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 cytos olic beta-glucosidases in jejunal mucosa. As expected, one was broad s pecificity beta-glucosidase that hydrolyzed aryl beta-D-glycosides but not pyridoxine beta-D-glucoside. We also found a previously unknown e nzyme, designated pyridoxine-beta-D-glucoside hydrolase, that efficien tly hydrolyzed pyridoxine beta-D-glucoside. These were separated and p urified as follows: broad specificity beta-glucosidase 1460-fold and p yridoxine-beta-D-glucoside hydrolase 36,500-fold, Purified pyridoxine- beta-D-glucoside hydrolase did not hydrolyze any of the aryl glycoside s tested but did hydrolyze cellobiose and lactose. Pyridoxine-beta-D-g lucoside hydrolase exhibited a pH optimum of 5.5 and apparent molecula r mass of 130 kDa by SDS-polyacrylamide gel electrophoresis and 160 kD a by nondenaturing gel filtration, in contrast to 60 kDa for native an d 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 inhibi tor of lysosomal acid beta-glucosidase, inhibited pyridoxine-beta-D-gl ucoside hydrolase but not broad spec- ificity beta-glucosidase, but bo th were inhibited by the mechanism-based inhibitor 2-deoxy-2-fluoro-be ta-D-glucosyl fluoride. Our findings indicate major differences betwee n these two cytosolic beta-glucosidases. Studies addressing the role o f vitamin B6 nutrition in regulating the activity and its consequences regarding pyridoxine glucoside bioavailability are in progress.