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Query: EC:3.2.1.20 (alpha-glucosidase)
 4,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The recent findings that alpha-glucosidase from human kidney was identical with one component (F1) of the alpha-glucosidases found in human urine suggested the idea that this enzyme might originate in the kidneys. The present study was performed to test this idea by immunological methods. Urine alpha-glucosidase F1 was isolated in the electrophoretically homogeneous state, and the antibody prepared in rabbits was purified by affinity chromatography after the antisera were fractionally precipitated with ammonium sulfate and chromatographed on diethylamino ethyl (DEAE)-cellulose. The staining of human kidney tissue sections was performed by the indirect method, using alpha-glucosidase F1 antibody and fluorescein-conjugated anti-rabbit immunoglobulin goat sera. The proximal convoluted portion (proximal tubules) with brush border and Henle's loops (late proximal) were stained clearly. Preincubation of intact antibody with purified antigen prevented specific staining of the proximal convoluted portion and Henle's loops. In contrast, all other tissues of kidney were stained less positively or negatively. These results indicate that alpha-glucosidase F1 originates in the kidney, and that glucosidase is specifically localized in the proximal convoluted portion and Henle's loops.
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PMID:On the origin of alpha-glucosidase in human urine. 618 15

Six glycoside hydrolases in the culture medium of Bacteroides fragilis--alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-L-fucosidase-were systematically purified by ammonium sulfate precipitation, gel filtration chromatography, and density gradient isoelectric focusing. The isoelectric focusing resolved the glycosidases into distinct, well-separated fractions and revealed three differently charged forms of beta-N-acetylglucosaminidase and of alpha-L-fucosidase. Furthermore, alpha-glucosidase and beta-N-acetylglucosaminidase were shown to possess dual affinities for the respective galactoside substrates, and beta-galactosidase also hydrolyzed beta-D-fucoside. alpha-Glucosidase was purified to homogeneity, as indicated by a thin-layer isoelectric focusing zymogram technique. The glycosidases, with exception of beta-glucosidase and the acid alpha-L-fucosidase, were each separated from other glycosidic activities to 99%. The molecular weights varied between 58,000 and 125,000. The pH optima ranged from 4.8 to 6.9.
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PMID:Purification of glycoside hydrolases from Bacteroides fragilis. 625 Apr 77

Mechanisms of the adverse effects of dietary Tween 20, Tween 60, Span 20, sodium taurocholate (NaTC), sodium deoxycholate (DOC), sodium laurylbenzene sulfonate (LBS) and sodium dodecyl sulfate (SDS), and of the ameliorating effect of the concurrent feeding of dietary fiber were investigated along with releases of the hydrolase activities, which were localized in the brush border membrane of rat small intestine, during a jejunum perfusion in vivo. The releases of sucrase, maltase and alkaline phosphatase activities from the jejunum with Ringer bicarbonate solution (RBS) perfusion for 150 min proceeded at a constant rate after RBS perfusion for the first 30 min. The detergents were perfused after RBS perfusion for 60 min. In Tween 20- or 60-RBS perfusion at the 2% level, the released sucrase activity gradually increased, reaching a level 3 times that with RBS perfusion 90 min after the beginning of Tween 20- or Tween 60-RBS perfusion. With NaTC- or DOC-RBS perfusion at the 0.5% or 0.2% level respectively, the released sucrase activity reached a level 3 to 4 times that with RBS perfusion within 30 min of the beginning of NaTC- or DOC-RBS perfusion, but that with Span 20-RBS perfusion at the 2% level was slightly lower compared with that with RBS perfusion. On the other hand, with SDS- or LBS-RBS perfusion at the 0.5% level, the released alkaline phosphatase activity rapidly reached a level 3 to 4 times as high as that with RBS perfusion. The inclusion of Gobo dietary fiber at the 0.04% level with Tween 20-RBS perfusion completely eliminated the releasing effect of Tween 20 on sucrase activity. These results suggest that the primary cause of the adverse effects of the feeding of these detergents is the exfoliating or releasing effect thereof on the brush border membrane, together with the inhibitory effect of some of these detergents on intestinal disaccharidase activities, and that the dietary fiber prevents the exfoliating or releasing effects of several detergents on the brush border membrane.
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PMID:Mechanisms of toxicities of some detergents added to a diet and of the ameliorating effect of dietary fiber in the rat. 629 91

Mammalian muscle acid alpha-glucosidase was highly purified for the first time from rabbit muscle by fractionation with ammonium sulfate, and chromatographies on Sephadex G-100, CM-TOYOPEARL and TOYOPEARL HW-55. The resulting preparation showed a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be 1.02 X 10(5) by SDS-electrophoresis. The optimum pH was found to be 4.5. The alpha-glucosidase showed relatively high activity not only toward maltose but also toward alpha-glucans, such as soluble starch, beta-limit dextrin, amylopectin, shellfish glycogen, and amylose. The Km values for maltose and glycogen were 6.3 mM and 12 mM (the concentration of non-reducing glucose units), respectively, and the ratio of the maximum velocities of hydrolyses of the two substrates was 100:66.7, in that order. Rabbit muscle acid alpha-glucosidase showed a wide specificity for various substrates. The Km values for maltose, maltotriose, -tetraose, -pentaose, -hexaose, -heptaose, and -octaose, and maltodextrins of average polymerization degrees of 13 and 17 were 6.3 mM, 2.6 mM, 5.9 mM, 3.0 mM, 5.9 mM, 5.9 mM, 5.9 mM, 7.7 mM, and 5.6 mM, respectively. The relative maximum velocities for maltooligosaccharides consisting of three or more glucose units were 43.5-89.3% of that for maltose. For disaccharides, the rate of hydrolysis decreased in the following order: maltose divided by nigerose greater than kojibiose greater than isomaltose. The purified enzyme was a typical acid alpha-glucosidase of mammalian origin, which hydrolyzed various substrates to produce alpha-glucose. The nature of the active site catalyzing the hydrolyses of maltose and glycogen was investigated by some kinetic methods. In experiments with mixed substrates, maltose and shellfish glycogen, the kinetic features agreed very closely with those theoretically predicted for a single site mechanism. The essential ionizable groups, 1 (on the acidic side) and 2 (on the alkaline side), were identified as -COO- and -COOH for the hydrolysis of both substrates. Cations, Na+, K+, and Mg2+, were about equally effective for stimulation of the enzyme actions on maltose and shellfish glycogen. Tris, turanose and erythritol inhibited not only maltase activity but also glucoamylase activity of the enzyme. From these results, it was concluded that rabbit muscle acid alpha-glucosidase attacks maltose and glycogen by a single active site mechanism.
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PMID:Kinetic studies on the substrate specificity and active site of rabbit muscle acid alpha-glucosidase. 639 1

Brush border membrane vesicles from rat small intestine were isolated by a Mg/EGTA precipitation method. Further fractionation either by free flow electrophoresis or by sucrose density gradient centrifugation leads to subfractions which differ with respect to enzyme enrichment factors, transport properties for D-glucose and protein pattern analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A relative enrichment of (Na+ + K+)-ATPase is found in one fraction, whereas in another fraction maltase, aminopeptidase M and alkaline phosphatase are relatively enriched. The fractions show different properties of D-glucose transport under tracer exchange conditions and a different inhibition of D-glucose transport by phlorizin and phloretin. These results indicate that the vesicles obtained from rat small intestine by this cation precipitation method are not homogeneous. The inhomogeneity cannot be due to a crosscontamination by membranes other than from the cell envelopment, as none of the fractions show a significant enrichment of succinate--cytochrome c oxidoreductase, KCN-resistant NADH oxidoreductase or glucosaminidase. The inhomogeneity might be due either to a crosscontamination by basal-lateral membranes or to membranes derived from epithelial cells not yet fully differentiated.
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PMID:Heterogeneity of brush-border-membrane vesicles from rat small intestine prepared by a precipitation method using Mg/EGTA. 641 69

Failure to develop clear-cut, distinguishing characteristics for hydrophobic and hydrophilic forms of maltase-glucoamylase led us to attempt the purification of the detergent-extracted enzyme in the continuous presence of protease inhibitors (phenylmethylsulfonyl fluoride and N-ethylmaleimide). The enzyme was purified by molecular exclusion, anion-exchange, and affinity column chromatography to a final specific maltase activity of 80 U/mg protein, comparable to previously solubilized enzymes. Both detergent (d-maltase) and proteolytically (p-maltase) solubilized enzymes had identical Km's for maltose and similar glycogenase activity. d-Maltase was clearly amphipathic. Whereas 95% of p-maltase was eluted with aqueous buffer from an octyl-Sepharose CL-4B column, the elution of d-maltase required solutions containing Triton X-100 and ethylene glycol. On density gradient centrifugation and sodium dodecyl sulfate (SDS)--polyacrylamide gels, p-maltase migrated as one high molecular weight species of 500,000. In contrast d-maltase migrated heterogeneously and the smallest maltase-active forms delineated by these two techniques, as well as by high pressure liquid chromatography, had molecular weights which ranged from 120,000 to 15,0000. Both p- and d-maltase were dissociated by heat in SDS, forming five prominent species as we have previously described. In contrast to p-maltase, in which the smallest species, band 1, equalled 36.7% of the total mass, band 1 of d-maltase accounted for 66.5%. Band 1 was separable when smaller amounts of enzyme were applied to slab gels and stained with silver, into two proteins of 130,000 and 145,000 daltons. The 145,000 dalton protein was absent in p-maltase and was replaced by a faint band of 140,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Rat intestinal maltase--glucoamylase. Purification of the detergent-solubilized enzyme in the presence of protease inhibitors: properties and identification of a protease-sensitive subunit. 642 12

Acid alpha-glucosidase has been purified from human placenta to a specific activity of approximately 6800, (4-methylumbelliferyl-alpha-D-glucoside as a substrate) or 55,400 mumol g-1 min-1 (glycogen or maltose as substrate). The purified enzyme gives rise to multiple protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), i.e., a major doublet of 82K and 69K , a minor doublet of 25K and 21K , and a faint band of 100K. All of the molecular weight species stained as glycoproteins with an intensity apparently proportional to their protein content, and were present in enzyme from individuals homozygous for the allozyme alpha-Glu 1. Isoelectric focusing revealed only enzymatically active proteins which, when analysed by SDS-PAGE, gave rise to multiple molecular weight species. Chromatography of I125-labeled, purified enzyme on Bio-Gel P-100 revealed only a radiolabeled, high-molecular-weight species which corresponded with enzyme activity. These findings suggest that, in the native state, the mature enzyme exists as a high-molecular-weight species, which is dissociable in SDS to several low-molecular-weight species. These results are consistent with reports that a 100K primary product of translation is post-translationally modified to yield polypeptides of lower molecular weights, and that all of the molecular species are absent in cells genetically deficient for acid alpha-glucosidase. The possibility that the low-molecular-weight (20- 25K ) protein bands in SDS-gels corresponded to a previously reported low-molecular-weight species generated by treatment with guanidine-HCl was investigated. The I125-labeled, purified acid maltase was dissociated by guanidine into two equal peaks of approximately 64K and 28K molecular weight. Surprisingly, both peaks, when analyzed on SDS-gels, yielded identical and equally intensely staining bands of 64K molecular weight. These results suggest that the mature acid alpha-glucosidase is made up of polypeptides which are bonded in the native state by at least two different types of interaction, one type which is dissociable in SDS and one type which is dissociable in guanidine but not in SDS. The nature and possible function of the 25K polypeptide generated only by guanidine-HCl remains to be determined.
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PMID:Further studies of the structure of human placental acid alpha-glucosidase. 642 17

Both the common and a variant isozyme of acid alpha-glucosidase have been purified from a heterozygous placenta with CM-Sephadex, ammonium sulfate precipitation, dialysis, Amicon filtration, affinity chromatography by Sephadex G-100, and DEAE-cellulose chromatography. Three and two activity peaks, from the common and variant isozymes, respectively, were obtained by DEAE-cellulose chromatography using a linear NaCl gradient. The three peaks of activity of the common isozyme were eluted with 0.08, 0.12, and 0.17 M NaCl, whereas the two peaks of the variant, with 0.01 and 0.06 M NaCl. The pH optimum and thermal denaturation at 57 degrees C were the same in all enzyme peaks of both isozymes. Rabbit antiacid alpha-glucosidase antibodies produced against the common isozyme were found to cross-react with both peaks of the variant isozyme. The two isozymes shared antigenic identity and had similar Km's with maltose as substrate. Normal substrate saturation kinetics were observed with the common isozyme when glycogen was the substrate, but the variant produced an S-shaped saturation curve indicating a phase of negative and positive cooperativity at low and high glycogen concentrations, respectively. The activity of the variant was only 8.6% and 19.2% of the common isozyme when assayed with nonsaturating and saturating concentrations of glycogen, respectively. A similar rate of hydrolysis of isomaltose by both isozymes was found indicating that the reduced catalytic activity of the variant isozyme toward glycogen is not the result of a reduced ability of this enzyme to cleave the alpha-1,6 linkages of glycogen.
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PMID:An isozyme of acid alpha-glucosidase with reduced catalytic activity for glycogen. 677 Jun 74

Diamine oxidase (histaminase) is an enzyme found in high concentrations in the intestinal mucosa of humans and other mammalian species. We investigated whether plasma and mucosal levels of diamine oxidase activity reflect both the maturational status of the mucosa during its development in the newborn rate and the degree of mucosal damage during its injury in the adult rat. Litter mates were reared under identical conditions and killed at different ages from day 0 to day 40 after birth. Diamine oxidase in the small intestine was low at birth, increased gradually with age, reached a peak at 22 d, and then remained at normal adult levels, similar to the developmental patterns of maltase and sucrase. Plasma diamine oxidase rose in parallel with intestinal levels (n = 500, r = 0.84, P less than 0.001), reached a peak at 24 d, and then remained at normal adult levels. Diamine oxidase activity in 15 nonintestinal tissues was less than 5% of ileal mucosal activity, and no nonintestinal activities showed increase with age. Adult rat intestinal loops were perfused with hyperosmolar sodium sulfate solutions to produce selective damage to villus mucosa. With increasing mucosal damage, there was a progressive decrease in the enzyme activities studied; first, lactase levels fell, then maltase and sucrase, and finally mucosal and plasma diamine oxidase activity levels fell. The decrease in plasma diamine oxidase reflected the degree of mucosal damage (n = 29, P less than 0.04). Diamine oxidase activity is thus unique among intestinal mucosal enzymes studied to date in that circulating levels can serve as a marker of mucosal maturation and integrity.
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PMID:Diamine oxidase (histaminase). A circulating marker for rat intestinal mucosal maturation and integrity. 677 69

alpha-Glucosidase was extracted from a homogenate of human kidney, initially with 0.02 M Tris-HCl buffer, pH 7.6, and subsequently with a mixture of 0.5% cholate and 0.5% Triton X-100 in the same buffer, pH 7.6. The enzyme in each of these two fractions was purified to the electrophoretically pure state by fractional precipitation with ammonium sulfate, column chromatographies on DEAE-cellulose, hydroxyapatite, Bio Gel A-1.5 m and affinity chromatography on heated glutinous rice. The two purified alpha-glucosidase preparations obtained were the same in enzymatic and proteochemical properties, and the molecular weight and isoelectric point estimated were 3 x 10(5) and 4.2, respectively. No evidence for subunit structure was obtained. The optimum pH for activity was 5.6 and the activity was drastically inhibited by Nojirimycin. The alpha-glucosidase readily hydrolyzed maltose, starch, and glycogen, producing only glucose. It hydrolyzed maltotriitol to split the non-reducing end glucose, but scarcely hydrolyzed maltitol or various other heteroglucosides examined. All these proteochemical and enzymatic properties of kidney alpha-glucosidase were the same as those of urine F-1 alpha-glucosidase. Also, kidney tissue alpha-glucosidase produced a clear precipitin line with antisera against urine F-1 alpha-glucosidase. These facts suggest that F-1 alpha-glucosidase in urine originates from kidney tissue.
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PMID:Identity of alpha-glucosidase of human kidney with urine F-1 alpha-glucosidase. 680 53


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