaccharide from the
enzyme reaction mixture was utilized. This pyridoxine disaccharide has
a pyridoxine molecule with the 5'- The finding of PNG as a novel substrate for LPH has extended our
understanding of mechanisms governing vitamin B6
bioavailability and has added to the growing evidence that LPH
catalyzes the hydrolysis of not only lactose but other LPH was purified from 20-day-old rats to ensure reasonable lactase
activity with an adequate amount of starting tissue. The recognition of
both the mature and precursor forms of LPH by our antibody in the
Western analysis allowed us to verify the qualitative abundance of our
purified enzyme. This recognition was sequence-specific, since the
pre-immune serum and peptide-competed antibody did not detect the high
molecular mass bands that were recognized by the whole immune serum.
Values of Km and Vmax
(lactose) calculated for purified rat LPH were consistent with
published values (27, 34) (Table I). The kinetic parameters (for
lactose and PNG) appeared to be representative of the concentrations of
substrates that would be encountered physiologically by LPH. However,
under typical dietary conditions, the amount of PNG in digesta entering the small intestine would be in the micromolar concentration range, whereas the concentration of lactose (millimolar range) would greatly
exceed that of PNG. The relatively high Km value for
PNG (1.0 mM) suggests that LPH has the capacity to
hydrolyze physiological concentrations of PNG, but fractional
saturation of substrate of the enzyme would be low. Similarly, kinetic
ratios also indicated that LPH favors the hydrolysis of lactose over PNG.
Although LPH-catalyzed PNG hydrolysis was inhibited by one of its
products, glucose, the concentration at which inhibition occurred may
not be physiologically relevant ( Cellobiose, although not present in appreciable amounts in the human
diet, also is a substrate for LPH (17, 35); therefore, it was not
unexpected that this Fluoroglucosides are used widely as general inhibitors of
Various pyridoxine glucosides, including several pyridoxine
oligosaccharide derivatives, are known to exist widely in nature (37);
however, the formation of pyridoxine disaccharide catalyzed by enzymes
in the small intestine has not been reported. Future research will
address the nutritional significance of LPH hydrolysis of PNG, with
particular attention to the vitamin in human LPH expression and
activity and the inhibition of PNG hydrolysis by lactose.
*
This work was supported by National Institutes
of Health Grants DK 37481, T32 DK07667, and RR-00082 and is Florida
Agricultural Experiment Station Journal Series No. R-08682.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Food Science and
Human Nutrition Dept., P.O. Box 110370, University of Florida, Gainesville, FL 32611-0370. Tel.: 352-392-1991 (ext. 225); Fax: 352-392-4515; E-mail: jfgy@ufl.edu.
Published, JBC Papers in Press, May 21, 2002, DOI 10.1074/jbc.M201774200
2
C. W. Tseung, L. G. McMahon, and
J. F. Gregory, unpublished data.
3
A. D. Mackey, S. O. Lieu, and J. F. Gregory, unpublished data.
The abbreviations used are:
PNG, pyridoxine-5'-
Enzymatic Hydrolysis of Pyridoxine-5'-
-D-glucoside
Is Catalyzed by Intestinal Lactase-Phlorizin Hydrolase*
,¶
Food Science and Human Nutrition Department,
Institute of Food and Agricultural Sciences, University of Florida,
Gainesville, Florida 32611 and the § Division of
Endocrinology and Metabolism, Department of Medicine, College of
Medicine, University of Florida, Gainesville, Florida 32610
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucoside (PNG) is
the intestinal hydrolysis of its -glucosidic bond that releases
pyridoxine (PN). This laboratory previously reported the purification
and partial characterization of a novel cytosolic enzyme, designated
PNG hydrolase, which hydrolyzed PNG. An investigation of the
subcellular distribution of intestinal PNG hydrolysis found substantial
hydrolytic activity in the total membrane fraction, of which 40-50%
was localized to brush border membrane. To investigate the possible
role of a brush border -glucosidase in the hydrolysis of PNG,
lactase phlorizin hydrolase (LPH) was purified from rat small
intestinal mucosa. LPH hydrolyzed PNG with a Km of
1.0 ± 0.1 mM, a Vmax of
0.11 ± 0.01 µmol/min·mg protein, and a
kcat of 1.0 s
1. LPH-catalyzed PNG
hydrolysis was inhibited by glucose, lactose, and cellobiose but not by
PN. Specific blockage of the phlorizin hydrolase site of LPH using
2',4'-dintrophenyl-2-fluoro-2-deoxy--D-glucopyranoside did not reduce PNG hydrolysis. Evidence of transferase activity was
also obtained. Reaction mixtures containing LPH, PNG, and lactose
yielded the formation of another PN derivative that was identified as a
pyridoxine disaccharide. These results indicate that LPH may
play an important role in the bioavailability of PNG, but further
characterization is needed to assess its physiological function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucoside (PNG).1 PNG, found
predominantly in foods of plant origin, provides 15% of total vitamin
B6 in a mixed diet, depending on food selection (1, 2). PNG
exhibits ~50% bioavailability in humans (3, 4) and 25-30% in
rodents (5-8) in comparison to pyridoxine, the metabolically usable
form of vitamin B6. The rate-limiting step in the
utilization of PNG is not its intestinal absorption but rather the
enzymatic hydrolysis of the -glucosidic linkage to release glucose
and pyridoxine in the intestine (6-8). Although PNG can be absorbed
intact, this form is not metabolized or retained by the liver (6-8)
and can antagonize the metabolism of nonglycosylated forms of vitamin
B6 (9, 10). The remainder of PNG that is not absorbed is
likely to be accounted for through fecal losses.
-glucosidase (BSG) (EC 3.2.1.21)
(11), a cytosolic enzyme found in the intestine and liver (12-14).
Cytosolic PNG hydrolysis was also reported to be inversely related to
vitamin B6 nutritional status in rodents (13, 14). To
further study cytosolic PNG hydrolysis, this laboratory purified BSG
from pig intestinal mucosa, which led to the identification and
subsequent purification of a distinct and novel cytosolic
-glucosidase, designated pyridoxine-5'--D-glucoside
hydrolase (PNG hydrolase) (15). It was found that PNG hydrolase, not
BSG, was responsible for the cytosolic cleavage of PNG. Partial
characterization of cytosolic PNG hydrolase revealed its ability to
hydrolyze PNG as well as lactose and cellobiose but not sucrose (15).
Partial amino acid analysis showed substantial sequence homology
but revealed significant differences between PNG hydrolase and the
brush border membrane enzyme lactase phlorizin hydrolase (LPH) (EC
3.2.1.23, EC 3.2.1.62, and EC 3.2.1.108) (16, 17), the enzyme primarily responsible for the hydrolysis of dietary
lactose.2
-glucosides were hydrolyzed by purified sheep LPH prior to absorption. LPH is the only
mammalian brush border -glucosidase; thus, we hypothesize that LPH
is responsible for the hydrolysis of PNG, a -glucoside. LPH has two
distinct catalytic active sites, one for the hydrolysis of lactose and
flavonoid glucosides and another, phlorizin hydrolase, for the
hydrolysis of phlorizin and -glucosylceramides (19, 20). As reported
by Day et al. (18), the site of catalysis for flavonoid and
isoflavonoid glucoside hydrolysis was assigned experimentally to the
lactase active site, not to the phlorizin hydrolase site.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside (2F-DNPGlc), D-glucal, protease inhibitor mixture,
goat-anti-rabbit IgG horseradish peroxidase antibody, and a glucose
detection kit were obtained from Sigma. Prestained protein
molecular mass markers were obtained from Invitrogen. DEAE cellulose
was obtained from Whatman, and Simply Blue SafeStain was purchased from
Invitrogen. Maleimide-activated keyhole limpet hemocyanin (KLH) was
obtained from Pierce. Polyvinyldifluoride (Immobilon-P) was
obtained from Millipore Corporation (Bedford, MA). ECL Plus
chemiluminescent reagent was purchased from Amersham Biosciences.
Pyridoxine-5'--D-glucoside was prepared by biological
synthesis from pyridoxine using germinating alfalfa seeds and was
purified chromatographically (14, 21).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
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Fig. 1.
Purification of LPH from rat intestine.
A, SDS-PAGE of purified LPH preparation. The gel (8% w/v
acrylamide) has two lanes, one large lane containing enzyme preparation
and another for the molecular mass marker. Purified enzyme (20 µg)
was loaded into one large well. Enzyme preparation was electrophoresed
at 130 V for 2 h. The gel was stained with commercially available
formulation of Coomassie Blue stain (Simply Blue SafeStain). Molecular
mass markers ranged from 10-173 kDa. The protein band at 135-140 kDa
corresponds to LPH. B, Western analysis of purified LPH
preparation. Protein (20 µg) was separated by SDS-PAGE (8% w/v
acrylamide) and transferred to polyvinyldifluoride membrane. Membrane
was cut into strips and subjected to various antibody treatments.
Lane 1, no antibody applied; lane 2, secondary
antibody (goat-anti-rabbit horseradish peroxidase) only; lane
3, whole immune serum and secondary antibody; lane 4,
peptide-competed serum and secondary antibody; lane 5,
pre-immune sera and secondary antibody. Application of
chemiluminescent/fluorescent substrate reagent (ECL Plus) was followed
by detection using a fluorescent optical scanner.
Kinetic parameters of purified rat LPH using PNG and lactose as
substrates
25 mM (Ki equal
to 64 ± 5 mM). The primary substrate for LPH, lactose
(25-100 mM), inhibited PNG hydrolysis competitively with a
Ki equal to 56 ± 8 mM (Fig.
3). This suggests that the two substrates
(PNG and lactose) are hydrolyzed at the same active site. Cellobiose,
maltose, lactose, sucrose, and trehalose (25-100 mM) also
were added individually to standard reaction mixtures containing PNG
(0.01-0.25 mM) and purified LPH. Although sucrose and
trehalose did not affect PNG hydrolysis, cellobiose and maltose
inhibited the enzymatic release of PN. The inhibition of PNG hydrolysis
by maltose was reduced by 35-40% in the presence of 2.0 µM 1-deoxynojirimycin, an inhibitor of
maltase-glucoamylase activity (30).
View larger version (10K):
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Fig. 2.
Effect of substrate concentration on the
activity of LPH-catalyzed PNG hydrolysis. Comparison of rat small
intestine brush border membrane and purified LPH is shown.
View larger version (16K):
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Fig. 3.
Lineweaver-Burk and Michaelis-Menten
(inset) plots of LPH-catalyzed hydrolysis of PNG with
increasing amounts of lactose as inhibitor.
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Fig. 4.
Identification of the site of PNG hydrolysis
by LPH. Protection of the active site with D-glucal
prior to addition of 2F-DNPGlc (inhibitor) selectively inhibited the
phlorizin hydrolase active site. Completely inhibited enzyme (with
2F-DNPGlc) exhibited 10% of the PNG hydrolytic activity of the
glucal-protected enzyme.
-D-glucoside and
another glucose or galactose moiety. Similar to the observation of the
pyridoxine disaccharide formed in the presence of lactose, two other
disaccharides tested in the LPH inhibition experiments, cellobiose and
maltose, also formed fluorescent compounds that exhibited retention
times different from that formed with lactose. These compounds,
although detectable, were minor in relation to the amount of the
PN-disaccharide formed in the presence of lactose and were independent
of disaccharide concentration.
View larger version (15K):
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Fig. 5.
Sample chromatograms of enzyme reaction
mixtures of LPH-catalyzed PNG hydrolysis. A,
chromatogram from LPH-catalyzed PNG hydrolysis without added lactose.
B, chromatogram from LPH-catalyzed PNG hydrolysis with
added lactose (25 mM). Retention times are PN,
3.3 min; PNG, 4.3 min; PN-disaccharide, 5.3 min.
Assays were conducted using purified rat LPH.
View larger version (18K):
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Fig. 6.
LC-MS/MS analysis in the APCI positive ion
mode (MS/MS experiment on the ion at m/e
494; collision energy, 30 V) of the PN-disaccharide product
formed during the incubation of LPH with PNG and lactose. The
predominant ions are parent molecular ion (M+1) at
m/e 494, M+1 ion of pyridoxine glucoside at
m/e 332, and M+1 ion of pyridoxine at
m/e 170. The other significant ions at
m/e 476, 314, and 152 are produced by the loss of
water from the respective M+1 ions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucosides
such as plant -glucans, flavonoid glucosides, and isoflavonoid
glucosides (18, 33). Dietary PNG exhibits only 50-60% of the
bioavailability of free PN as a source of vitamin B6 for
humans (3, 4), which is due to incomplete hydrolysis of the glycosidic
linkage of PNG. The results reported in the present study suggest that
the digestion of PNG may commence at the intestinal brush border where
LPH can catalyze the hydrolysis of PNG to release free PN, which is
then passively absorbed. This extends our previous findings of the hydrolysis of PNG that can occur immediately after absorption into the
cytosolic compartment by PNG hydrolase. This LPH-catalyzed hydrolysis
may be an important determinant of the bioavailability of PNG. In
populations with lactase deficiency, the hydrolysis, and hence the
nutritional utilization of PNG, may be less efficient than in
populations that exhibit lactase persistence.
25 mM). Accounting for
the stoichiometry of the hydrolytic reaction of PNG, the amount of
glucose released (micromolar concentration) would not be sufficient to
cause product inhibition. However, the glucose released from the
hydrolysis of other disaccharides such as lactose may contribute to the
observed inhibition by glucose. The observed competitive inhibition of
lactose on LPH-catalyzed PNG hydrolysis is consistent with the fact
that PNG and lactose are hydrolyzed at the same active site. The
inhibition may not have a dramatic effect on bioavailability because
foods that contain PNG are of plant origin, which are devoid of
lactose. Unless a dairy product and plant-derived food were consumed
together, PNG should be at least partially hydrolyzed by LPH. The
Ki for lactose (56 ± 8 mM) did not
agree with the calculated Km for lactose (16 ± 1 mM). One possible explanation for this discrepancy may be
in the formation of alternative pyridoxine glucosides. Some PNG may be diverted from its -glucosidic hydrolysis process to a
trans-glycosylation reaction that yields a pyridoxine disaccharide and
a reduction in free PN. Alternatively, PNG may be hydrolyzed to
liberate PN, which may undergo enzymatic modification to form PNG or
PN-disaccharide. The mechanism of the hydrolysis/trans-glycosylation
was not examined further in this study.
-linked disaccharide would also inhibit PNG
hydrolysis. The 
-disaccharide, maltose, also inhibited the release
of PN by LPH, which was not anticipated. The subsequent detection of
maltase-glucoamylase activity (EC 3.2.1.20 and EC 3.2.1.3) (36) as a
contaminant of the LPH preparation is consistent with the presence of
minor high molecular mass protein bands (200-220 kDa) faintly stained
in the SDS-PAGE. Western analysis using our antibody that was
sequence-specific to LPH but not maltase-glucoamylase showed that the
more prominently stained high molecular mass band was likely to be a
precursor form of LPH and not maltase-glucoamylase. Despite the
relatively low abundance of the maltase-glucoamylase contaminant, its
hydrolytic activity toward maltose had an effect on LPH-catalyzed PNG
hydrolysis. What appeared to be the inhibition of PN release actually
may have been the LPH-catalyzed formation of another maltose- or
glucose-derived pyridoxine oligosaccharide from PN, PNG, or both.
Alternatively, the hydrolysis of maltose released monosaccharide units
(D-glucose), which could inhibit PNG hydrolysis as
catalyzed by LPH. Maltase-glucoamylase activity was reduced by 63% in
the presence of 2.0 µM 1-deoxynojirimycin without
affecting lactose hydrolysis. In reaction mixtures containing LPH, PNG,
maltose (25 mM), and deoxynojirimycin, the release of PN
was increased by an average of 35%, and the formation of other
pyridoxine disaccharides was decreased compared with reactions without
deoxynojirimycin. Increasing the concentration of 1-deoxynojirimycin likely may have negated this inhibition; however, at higher
concentrations deoxynojirimycin also is a competitive inhibitor of
lactase (19). Although maltase-glucoamylase was present in the purified
LPH preparation, it appears that it does not alter the kinetics of PNG
hydrolysis in the absence of -disaccharides. This is further supported by the inhibition experiment with -glucosidase inhibitor 2F-DNPGlc. When LPH is inhibited completely with this fluoroglucoside, the release of PN is negligible, indicating that maltase-glucoamylase alone is not capable of hydrolyzing PNG. Maltase-glucoamylase that had
been co-purified with lactase by papain solubilization from 15-day-old
rat intestine was reported to have a final specific activity of 36 µmol/min·mg protein in another study (34), which is ~10× the
specific activity measured in the current study.
-glucosidases (32). 2F-DNPGlc is a mechanism-based inhibitor of
-glucosidases, and it exerts its effect by forming a stable covalent
linkage of the 2'-position of the fluoroglucoside to glutamic acid
residues at the active sites of LPH. The use of glucal in conjunction
with the inhibitor in the protocol allows individual examination of the
lactase and phlorizin hydrolase active sites on LPH (20). The
deglycosylation of flavonoid and isoflavonoid glucosides was studied
using this same experimental procedure (18). Inhibition of the
phlorizin hydrolase site with 2F-DNPGlc did not diminish the hydrolysis
of the quercetin glucosides. Similar to the results reported for the
flavonoid and isoflavonoid glucosides (18), we concluded that PNG is
hydrolyzed at the lactase active site of LPH.
FOOTNOTES
ABBREVIATIONS
-D-glucoside;
PN, pyridoxine;
BSG, broad specificity -glucosidase;
LPH, lactase phlorizin hydrolase;
2F-DNPGlc, 2',4'-dinitrophenyl-2-fluoro-2-deoxy--D-glucopyranoside;
PBST, phosphate-buffered saline/Tween;
NFDM, nonfat dry milk;
HPLC, high pressure liquid chromatography;
LC-MS, liquid chromatography-mass
spectrometry;
APCI, atmospheric pressure chemical ionization.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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