Hydrolysis of Pyridoxine-5'- -d-Glucoside by a Broad-Specificity  -Glucosidase from Mammalian Tissues

Trumbo, Paula R.; Banks, Melanie A.; Gregory, Jesse F.

doi:10.3181/00379727-195-43142

Cited by 28 publications

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“…The specific activity of pyridoxine-␤-D-glucoside hydrolase in human jejunum is approximately 5.8-fold greater than that of rat intestine (12). This difference is consistent with the greater in vivo bioavailability of orally administered PNG in humans than rats.…”

Section: Figsupporting

confidence: 69%

“…We have shown in the present study that pyridoxine 5Ј-␤-D-glucoside is hydrolyzed by a specific enzyme present in pig intestinal mucosal cytosol but not by the cytosolic broad specificity ␤-glucosidase. We have also shown that pyridoxine-␤-D-glucoside hydrolase activity exists in rat, guinea pig, and human intestinal mucosal cytosolic fractions (12,54). As shown in the present study, our previous assumption that the intestinal cytosolic broad specificity ␤-glucosidase was solely responsible for hydrolysis of PNG in rat, guinea pig, and human intestine (12) is incorrect.…”

Section: Figcontrasting

confidence: 48%

“…It has been reported that cytosolic broad specificity ␤-glucosidase from liver is capable of hydrolyzing certain cyanogenic plant glucosides such as L-picein (10,11). Enzymatic activity capable of hydrolyzing PNG has been found in nonpurified supernatant fractions of small intestinal mucosa and attributed initially to broad specificity ␤-glucosidase (12). PNG hydrolyzing activity, presumably of microbial origin, also has been detected in small intestinal contents of rodents and may contribute to the in vivo hydrolysis of dietary PNG (13,14), although the role of microbial ␤-glucosidases in PNG hydrolysis in the human small intestine probably would be less significant.…”

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Cytosolic Pyridoxine-β-d-Glucoside Hydrolase from Porcine Jejunal Mucosa

McMahon

Nakano

Levy

et al. 1997

Journal of Biological Chemistry

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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 ␤-Dglucoside. 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.A naturally occurring glycosylated derivative of vitamin B6, pyridoxine 5Ј-␤-D-glucoside (PNG), 1 was first isolated from rice bran (1). PNG is now known to exist in most fruits, vegetables, and cereal grains, in which it comprises from 5-75% of total vitamin B6 (2, 3). Because of the prevalence of PNG in plantderived foods, its bioavailability as a source of available vitamin B6 is a matter of nutritional concern. The bioavailability of this glycosylated form of vitamin B6, relative to pyridoxine (PN), is ϳ25% in rats (4, 5) and ϳ50% for humans (6, 7), as estimated from urinary excretion of 4-pyridoxic acid after oral administration of isotopically labeled PNG. The rate-limiting phase of PNG utilization in vitamin B6 metabolism is the hydrolysis of the ␤-glycosidic bond in both rats and humans. These findings indicate that the hydrolysis of PNG is a major factor governing its bioavailability.Glucosidases exist widely in nature. The primary ␤-glucosidases in mammalian tissues consist of a lysosomal membranebound acid ␤-glucosidase (EC 3.2.1.45; N-acylsphingosyl-␤-Dglucopyranoside glucohydrolase) which is responsible for the hydrolytic cleavage of glucosphingolipids (for review see Ref. 8), and a cytosolic ␤-glucosidase capable of hydrolyzing a variety of aryl ␤-D-glycosides (for review see Ref. 9). This cytosolic enzyme has been detected in a variety of mammalian tissues and has been designated broad specificity ␤-glucosidase. A...

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Section: Figsupporting

confidence: 69%

Section: Figcontrasting

confidence: 48%

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Cytosolic Pyridoxine-β-d-Glucoside Hydrolase from Porcine Jejunal Mucosa

McMahon

Nakano

Levy

et al. 1997

Journal of Biological Chemistry

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“…Although the intestinal B-glucosidase responsible for in vivo hydrolysis of PN-5'-B-D-glucoside has not been isolated and characterized, this enzyme has been shown to be highly similar to the cytosolic broad specificity B-glucosidase in other mammalian tissues (50). The intestinal B-glucosidase activity capable of hydrolyzing PN-5'-B-D-glucoside was mainly cytosolic, optimally active at pH 6, and was inhibited by sodium taurocholate.…”

Section: Nutritional Properties Of Vitamin B6-b-glucosidesmentioning

confidence: 98%

Nutritional Properties of Pyridoxine-β-D-Glucosides

Gregory

1993

ACS Symposium Series

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Several vitamin B6 derivatives, especially pyridoxine, exist in plants as ß-D-glucosides. The major form of glycosylated vitamin B6 is pyridoxine-5'-ß-D-glucoside (PNG) , which comprises between 5 and 80% of the total vitamin B6 in various plants. Isotopic studies have indicated that dietary PNG exhibits ~25% bioavailablity in rats and ~58% in humans, relative to simultaneously ingested pyridoxine. The rate limiting phase of PNG utilization in B6 metabolism is the action of mammalian or microbial ß -glucosidases. In view of this incomplete bioavailability, methods of food analysis should permit measurement of both glycosylated and nonglycosylated species.The generic term vitamin B6 refers to the group of 2-methyl,3-hydroxy,5-hydroxymethyl-pyridines that exhibit the biological activity of pyridoxine. These compounds also exist as 5'-phosphate esters and B-glucosides, as discussed below. Vitamin B6 functions coenzymically as pyridoxal 5'-phosphate in over 100 enzymic reactions involving the metabolism of amino acids, glycogen, lipids, and neurotransmitters. In addition, vitamin B6 functions in mamtaining the flux of substrate to energy metabolism during periods of caloric deprivation through glycogenolysis and amino acid interconversion and catabolism. Therefore, an adequate intake of vitamin B6 is essential for health, growth, cellular homeostasis, and many aspects of physiological function.B-Glucosidic conjugates of pyridoxine exist in many plants but are insignificant animal tissues and animal-derived food products. These compounds comprise a significant percentage of the total dietary vitamin B6 intake. Glycosylated vitamin B6 in common foods is largely comprised of pyridoxine-5'-B-D-glucoside (PN-5'-B-D-glucoside). Mammalian and, possibly, bacterial Bglucosidases release free pyridoxine (PN) which is then available for metabolic utilization. The extent of nutritional bioavailability of vitamin B6 from these conjugated forms depends directly on the extent of enzymatic hydrolysis. In addition, recent findings indicate that the fraction of PN-5'-B-D-glucoside that does not undergo hydrolysis can influence vitamin B6 metabolism by competitively inhibiting the cellular uptake and overall utilization of other nonglycosylated forms of the vitamin. (Abbreviations: pyridoxine-5'-B-D-0097-6156/93/0533-0113S06.00/0

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“…cytosolic) hydrolysis of PNG in mammalian small intestinal mucosa. Intestinal cytosolic PNG hydrolysis was initially thought to be catalyzed by a broad specificity ␤-glucosidase (BS␤G) (EC 3.2.1.21) (11), a cytosolic enzyme found in the intestine and liver (12)(13)(14). Cytosolic PNG hydrolysis was also reported to be inversely related to vitamin B 6 nutritional status in rodents (13,14).…”

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Enzymatic Hydrolysis of Pyridoxine-5′-β-d-glucoside Is Catalyzed by Intestinal Lactase-Phlorizin Hydrolase

Mackey¹,

Henderson²,

Gregory³

2002

Journal of Biological Chemistry

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An obligatory step in the mammalian nutritional utilization of pyridoxine-5-␤-D-glucoside (PNG) is the intestinal hydrolysis of its ␤-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 ␤-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 mol/min⅐mg 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-␤-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.A significant dietary source of vitamin B 6 for humans is provided by a glycosylated form of the vitamin, pyridoxine-5Ј-␤-D-glucoside (PNG).1 PNG, found predominantly in foods of plant origin, provides 15% of total vitamin B 6 in a mixed diet, depending on food selection (1, 2). PNG exhibits ϳ50% bioavailability in humans (3, 4) and 25-30% in rodents (5-8) in comparison to pyridoxine, the metabolically usable form of vitamin B 6 . The rate-limiting step in the utilization of PNG is not its intestinal absorption but rather the enzymatic hydrolysis of the ␤-glucosidic linkage to release glucose and pyridoxine in the intestine (6 -8). Although PNG can be absorbed intact, this form is not metabolized or retained by the liver (6 -8) and can antagonize the metabolism of nonglycosylated forms of vitamin B 6 (9, 10). The remainder of PNG that is not absorbed is likely to be accounted for through fecal losses.This laboratory has examined the intracellular (i.e. cytosolic) hydrolysis of PNG in mammalian small intestinal mucosa. Intestinal cytosolic PNG hydrolysis was initially thought to be catalyzed by a broad specificity ␤-glucosidase (BS␤G) (EC 3.2.1.21) (11), a cytosolic enzyme found in the intestine and liver (12-14). Cytosolic PNG hydrolysis was also reported to be inversely related to vitamin B 6 nutritional status in rodents (13,14). To further study cytosolic PNG hydrolysis, this laboratory purified BS␤G from pig intestinal mucosa, which led to the identification and subsequent purification of a distinct and novel cytosolic ␤-glucosidase, designated pyridoxine-5Ј-␤-D-glucoside hydrolase (PNG hydrolas...

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Hydrolysis of Pyridoxine-5'- -d-Glucoside by a Broad-Specificity -Glucosidase from Mammalian Tissues

Cited by 28 publications

References 0 publications

Cytosolic Pyridoxine-β-d-Glucoside Hydrolase from Porcine Jejunal Mucosa

Cytosolic Pyridoxine-β-d-Glucoside Hydrolase from Porcine Jejunal Mucosa

Nutritional Properties of Pyridoxine-β-D-Glucosides

Enzymatic Hydrolysis of Pyridoxine-5′-β-d-glucoside Is Catalyzed by Intestinal Lactase-Phlorizin Hydrolase

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