Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

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

Resident peritoneal macrophages of the mouse, cultivated for 3 d, have been studied by quantitative subcellular fractionation using differential centrifugation and density equilibration in linear gradients of sucrose. Density equilibration experiments were carried out on untreated cytoplasmic extracts, on cytoplasmic extracts treated with digitonin or sodium pyrophosphate, and on cytoplasmic extracts derived from cells cultivated for 24 h in the presence of Triton WR-1339. The enzyme distributions obtained distinguished six typical behaviors characteristic of distinct subcellular entities. Acid alpha-galactosidase and other acid hydrolases displayed the highest average velocity of sedimentation and equilibrium density. Culturing in a medium that contained Triton WR-1339 markedly decreased their density, most likely as a result of Triton WR-1339 accumulation within lysosomes. Cytochrome c oxidase and the sedimentable activity of malate dehydrogenase showed a narrow density distribution centered around 1.17, very similar under all the experimental situations; their rate of sedimentation fell within the range expected for mitochondria. Catalase was particle-bound and exhibited structure-linked latency (80 percent); it was released in soluble and fully active form by digitonin, but this required a much higher concentration than in the case of lysosomal enzymes. Differences relative to all the other enzymes studied suggest the existence of a particular species of organelles, distinctly smaller than mitochondria, and possibly related to peroxisomes. Many enzymes were microsomal in the sense that the specific activities, but not the yields, were greater in microsomes than in other fractions obtained by differential centrifugation. These enzymes were distinguished in three groups by their properties in density equilibration experiments. NAD glycohydrolase, alkaline phosphodiesterase I, and 5'-nucleotidase had low equilibrium densities but became noticeably more dense after addition of digitonin. The other microsomal enzymes were not shifted by digitonin, in particular N-acetylglucosaminyltransferase and galactosyltransferase, which otherwise equilibrated at the same position in the gradient. We assign the digitonin-sensitive enzymes to plasma membranes and possibly to related endomembranes of the cells, and the two glycosyltransferases to elements derived from the Golgi apparatus. Finally, alpha-glucosidase, sulphatase C, NADH cytochrome c reductase, NADPH cytochrome c reductase, and mannosyltransferase, equilibrated at a relatively high density but were shifted to lower density values after addition of sodium pyrophosphate. These properties support their association with elements derived from the endoplasmic reticulum.
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PMID:Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages. 630 Feb 79

The subcellular distribution of the Na+/H+ antiporter in renal proximal tubule cells was studied with differential and density gradient centrifugation. Enzyme markers for basolateral membranes [Na+/K+)-ATPase), brush border membranes (maltase), and a variety of intracellular organelles (NADPH cytochrome c reductase, thiamine pyrophosphatase, acid phosphatase, and succinate cytochrome c reductase) were simultaneously assayed in sucrose density gradients. Basolateral membranes (median rho = 1.150) were well separated from brush border membranes (median rho = 1.165) by this technique. Markers for other cellular organelles had intermediate or bimodal distributions. To determine the cellular location of the Na+/H+ antiporter, Na+-dependent collapse of preformed pH gradients was assayed in the sucrose density gradient fractions using acridine orange. Na+/H+ antiporter activity paralleled the distribution of the brush border membrane fractions; activity in the peak basolateral membrane fraction was less than 5% of that in the peak brush border fraction. To determine whether antiporter activity was potentially detectable in all cell fractions, nigericin was added to each fraction and K+/H+ exchange was assayed with acridine orange. Activity was present in all sucrose density gradient fractions. In addition, there was no alteration in Na+/H+ exchange activity measured in brush border membranes after mixing with cell sol or basolateral membranes, showing that neither inhibitors nor activators of the Na+/H+ antiporter were present in any of the cell fractions. These controls confirmed the finding that Na+/H+ antiporter activity was absent from basolateral membranes. The presence of the Na+/H+ antiporter in brush border membranes and its absence from basolateral membranes is consistent with its playing an important role in the vectorial transport of H+ from blood to tubular lumen in the renal proximal tubule.
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PMID:Asymmetric distribution of the Na+/H+ antiporter in the renal proximal tubule epithelial cell. 631 99

The hybridoma technique, originally developed by G. Kohler & C. Milstein, is a powerful new experimental approach for analysis of complex biological systems, and is particularly suited for identification and study of surface-membrane antigens. This technique has been used for the production of monoclonal antibodies to intestinal brush border membrane proteins. Spleen cells, obtained from BALB/c mice immunized with purified brush border membranes, were fused with NSI mouse myeloma cells, and hybrids were selected with a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Hybridoma cultures were screened for production of specific antibodies by radio-immunobinding assays and by immunofluorescent staining of intestinal frozen sections. Selected hybridoma cultures were cloned twice and used for the production of large amounts of antibodies, which were characterized. Nineteen monoclonal antibodies have been prepared to date, about half of them specifically staining the brush border membrane of mature enterocytes. Ten of the antibodies specifically immunoprecipitate surface-membrane proteins, which were analysed by sodium dodecyl sulphate slab-gel electrophoresis, by two-dimensional slab-gel electrophoresis, and by specific enzyme assays. Two antibodies were found to be specific for sucrase-isomaltase, one for an aminopeptidase, two for an isoenzyme of alkaline phosphatase that is present exclusively in the proximal small intestine, and one for maltase-glucoamylase. These monoclonal antibodies, and others prepared by similar techniques from mice immunized with a wide variety of intestinal subcellular fractions, should prove invaluable tools for the study of the biosynthesis of cell-surface proteins, the fetal and postnatal development of specific intestinal functions, and the process of cell differentiation in the intestinal epithelium.
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PMID:Use of monoclonal antibodies in the study of intestinal structure and function. 634 93

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

The influence of bile salts on the mucosal surface of rat jejunum was tested with an in vivo technique of segmental perfusion. Sodium taurocholate and chenodesoxycholate were applied in a concentration of 3 mmol/l. The release of 5 brush border membrane enzymes, 5 cytosolic, 1 mitochondrial, and 2 lysosomal enzymes during a perfusion time of 150 min as well as morphological alterations after bile salt treatment were investigated. Among the membrane enzymes, due to their superficial localization, the solubilization of enteropeptidase and alpha-1,4-glucosidase was highest both in the control perfusion and in the presence of bile salts. At the same time, cytoplasmic enzyme activities were liberated extensively whereas lysosomal and mitochondrial enzymes were scarcely detectable. This disproves any serious injury of the enterocytes. Electronmicroscopic results supported this suggestion. After administration of taurocholate (in physiological concentration), only an occasional diminution of the glycocalyx was observed and even chenodeoxycholate (in an unphysiological concentration) caused only negligible destructions of intestinal brush borders. Investigations with ruthenium red to contrast the glycocalyx showed a partially unchanged structure. Microvesiculation from the microvilli was observed in many electron microscopic photographs. That is a possibility for the release of membrane-bound and cytosolic enzymes without destruction of enterocytes.
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PMID:[Biochemical and morphologic studies on the effect of bile acids on the epithelium of the rat jejunum]. 643 37

The effect of dietary thiamin deficiency has been studied on intestinal functions and chemical composition of brush border membranes in rats. Intestinal uptake of glucose, glycine, alanine, and leucine was significantly stimulated in thiamin deficiency compared to pair-fed control group. Studies with glucose and glycine revealed that stimulation of the absorption process occurs only in the presence of Na+ but not in its absence. Km measured in the presence of 140 mM Na+ for glucose and glycine uptakes was reduced by 56 and 41%, respectively, but Vmax remained unaltered in vitamin deficiency. There was no change in these parameters in Na+-free medium (Km = 31.3 and 23.3 mM; Vmax = 17.2 to 19.7 and 13.5 to 16.4 mumol/10 min/g wet tissue, respectively) under these conditions. The activities of brush border sucrase, lactase, maltase, alkaline phosphatase, and leucine aminopeptidase were reduced by 42 to 66% in thiamin deficiency, compared to pair-fed controls. Kinetic studies with sucrase and alkaline phosphatase evinced that a decrease in Vmax (61 and 64%, respectively) with no change in Km (33.8 and 4.3 mM, respectively) was responsible for observed impairment in the enzyme activities in thiamin deficiency. Microvillus membrane proteins expressed on dry membrane basis were reduced by 20% in thiamin-deficient intestine. There was no difference in membrane sialic acid, cholesterol, phospholipids, and triglycerides fractions under these conditions. It is suggested that thinning of the microvillus membrane may be implicated in observed aberrations of intestinal functions in thiamin-deprived animals.
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PMID:Effect of dietary thiamin deficiency on intestinal functions in rats. 646 54


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