title: Enzymatic hydrolysis of pyridoxine-5'-beta-D-glucoside is catalyzed by intestinal lactase-phlorizin hydrolase.
abstract: An obligatory step in the mammalian nutritional utilization of pyridoxine-5'-beta-D-glucoside (PNG) is the intestinal hydrolysis of its beta-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 beta-glucosidase in the hydrolysis of PNG, lactase phlorizin hydrolase (LPH) was purified from rat small intestinal mucosa. LPH hydrolyzed PNG with a K(m) of 1.0 +/- 0.1 mm, a V(max) of 0.11 +/- 0.01 micromol/min.mg protein, and a k(cat) 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-beta-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.
authors.affiliation: Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Amy D
authors.initials: AD
authors.lastname: Mackey
authors.forename: George N
authors.initials: GN
authors.lastname: Henderson
authors.forename: Jesse F
authors.initials: JF
authors.lastname: Gregory
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000378:metabolism; D007421:Intestine, Small / Q000378:metabolism; D007700:Kinetics; D010694:Lactase-Phlorizin Hydrolase / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000378:metabolism; D011736:Pyridoxine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013997:Time Factors
chemical_list: D005960:Glucosides; D008070:Lipopolysaccharides; C022945:5'-O-(glucopyranosyl)pyridoxine; D010694:Lactase-Phlorizin Hydrolase; D011736:Pyridoxine