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Volume 272, Number 51, Issue of December 19, 1997 pp. 32025-32033

Cytosolic Pyridoxine--D-Glucoside Hydrolase from Porcine Jejunal Mucosa
PURIFICATION, PROPERTIES, AND COMPARISON WITH BROAD SPECIFICITY -GLUCOSIDASE

(Received for publication, August 5, 1997)

From the Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611-0370

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. As expected, one was broad specificity -glucosidase that hydrolyzed aryl -D-glycosides but not pyridoxine -D-glucoside. We also found a previously unknown enzyme, designated pyridoxine--D-glucoside hydrolase, that efficiently hydrolyzed pyridoxine -D-glucoside. These were separated and purified as follows: broad specificity -glucosidase 1460-fold and pyridoxine--D-glucoside hydrolase 36,500-fold. Purified pyridoxine--D-glucoside hydrolase did not hydrolyze any of the aryl glycosides tested but did hydrolyze cellobiose and lactose. Pyridoxine--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 -glucosidase. Glucono--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 -glucosidase, inhibited pyridoxine--D-glucoside hydrolase but not broad specificity -glucosidase, but both were inhibited by the mechanism-based inhibitor 2-deoxy-2-fluoro--D-glucosyl fluoride. Our findings indicate major differences between these two cytosolic -glucosidases. Studies addressing the role of vitamin B6 nutrition in regulating the activity and its consequences regarding pyridoxine glucoside bioavailability are in progress.

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