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* 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.
An obligatory step in the mammalian nutritional utilization of pyridoxine-5′-β-d-glucoside (PNG) is the intestinal hydrolysis of its β-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 β-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 akcat 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-β-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.
A significant dietary source of vitamin B6 for humans is provided by a glycosylated form of the vitamin, pyridoxine-5′-β-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 (
) 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 β-glucosidic linkage to release glucose and pyridoxine in the intestine (
). The remainder of PNG that is not absorbed is likely to be accounted for through fecal losses.
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 β-glucosidase (BSβG) (EC 126.96.36.199) (
). To further study cytosolic PNG hydrolysis, this laboratory purified BSβG from pig intestinal mucosa, which led to the identification and subsequent purification of a distinct and novel cytosolic β-glucosidase, designated pyridoxine-5′-β-d-glucoside hydrolase (PNG hydrolase) (
). It was found that PNG hydrolase, not BSβG, 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 (
C. W. Tseung, L. G. McMahon, and J. F. Gregory, unpublished data.
2C. W. Tseung, L. G. McMahon, and J. F. Gregory, unpublished data.
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.
A. D. Mackey, S. O. Lieu, and J. F. Gregory, unpublished data.
3A. D. Mackey, S. O. Lieu, and J. F. Gregory, unpublished data.
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. (
) found that quercetin, genistein, and daidzein β-glucosides were hydrolyzed by purified sheep LPH prior to absorption. LPH is the only mammalian brush border β-glucosidase; thus, we hypothesize that LPH is responsible for the hydrolysis of PNG, a β-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 β-glucosylceramides (
), the site of catalysis for flavonoid and isoflavonoid glucoside hydrolysis was assigned experimentally to the lactase active site, not to the phlorizin hydrolase site.
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.
Pyridoxine (PN) hydrochloride, lactose, glucose, phloridizin (phlorizin), phloretin, Sephacryl S400, Sephacryl S200, 2′,4′-dinitrophenyl-2-fluoro-2-deoxy-β-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′-β-d-glucoside was prepared by biological synthesis from pyridoxine using germinating alfalfa seeds and was purified chromatographically (
) 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. (
). 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 (
) 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 (
). 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 (
). 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 (
) 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 (
). Standard assay mixtures contained 100 mmphlorizin 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 Dayet al. (
). Other potential inhibitors as specified were added to standard reaction mixtures without preincubation of enzyme. Kinetic constants (Km, Vmax, andKi) 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 (
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) ofm/e 494.
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) (
). 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 (
). 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 (
). 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 188.8.131.52 and 3) at a specific activity of 3.8 μmol/min·mg protein. This activity was controlled for in later kinetic characterization studies.
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 forkcat andVmax/Km indicated that lactose hydrolysis was catalyzed more efficiently than the hydrolysis of PNG (Table I).
Table IKinetic parameters of purified rat LPH using PNG and lactose as substrates
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 Kmwere 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 ≥ 25 mm (Ki equal to 64 ± 5 mm). The primary substrate for LPH, lactose (25–100 mm), inhibited PNG hydrolysis competitively with aKi 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 (
). 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′-β-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.
The finding of PNG as a novel substrate for LPH has extended our understanding of mechanisms governing vitamin B6bioavailability and has added to the growing evidence that LPH catalyzes the hydrolysis of not only lactose but other β-glucosides such as plant β-glucans, flavonoid glucosides, and isoflavonoid glucosides (
), 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.
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 (
) (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 (≥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. TheKi 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 β-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.
Cellobiose, although not present in appreciable amounts in the human diet, also is a substrate for LPH (
); therefore, it was not unexpected that this β-linked disaccharide would also inhibit PNG hydrolysis. The α-disaccharide, maltose, also inhibited the release of PN by LPH, which was not anticipated. The subsequent detection of maltase-glucoamylase activity (EC 184.108.40.206 and EC 220.127.116.11) (
) 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 (
). 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 α-disaccharides. This is further supported by the inhibition experiment with β-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 (
). 2F-DNPGlc is a mechanism-based inhibitor of β-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 (
). 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 (
); 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.