The preparation of lactase and glucoamylase of rat small intestine

Schlegel‐Haueter, Susanna E.; Hore, P.; Kerry, Knowles; Semenza, Giorgio

doi:10.1016/0005-2744(72)90242-2

Cited by 124 publications

(45 citation statements)

References 31 publications

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“…This recognition was sequence-specific, since the pre-immune serum and pep- tide-competed antibody did not detect the high molecular mass bands that were recognized by the whole immune serum. Values of K m and V max (lactose) calculated for purified rat LPH were consistent with published values (27,34) (Table I). The kinetic parameters (for lactose and PNG) appeared to be representative of the concentrations of substrates that would be encountered physiologically by LPH.…”

Section: Discussionsupporting

confidence: 87%

“…When LPH is inhibited completely with this fluoroglucoside, the release of PN is negligible, indicating that maltase-glucoamylase alone is not capable of hydrolyzing PNG. Maltase-glucoamylase that had been co-purified with lactase by papain solubilization from 15-day-old rat intestine was reported to have a final specific activity of 36 mol/min⅐mg protein in another study (34), which is ϳ10ϫ the specific activity measured in the current study.…”

Section: Discussionsupporting

confidence: 61%

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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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Section: Discussionsupporting

confidence: 87%

Section: Discussionsupporting

confidence: 61%

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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“…The enzyme sediments faster (Eggermont & Hers, 1969) and is more heat-stable (Auricchioetal., 1965)than the other disaccharidases. The enzyme has been partially purified from the intestine of the monkey (Seetharam et al, 1970), suckling rat (Schlegel-Haueter et al, 1972), adult rat (Kolinska & Kraml, 1972), human (Kelly & Alpers, 1973) and rabbit (Sivakami & Radhakrishnan, 1973), and kinetic studies have been performed by Kelly & Alpers (1973) and by Sivakami & Radhakrishnan (1976). It is a glycoprotein (Forstner, 1971;Kelly & Alpers, 1973;Forstner & Galand, 1976) and appears to be the largest component of the brushborder membrane from rat (Galand & Forstner, 1974b;Forstner & Galand, 1976), hamster (Critchley et al, 1975) and human (Maestracci et al, 1975) intestine.…”

mentioning

confidence: 99%

Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH

Flanagan¹,

Forstner²

1978

Biochemical Journal

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Maltase-glucoamylase, a microvillous membrane ectoenzyme, was solubilized from rat intestinal mucosa by digestion with papain and subsequently purified to homogeneity with an overall yield of 10–20%. An antibody to the purified enzyme formed a single precipitin line in immunodiffusion experiments with an intestinal homogenate. The enzyme was shown to be an acidic glycoprotein (20% sugar by weight) which contained low amounts of cysteine and no sialic acid. At pH3–6, maltase activity was slowly lost, but the enzyme was re-activated by re-adjustment of the pH to neutrality. However, in the presence of sodium dodecyl sulphate, acid pH values inactivated maltase irreversibly, and at the same time converted the enzyme (mol.wt. 500000 approx.) into five new species with apparent molecular weights ranging from 134000 to 480000 as judged by polyacrylamide-gel electrophoresis. The same five fragments were also formed by boiling the enzyme for brief periods in the presence of sodium dodecyl sulphate or urea either with or without reducing agents. The dissociated species were stable on re-electrophoresis, and amino acid analysis showed them to be very similar to each other and to the original enzyme. The bands migrated anomalously on polyacrylamide gels of different concentration, thereby preventing the assignment of precise molecular weights. It is possible that the five species may represent stable aggregates of a common monomer of the enzyme.

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“…Lactase is detected in early stages of development and gradually increases until term. In other species, it is only in late gestation that lactase activity rapidly increases to newborn levels (Alvarez & Sas, 1961;Dahlqvist & Lindberg, 1966 (Dahlqvist, 1961;Kraml et al, 1972;Schlegel-Haueter et al, 1972;Birkenmeier & Alpers, 1974 (Roberts, 1972) (Timiras, 1972). Disaccharidase activities in fetal sheep meconium were 55-to 190-fold lower than in jejunal mucosa, indicating that these enzymes are considerably degraded, presumably by pancreatic proteases present in meconium (Eggermont, 1966), and/or that large amounts of protein from swallowed amniotic fluid have been incorporated into meconium.…”

Section: Discussionmentioning

confidence: 99%

“…In mammals, this enzyme is really a hetero-ß-glycosidase since it also exerts its hydrolytic activity on cellobiose and on 4-methylumbelliferyl (or nitrophenyl) derivatives of ß-galactose and ß-glucose (Dahlqvist, 1961;Kraml, Kolinska, Ellenderova & Hirsova, 1972;Schlegel-Haueter, Hore, Kerry & Semenza, 1972;Birkenmeier & Alpers, 1974 …”

Section: Introductionmentioning

confidence: 99%

Origin and developmental patterns of lactase and other glycosidases in sheep amniotic and allantoic fluid

Potier¹,

Guay²,

Lamothe³

et al. 1979

Reproduction

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Summary. Intestinal lactase activity (with its associated cellobiase, 4\ x=r eq-\ methylumbelliferyl-\g=b\-galactosidase and -\g=b\-glucosidaseactivities) was used as a specific intestinal marker enzyme to study the release of protein and enzymes of intestinal origin in sheep amniotic fluid during gestation. In amniotic fluid, intestinal lactase activity peaked at 66\p=n-\85days of gestation and then decreased with gestation. This enzyme activity was very low or absent in allantoic fluid throughout gestation suggesting that there is no important transfer of amniotic fluid lactase towards the allantoic cavity. Maltase and 4-methylumbelliferyl-\g=a\-glucosidase showed no statistically significant variation with gestation in both amniotic and allantoic fluid whereas \g=a\-galactosidase and N-acetyl-\g=b\-hexosaminidase which were first higher in allantoic than in amniotic fluid increased in amniotic fluid to reach allantoic fluid levels near term. Such patterns are consistent with the suggestion that the fetal urine is a source of \g=a\-galactosidase and N-acetyl-\g=b\-hexosaminidase activities and that sheep urine is first accumulated in the allantoic sac via the urachus up to 86\p=n-\90 days of gestation and thereafter passes more and more into the amniotic sac.

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The preparation of lactase and glucoamylase of rat small intestine

Cited by 124 publications

References 31 publications

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

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

Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH

Origin and developmental patterns of lactase and other glycosidases in sheep amniotic and allantoic fluid

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