Abstract
During studies of the nutritional utilization of pyridoxine 5'-beta-D-glucoside, a major form of vitamin B6 in plants, we detected two cytosolic beta-glucosidases in jejunal mucosa. As expected, one was broad specificity beta-glucosidase that hydrolyzed aryl beta-D-glycosides but not pyridoxine beta-D-glucoside. We also found a previously unknown enzyme, designated pyridoxine-beta-D-glucoside hydrolase, that efficiently hydrolyzed pyridoxine beta-D-glucoside. These were separated and purified as follows: broad specificity beta-glucosidase 1460-fold and pyridoxine-beta-D-glucoside hydrolase 36,500-fold. Purified pyridoxine-beta-D-glucoside hydrolase did not hydrolyze any of the aryl glycosides tested but did hydrolyze cellobiose and lactose. Pyridoxine-beta-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 beta-glucosidase. Glucono-delta-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 beta-glucosidase, inhibited pyridoxine-beta-D-glucoside hydrolase but not broad specificity beta-glucosidase, but both were inhibited by the mechanism-based inhibitor 2-deoxy-2-fluoro-beta-D-glucosyl fluoride. Our findings indicate major differences between these two cytosolic beta-glucosidases. Studies addressing the role of vitamin B6 nutrition in regulating the activity and its consequences regarding pyridoxine glucoside bioavailability are in progress.
Highlights
During studies of the nutritional utilization of pyridoxine 5--D-glucoside, a major form of vitamin B6 in plants, we detected two cytosolic -glucosidases in jejunal mucosa
This observation led us to undertake the isolation of the enzyme that is responsible for the hydrolysis of pyridoxine 5Ј--D-glucoside (PNG)
Because we did not anticipate the existence of a novel enzyme that is responsible for the hydrolysis of PNG, we initially attempted a purification of broad specificity -glucosidase from the cytosolic fraction of jejunal mucosa and monitored the purification using a conventional assay with a nonphysiological substrate often used for this purpose
Summary
Materials—Pyridoxine (PN) hydrochloride, p-nitrophenyl -D-glucoside (p-NPGlc), p-nitrophenyl(p-NP)--D-galactoside, p-NP--D-fucoside, p-NP--D-xyloside, p-NP-N-acetyl--D-glucosaminide, n-octyl--Dglucoside, n-amyl--D-glucoside, conduritol B epoxide, taurocholic acid, deoxycholic acid, glucono-␦-lactone, N-ethylmaleimide, p-hydroxymercuribenzoic acid, octyl-Sepharose, and protein molecular weight standards were obtained from Sigma. R. Grace and Co., Danvers, MA), purified further by chromatography on a Pharmacia Superdex 200 column (10 mm inner diameter ϫ 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 ϫ 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. Kinetic Analysis—Kinetic constants (Km, Vmax, and Ki) were calculated by nonlinear regression using EZ-FIT software [27]
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