Cytosolic Pyridoxine-β-d-Glucoside Hydrolase from Porcine Jejunal Mucosa
1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês
10.1074/jbc.272.51.32025
ISSN1083-351X
AutoresLaura G. McMahon, Hideko Nakano, Marc-David Levy, Jesse F. Gregory,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoDuring studies of the nutritional utilization of pyridoxine 5′-β-d-glucoside, a major form of vitamin B6 in plants, we detected two cytosolic β-glucosidases in jejunal mucosa. As expected, one was broad specificity β-glucosidase that hydrolyzed aryl β-d-glycosides but not pyridoxine β-d-glucoside. We also found a previously unknown enzyme, designated pyridoxine-β-d-glucoside hydrolase, that efficiently hydrolyzed pyridoxine β-d-glucoside. These were separated and purified as follows: broad specificity β-glucosidase 1460-fold and pyridoxine-β-d-glucoside hydrolase 36,500-fold. Purified pyridoxine-β-d-glucoside hydrolase did not hydrolyze any of the aryl glycosides tested but did hydrolyze cellobiose and lactose. Pyridoxine-β-d-glucoside hydrolase exhibited a pH optimum of 5.5 and apparent molecular mass of 130 kDa by SDS-polyacrylamide gel electrophoresis and 160 kDa by nondenaturing gel filtration, in contrast to 60 kDa for native and denatured broad specificity β-glucosidase. Glucono-δ-lactone was a strong inhibitor of both enzymes. Ionic and nonionic detergents were inhibitory for each enzyme. Conduritol B epoxide, a potent inhibitor of lysosomal acid β-glucosidase, inhibited pyridoxine-β-d-glucoside hydrolase but not broad specificity β-glucosidase, but both were inhibited by the mechanism-based inhibitor 2-deoxy-2-fluoro-β-d-glucosyl fluoride. Our findings indicate major differences between these two cytosolic β-glucosidases. Studies addressing the role of vitamin B6 nutrition in regulating the activity and its consequences regarding pyridoxine glucoside bioavailability are in progress. During studies of the nutritional utilization of pyridoxine 5′-β-d-glucoside, a major form of vitamin B6 in plants, we detected two cytosolic β-glucosidases in jejunal mucosa. As expected, one was broad specificity β-glucosidase that hydrolyzed aryl β-d-glycosides but not pyridoxine β-d-glucoside. We also found a previously unknown enzyme, designated pyridoxine-β-d-glucoside hydrolase, that efficiently hydrolyzed pyridoxine β-d-glucoside. These were separated and purified as follows: broad specificity β-glucosidase 1460-fold and pyridoxine-β-d-glucoside hydrolase 36,500-fold. Purified pyridoxine-β-d-glucoside hydrolase did not hydrolyze any of the aryl glycosides tested but did hydrolyze cellobiose and lactose. Pyridoxine-β-d-glucoside hydrolase exhibited a pH optimum of 5.5 and apparent molecular mass of 130 kDa by SDS-polyacrylamide gel electrophoresis and 160 kDa by nondenaturing gel filtration, in contrast to 60 kDa for native and denatured broad specificity β-glucosidase. Glucono-δ-lactone was a strong inhibitor of both enzymes. Ionic and nonionic detergents were inhibitory for each enzyme. Conduritol B epoxide, a potent inhibitor of lysosomal acid β-glucosidase, inhibited pyridoxine-β-d-glucoside hydrolase but not broad specificity β-glucosidase, but both were inhibited by the mechanism-based inhibitor 2-deoxy-2-fluoro-β-d-glucosyl fluoride. Our findings indicate major differences between these two cytosolic β-glucosidases. Studies addressing the role of vitamin B6 nutrition in regulating the activity and its consequences regarding pyridoxine glucoside bioavailability are in progress. A naturally occurring glycosylated derivative of vitamin B6, pyridoxine 5′-β-d-glucoside (PNG), 1The abbreviations used are: PNG, pyridoxine 5′-β-d-glucoside; PN, pyridoxine; p-NP,p-nitrophenyl; p-NPGlc,p-nitrophenyl-β-d-glucoside; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]glycine. 1The abbreviations used are: PNG, pyridoxine 5′-β-d-glucoside; PN, pyridoxine; p-NP,p-nitrophenyl; p-NPGlc,p-nitrophenyl-β-d-glucoside; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]glycine. was first isolated from rice bran (1Yasumoto K. Iwami K. Mitsuda H. Agric. Biol. Chem. 1977; 41: 1061-1067Crossref Scopus (12) Google Scholar). PNG is now known to exist in most fruits, vegetables, and cereal grains, in which it comprises from 5–75% of total vitamin B6 (2Kabir H. Leklem J.E. Miller L.T. J. Food Sci. 1983; 48: 1422-1425Crossref Scopus (56) Google Scholar, 3Gregory J.F. Ink S.L. J. Agric. Food Chem. 1987; 35: 76-82Crossref Scopus (69) Google Scholar). Because of the prevalence of PNG in plant-derived foods, its bioavailability as a source of available vitamin B6 is a matter of nutritional concern. The bioavailability of this glycosylated form of vitamin B6, relative to pyridoxine (PN), is ∼25% in rats (4Ink S.L. Gregory J.F. Sartain D.B. J. Agric. Food Chem. 1986; 34: 857-862Crossref Scopus (38) Google Scholar, 5Trumbo P.R. Gregory J.F. Sartain D.B. J. Nutr. 1988; 118: 175-179Google Scholar) and ∼50% for humans (6Gregory III, J.F. Trumbo P.R. Bailey L.B. Toth J.P. Baumgartner T.G. Cerda J.J. J. Nutr. 1991; 121: 177-186Crossref PubMed Scopus (56) Google Scholar, 7Nakano H. McMahon L.G. Gregory III, J.F. J. Nutr. 1997; 127: 1508-1513Crossref PubMed Scopus (37) Google Scholar), as estimated from urinary excretion of 4-pyridoxic acid after oral administration of isotopically labeled PNG. The rate-limiting phase of PNG utilization in vitamin B6 metabolism is the hydrolysis of the β-glycosidic bond in both rats and humans. These findings indicate that the hydrolysis of PNG is a major factor governing its bioavailability. Glucosidases exist widely in nature. The primary β-glucosidases in mammalian tissues consist of a lysosomal membrane-bound acid β-glucosidase (EC 3.2.1.45;N-acylsphingosyl-β-d-glucopyranoside glucohydrolase) which is responsible for the hydrolytic cleavage of glucosphingolipids (for review see Ref. 8Grabowski G.A. Berg-Fussman A. Grace M. Esen A. β-Glucosidases: Biochemistry and Molecular Biology. American Chemical Society, Washington, D. C.1993: 66-82Google Scholar), and a cytosolic β-glucosidase capable of hydrolyzing a variety of aryl β-d-glycosides (for review see Ref. 9Glew R.H. Gopalan V. Forsyth G.W. VanderJagt D.J. Esen A. β-Glucosidases: Biochemistry and Molecular Biology. American Chemical Society, Washington, D. C.1993: 83-112Google Scholar). This cytosolic enzyme has been detected in a variety of mammalian tissues and has been designated broad specificity β-glucosidase. Although broad specificity β-glucosidase has been purified from several organs, cloned, sequenced, and intensively studied, the physiological function of this enzyme remains unclear. It has been reported that cytosolic broad specificity β-glucosidase from liver is capable of hydrolyzing certain cyanogenic plant glucosides such as l-picein (10LaMarco K.L. Glew R.H. Biochem. J. 1986; 237: 469-476Crossref PubMed Scopus (26) Google Scholar,11Gopalan V. Pastuszyn A.P. Galey Jr., W.R. Glew R.H. J. Biol. Chem. 1992; 267: 14027-14032Abstract Full Text PDF PubMed Google Scholar). Enzymatic activity capable of hydrolyzing PNG has been found in nonpurified supernatant fractions of small intestinal mucosa and attributed initially to broad specificity β-glucosidase (12Trumbo P.R. Banks M.A. Gregory J.F. Proc. Soc. Exp. Biol. Med. 1990; 195: 240-246Crossref PubMed Scopus (25) Google Scholar). PNG hydrolyzing activity, presumably of microbial origin, also has been detected in small intestinal contents of rodents and may contribute to the in vivo hydrolysis of dietary PNG (13Banks M.A. Porter D.W. Martin W.G. Gregory J.F. J. Nutr. Biochem. 1994; 5: 238-242Crossref Scopus (7) Google Scholar, 14Nakano H. Gregory J.F. J. Nutr. 1995; 125: 2751-2762PubMed Google Scholar), although the role of microbial β-glucosidases in PNG hydrolysis in the human small intestine probably would be less significant. Hydrolysis, rather than intestinal absorption, is a rate-limiting factor for the utilization of PNG (4Ink S.L. Gregory J.F. Sartain D.B. J. Agric. Food Chem. 1986; 34: 857-862Crossref Scopus (38) Google Scholar), and in vivo studies in rats and humans have indicated that much of the hydrolysis of PNG is associated with the intestine (6Gregory III, J.F. Trumbo P.R. Bailey L.B. Toth J.P. Baumgartner T.G. Cerda J.J. J. Nutr. 1991; 121: 177-186Crossref PubMed Scopus (56) Google Scholar, 15Trumbo P.R. Gregory J.F. J. Nutr. 1988; 118: 1336-1342Crossref PubMed Scopus (28) Google Scholar). To obtain a better understanding of the biochemistry of PNG hydrolysis, we initiated purification of broad specificity β-glucosidase from jejunal mucosa of the pig, a monogastric animal physiologically similar to the human. After purification of broad specificity β-glucosidase from porcine intestinal mucosa using procedures of Glew and associates (9Glew R.H. Gopalan V. Forsyth G.W. VanderJagt D.J. Esen A. β-Glucosidases: Biochemistry and Molecular Biology. American Chemical Society, Washington, D. C.1993: 83-112Google Scholar) and DePetro (16.DePetro, J. J., Characterization of the Interaction between a Mammalian Liver β Glucosidase and Amphipathic Organic Compounds. M.Sc. thesis, 1987, University of Pittsburgh.Google Scholar) with p-nitrophenyl-β-d-glucoside as a routine substrate, we observed that the purified enzyme does not catalyze the hydrolysis of PNG nor does PNG inhibit the hydrolysis of this aryl β-d-glucoside. This observation led us to undertake the isolation of the enzyme that is responsible for the hydrolysis of PNG. Using PNG as substrate, we have succeeded in purifying a previously unknown enzyme that we designate pyridoxine-β-d-glucoside hydrolase from porcine jejunal mucosal cytosol. In this paper we report (a) the purification of these two cytosolic β-glucosidases from porcine intestinal mucosa, (b) initial characterization of pyridoxine-β-d-glucoside hydrolase, and (c) comparison of the catalytic properties of these enzymes. Concurrent with the discovery of the distinct pyridoxine-β-d-glucoside hydrolase in porcine intestine, this laboratory has reported that vitamin B6 nutritional status regulates the activity of mucosal cytosolic β-glucosidases (13Banks M.A. Porter D.W. Martin W.G. Gregory J.F. J. Nutr. Biochem. 1994; 5: 238-242Crossref Scopus (7) Google Scholar, 14Nakano H. Gregory J.F. J. Nutr. 1995; 125: 2751-2762PubMed Google Scholar). Vitamin B6 deficiency causes elevation in mucosal activities of both broad specificity β-glucosidase and pyridoxine-β-d-glucoside hydrolase. A long term objective is to determine the mechanism and metabolic consequences of the nutritional regulation of these enzymes. Pyridoxine (PN) hydrochloride,p-nitrophenyl β-d-glucoside (p-NPGlc),p-nitrophenyl(p-NP)-β-d-galactoside,p-NP-β-d-fucoside,p-NP-β-d-xyloside,p-NP-N-acetyl-β-d-glucosaminide,n-octyl-β-d-glucoside,n-amyl-β-d-glucoside, conduritol B epoxide, taurocholic acid, deoxycholic acid, glucono-δ-lactone,N-ethylmaleimide, p-hydroxymercuribenzoic acid, octyl-Sepharose, and protein molecular weight standards were obtained from Sigma. Prestained protein molecular weight standards were obtained from Bio-Rad. DE52 cellulose was obtained from Whatman, and Bio-Gel HTP and Econo-Pac t-Butyl HIC columns were from Bio-Rad; Superdex 200 10/30 gel filtration columns were from Pharmacia Biotech Inc., and the Hydropore AX anion exchange HPLC column was from Rainin Instruments (Woburn, MA). 2-Deoxy-2-fluoro-β-d-glucosyl fluoride was provided by Stephen Withers (University of British Columbia, Vancouver, British Columbia). Pyridoxine 5′-β-d-glucoside was prepared by biological synthesis from pyridoxine using germinating alfalfa seeds and purified chromatographically (15Trumbo P.R. Gregory J.F. J. Nutr. 1988; 118: 1336-1342Crossref PubMed Scopus (28) Google Scholar, 17Gregory J.F. Nakano H. Methods Enzymol. 1997; 280: 58-65Crossref PubMed Scopus (10) Google Scholar). During this purification, all materials were kept on ice or 4 °C. Pig jejunum was obtained immediately following slaughter at the University of Florida Animal Science Department. Sections were cut longitudinally and washed with 0.9% (w/v) NaCl and then stored at −80 °C until use. Frozen intestine strips (approximately 200 g) were allowed to thaw on ice overnight, and mucosa was scraped off using a glass slide, yielding approximately 60 g of mucosa. The mucosa was homogenized in 180 ml of homogenization buffer comprised of 10 mm sodium phosphate, pH 7, containing 10 mm 2-mercaptoethanol and 1 mm phenylmethylsulfonyl fluoride using a Polytron PT 10–35 device (Brinkmann Instruments, Inc., Westbury, NY). The homogenate was centrifuged at 183,000 × g for 30 min at 4 °C, and the pH of the supernatant was adjusted to 6 with 0.2 macetic acid followed by centrifugation at 20,000 × gfor 20 min at 4 °C (14Nakano H. Gregory J.F. J. Nutr. 1995; 125: 2751-2762PubMed Google Scholar, 16.DePetro, J. J., Characterization of the Interaction between a Mammalian Liver β Glucosidase and Amphipathic Organic Compounds. M.Sc. thesis, 1987, University of Pittsburgh.Google Scholar). The supernatant from acid precipitation was applied to a DE52 cellulose column (2.5 × 25 cm) equilibrated with 10 mm sodium phosphate, pH 6. The column was washed with 1 liter of the starting buffer and the enzyme eluted with a linear gradient of 0 to 0.4m NaCl in the same buffer (total volume of 2 liters). The fractions containing activity were pooled, concentrated by ultrafiltration in a stirred-cell apparatus (Amicon Diaflo 10 PM30 membrane, W. R. Grace and Co., Danvers, MA), purified further by chromatography on a Pharmacia Superdex 200 column (10 mm inner diameter × 30 cm), equilibrated with 10 mm sodium phosphate, pH 6, containing 50 mm NaCl. Fractions that contained PNG hydrolase activity were pooled, concentrated by ultrafiltration (Ultrafree-15 centrifugal filter device, Biomax-30K NMWL membrane, 15-ml volume, Millipore Corp., Bedford, MA), and subjected to chromatography on a Rainin Hydropore AX anion exchange column (polyethyleneimine with mixed primary, secondary, and tertiary amino sites, 4.6 mm inner diameter × 25 cm, Rainin Instruments, Woburn, MA) in the same buffer at a flow rate of 1 ml/min using a linear gradient of 0–0.4 m NaCl over 30 min. Fractions that contained activity were again concentrated by ultrafiltration (Ultrafree-15 centrifugal filter device, Biomax-30K NMWL membrane, 15-ml volume, Millipore Corp., Bedford, MA) and then applied to a Bio-Rad Econo-Pac t-Butyl HIC cartridge (Macro-Prep t-Butyl HIC support, 5-ml bed volume, Bio-Rad) equilibrated in 10 mm sodium phosphate, pH 6, with 2.4 mammonium sulfate. Elution was accomplished using a linear gradient from 2.4 to 0 m ammonium sulfate over 30 min at a flow rate of 1 ml/min. Fractions that contained pyridoxine-β-d-glucoside hydrolase activity were pooled and stored as small portions at −80 °C. Initial attempts to purify pyridoxine-β-d-glucoside hydrolase involved application of active fractions from DE52 cellulose to a hydroxyapatite column (2.5 × 18 cm; Bio-Gel HTP, Bio-Rad) equilibrated with 10 mm sodium phosphate, pH 6, without dialysis to remove salt. Nonretained effluent fractions exhibiting activity were pooled and passed through another hydroxyapatite column under the same conditions. Where noted, initial studies investigating factors affecting pyridoxine-β-d-glucoside hydrolase activity were conducted with this highly active, partially purified preparation (275-fold purification; 98.5% reduction in broad specificity β-glucosidase activity). Chromatographic purification of broad specificity β-glucosidase was conducted by a minor modification of the procedures of Glew and associates (9Glew R.H. Gopalan V. Forsyth G.W. VanderJagt D.J. Esen A. β-Glucosidases: Biochemistry and Molecular Biology. American Chemical Society, Washington, D. C.1993: 83-112Google Scholar) and DePetro (16.DePetro, J. J., Characterization of the Interaction between a Mammalian Liver β Glucosidase and Amphipathic Organic Compounds. M.Sc. thesis, 1987, University of Pittsburgh.Google Scholar). The pH 6 supernatant prepared and described above was applied to a DE52 cellulose column (2.5 × 25 cm) previously equilibrated with 10 mmsodium phosphate, pH 6. After the column was washed with 1 liter of the starting buffer, the enzyme was eluted with a linear gradient of 0–0.5m NaCl in the same buffer (total volume of 1 liter). The fractions containing the activity were pooled and applied to a hydroxyapatite column (2.5 × 18 cm) equilibrated with 10 mm sodium phosphate, pH 6, without dialysis to remove salt. The column was washed with five bed volumes of the same buffer, and then the enzyme was eluted with a 500-ml gradient of 10 mmto 0.1 m sodium phosphate, pH 6, followed by 200 ml of 0.1m sodium phosphate, pH 6. The fractions that had activity were collected and applied to an octyl-Sepharose column (1.5 × 25 cm) equilibrated with 10 mm sodium phosphate, pH 6, containing 0.5 m(NH4)2SO4. The column was washed with 200 ml of the same buffer and then the enzyme was eluted with 10 mm sodium phosphate, pH 6, containing 60% (v/v) ethylene glycol. The fractions containing the activity were pooled and stored as small portions at −20 °C until further analysis. The standard assays for broad specificity β-glucosidase and pyridoxine-5′-β-d-glucoside hydrolase were performed according to Nakano and Gregory (14Nakano H. Gregory J.F. J. Nutr. 1995; 125: 2751-2762PubMed Google Scholar, 17Gregory J.F. Nakano H. Methods Enzymol. 1997; 280: 58-65Crossref PubMed Scopus (10) Google Scholar). The activity of broad specificity β-glucosidase was determined with 2 mm p-NPGlc and pyridoxine-5′-β-d-glucoside hydrolase with 0.2 mm PNG and 0.2 m sodium acetate buffer, pH 6.0. All incubations were conducted at 37 °C under conditions that allowed measurement of initial rate. One unit of broad specificity β-glucosidase activity was defined as 1 nmol of p-nitrophenol released from the substrate per hour, using the molar absorptivity coefficient at 400 nm (18,300m−1 cm−1; 18). One unit of pyridoxine-5′-β-d-glucoside hydrolase activity was defined as the release of 1 nmol PN from PNG per h. For the pH dependence studies, reactions were conducted using 0.2m sodium acetate buffer at pH 4.5–6.0, 0.1 msodium phosphate buffer at pH 6.5–7.5, and 0.1 m Tris-HCl buffer at pH 8.0–9.0. For kinetic studies, standard substrates were replaced by various alternate β-d-glycosides at concentrations specified in the text and tables. Alternatively, a variety of concentrations of potential inhibitors or stimulating compounds as specified were added to the standard mixtures without preincubation of the enzyme. Purified PNG hydrolase was evaluated for disaccharidase activity using 1 mm lactose, cellobiose, or sucrose individually as substrate. Incubations were conducted at 37 °C and then stopped by incubating for 3 min in boiling water. Parallel blank reactions were conducted without enzyme added. Samples were analyzed for the residual disaccharides and formation of monosaccharide products by liquid chromatography (19Rocklin R.D. Pohl C.A. J. Liq. Chromatogr. 1983; 6: 1577-1590Crossref Scopus (402) Google Scholar) using a Dionex DX500 HPLC system and Dionex ED400 electrochemical detection system with pulsed amperometric detector (Dionex Corp., Sunnyvale, CA). Separations were performed using a Dionex CarboPac PA10 column eluted isocratically with 14 mmNaOH for 22 min at 1 ml/min and an additional 18 min at 100 mm NaOH. Retention times were determined and calibration curves established with authentic standards. Protein concentration during chromatography was monitored by absorbance at 280 nm. Determination of protein concentration in the isolation of broad specificity β-glucosidase was determined according to Bradford (20Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214435) Google Scholar), and the method of Markwell et al. (21Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5307) Google Scholar) was used in the isolation of PNG hydrolase for its lower detection limits. Bovine serum albumin was the standard in each procedure. SDS-polyacrylamide gel electrophoresis was performed in 5% (w/v) polyacrylamide gels according to Laemmli (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206620) Google Scholar). The molecular weight standards used were obtained in prestained form from Bio-Rad, which included myosin heavy chain (213 kDa), β-galactosidase (119 kDa), bovine serum albumin (83 kDa), and ovalbumin (47 kDa). The gels were stained with Coomassie Blue R-250. Isoelectric focusing was performed in 5% (w/v) polyacrylamide gels as described by Garfin (23Garfin D.E. Methods Enzymol. 1990; 182: 459-477Crossref PubMed Scopus (60) Google Scholar). Standard proteins (Sigma) used were soybean trypsin inhibitor (pI 4.6), bovine β-lactoglobulin A (pI 5.1), bovine carbonic anhydrase II (pI 5.9), and human carbonic anhydrase I (pI 6.6). pH gradients were prepared using a 60:40 (v/v) mixture of pH 3–10 and pH 2.5–5 ampholyte solutions (Pharmacia Biotech Inc.). In a parallel gel that was not fixed or stained, gel slices (3 mm) of the PNG hydrolase lane were assayed for enzymatic activity. In this procedure the gel segments were incubated in 0.2 m sodium acetate buffer, pH 5.5, for 10 h and then incubated with 125 μm PNG at 37 °C for 3 h. Gel filtration chromatography was performed as described above using Pharmacia Superdex 200 column for determination of molecular mass under nondenaturing conditions. Molecular weight standards (Sigma) were cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), and β-amylase (200 kDa). The absorbance of the column effluent was monitored at 280 nm. The purified enzyme preparations were subjected to SDS-polyacrylamide gel electrophoresis in a Tris-Tricine discontinuous buffer system (24Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10457) Google Scholar) and then electrophoretically blotted to polyvinylidene difluoride membranes and stained with Coomassie Blue R-250 (25Ploug M. Jensen A.L. Barkholt V. Anal. Biochem. 1989; 181: 33-39Crossref PubMed Scopus (147) Google Scholar). The band corresponding to each enzyme was excised and subjected to gas-phase acid hydrolysis (26Ozols J. Methods Enzymol. 1990; 182: 587-601Crossref PubMed Scopus (184) Google Scholar) followed by amino acid analysis as the phenylisothiocarbamate derivative using an Applied Biosystems 420A instrument (Foster City, CA). Kinetic constants (K m, V max, and K i) were calculated by nonlinear regression using EZ-FIT software (27Perrella F.W. Anal. Biochem. 1988; 174: 1983-1989Crossref Scopus (280) Google Scholar). The separation of pyridoxine-β-d-glucoside hydrolase and broad specificity β-glucosidase activities by ion exchange on DE52 cellulose provided clear evidence of the existence of a distinct enzyme able to hydrolyze PNG (Fig. 1). The subsequently developed purification scheme of four consecutive chromatographic steps is shown in Fig. 2, and a summary of the purification of pyridoxine-β-d-glucoside hydrolase is shown in Table I. HPLC gel filtration and ion exchange steps each yielded further purification, and the final hydrophobic interaction chromatography step yielded an essentially pure enzyme with 36,500-fold purification relative to the supernatant from acid precipitation. This procedure has been repeated several times with consistent results.Figure 2Chromatographic purification of pyridoxine-β-d-glucoside hydrolase. A, anion exchange chromatography on DE52 cellulose. Mucosal cytosolic fraction following acid precipitation was applied to a DE52 diethylaminoethyl cellulose column. Protein concentration was monitored as absorbance at 280 nm. Elution was accomplished using a 0–0.4 m NaCl gradient in 10 mm sodium phosphate buffer, pH 6, following a 1-liter wash. Fractions (15 ml) were collected throughout the NaCl gradient only. B, gel filtration chromatography on Superdex 200. Fractions with activity from the previous column were concentrated and then separated (750-μl injections) at 0.5 ml/min flow rate and 1 ml/fraction. Absorbance (solid line) was measured continuously at 280 nm. C, anion exchange HPLC on Hydropore AX column. Fractions with activity from the previous column were concentrated and then separated (750-μl injections) at 1 ml/min with a gradient of 0–0.4 m NaCl in 10 mm sodium phosphate buffer, pH 6, in 30 min. Absorbance (solid line) was measured continuously at 280 nm. Fractions of 1 min/tube were collected. D, hydrophobic interaction chromatography on Econo-Pac t-Butyl HIC column. Fractions with activity from the previous column were concentrated and then separated (100-μl injections) at 1 ml/min using a linear gradient from 2.4 to 0m ammonium sulfate over 60 min in 10 mm sodium phosphate, pH 6. The column was initially equilibrated in this buffer containing 2.4 m ammonium sulfate. Absorbance (solid line) was measured continuously at 280 nm. Fractions of 1 min/tube were collected.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IPurification of broad specificity β-glucosidase from pig intestinal mucosaFractionFraction volumeTotal proteinTotal activitySpecific activityYieldPurificationmlmgunitsunits/mg%-foldSupernatant from acid precipitation112613223,0003631001DE52 cellulose eluent10554.9242,000440010912Hydroxyapatite eluent880.74132,000178,00059490Octyl-Sepharose eluent320.19103,000530,000461460 Open table in a new tab Although PNG hydrolase activity is quite stable in frozen intestine, the fully purified enzyme exhibits much less stability. Only about 25% retention of activity is observed following a single cycle of frozen storage at −80 °C (2 days) followed by thawing and assay. In contrast, the purified enzyme loses about 15% activity per day at 4 °C. A summary of the purification is shown in Table II. The enzyme was purified ∼1,460-fold relative to the supernatant from acid precipitation.Table IIPurification of pyridoxine-β-d-glucoside hydrolase from pig intestinal mucosaFractionFraction volumeTotal proteinTotal activitySpecific activityYieldPurificationmlmgunitsunits/mg%-foldSupernatant from acid precipitation8295044104.641001DE52 cellulose eluent206222337015.2773Hydroxyapatite effluent 1543.4249073656159Hydroxyapatite effluent 2581.51940127544275 Open table in a new tab The enzyme activity was stable in 10 mm sodium phosphate, pH 6, containing 60% (v/v) ethylene glycol at −20 or −80 °C for over 2 months. The activity toward p-NPGlc did not change in the presence of 0.1% (w/v) taurocholic acid with or without 0.05% (v/v) Triton X-100 in the assay medium. SDS-polyacrylamide gel electrophoresis analysis indicated that broad specificity β-glucosidase and pyridoxine-β-d-glucoside hydrolase had been purified to homogeneity (Fig.3). The apparent molecular mass under denaturing conditions as determined by SDS-polyacrylamide gel electrophoresis was 60 kDa for broad specificity β-glucosidase and 130 kDa for pyridoxine-β-d-glucoside hydrolase. Determination of native molecular mass by gel filtration chromatography purification indicated 57 kDa for broad specificity β-glucosidase and 160 kDa for pyridoxine-β-d-glucoside hydrolase (Fig.4). The isoelectric point of broad specificity β-glucosidase was 4.8, and pyridoxine-β-d-glucoside hydrolase exhibited a cluster of at least 4 bands all with pI ≤ 4.8 (Fig.5). Direct assay of gel slices for PNG hydrolase activity revealed activity across this cluster, which confirmed the heterogeneity of charged species in this preparation.Figure 4Determination of molecular weight by gel filtration. The calibration curve for the estimation of molecular weight using Superdex 200 chromatography. Standard proteins used were cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), and β-amylase (200 kDa). The positions of broad specificity β-glucosidase and pyridoxine-β-d-glucoside hydrolase are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Determination of isoelectric point by isoelectric focusing. Lane 1, human carbonic anhydrase I standard (pI 6.6); lane 2, bovine carbonic anhydrase II standard (pI 5.9); lane 3, bovine β-lactoglobulin A standard (pI 5.1); lane 4, soybean trypsin inhibitor standard (pI 4.6); lanes 5 and 6, purified pyridoxine-β-d-glucoside hydrolase; lane 7, purified broad specificity β-glucosidase. The gel was stained with Coomassie Blue. pI values for the standards are indicated on the left of the gel. Relative pyridoxine-β-d-glucoside hydrolase activity of gel slices (3 mm each, shown by brackets, determined in a replicate gel) is shown by + symbols. Where not designated, the activity was not detected.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Broad specificity β-glucosidase showed a pH optimum of approximately 6.5, and pyridoxine-β-d-glucoside hydrolase exhibited a pH optimum of approximately 5.5 (Fig. 6). The results presented in TableIII indicate similarity but also substantial compositional differences between these two enzymes. Most notably, broad specificity β-glucosidase exhibited ∼2-fold greater proline and ∼30% greater serine, whereas pyridoxine-β-d-glucoside hydrolase exhibited ∼80% greater lysine and over 40% greater alanine content. Both enzymes were high in glycine (∼12 mol%) and Asx + Glx (18–21 mol%).Table IIIAmino acid composition of purified pyridoxine-β-d-gl
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