Cytosolic pyridoxine-beta-D-glucoside hydrolase from porcine jejunal mucosa. Purification, properties, and comparison with broad specificity beta-glucosidase.
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.
McMahon
L G
LG
Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611-0370, USA.
Nakano
H
H
Levy
M D
MD
Gregory
J F
JF
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000201:enzymology; D007583:Jejunum / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D006026:Glycoside Hydrolases; C107260:pyridoxine-5'-glucoside hydrolase; D001617:beta-Glucosidase