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)

Cell-associated oligo-1,6-alpha-glucosidase (EC 3.2.1.10) was isolated from Thermoanaerobium Tok6-B1 grown on starch-containing medium. Activity was purified 11.4-fold by salt precipitation, gel filtration, hydroxyapatite and anion-exchange chromatography. Molecular mass was determined as 30,000 by SDS/polyacrylamide-gel electrophoresis and 33,000 by analytical gel filtration. The probable order of specificity was p-nitrophenyl-alpha D-glucose greater than-isomaltose greater than-isomaltotriose greater than-panose greater than-nigerose and no activity was shown against malto-oligosaccharides, melezitose, melibiose, raffinose, cellobiose, sophorose, gentiobiose, lactose, pullulan, dextran or amylose. The optima for activity and stability were between pH 5.6 and 7.0 and the half-life at pH 6.5 was 1000 min at 70 degrees C and 20 min at 76 degrees C. Activity was stabilized by substrate, Mg2+, Mn2+ and Ca2+, but was destabilized by Zn2+ and EDTA. N-Ethylmaleimide, glucose and 1-O-methyl-alpha D-glucose were inhibitory but 1-O-methyl-beta D-glucose stimulated activity. The activation energy (Ea) was 109 kJ/mol.
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PMID:A cell-associated oligo-1,6-alpha-glucosidase from an extremely thermophilic anaerobic bacterium, Thermoanaerobium Tok6-B1. 321 28

Procedures have been validated for the investigation of the physical properties of canine microvillar membrane proteins by SDS-polyacrylamide gel electrophoresis. These have been used to examine mucosal samples from eight control dogs and from five dogs with naturally occurring exocrine pancreatic insufficiency (EPI) in order to evaluate the potential role of the pancreas in the normal turnover of microvillar membrane proteins in the dog. Gel scanning showed that the proportion of total membrane protein in bands corresponding to a molecular mass greater than 200 kDa was up to 20-times higher in dogs with EPI than in control dogs. In particular, a band of apparent molecular mass 218 kDa represented between 8 and 28% of membrane protein in all affected dogs, compared with only 0.5 to 1.8% in controls, and is most likely to contain single chains of both pro-maltase-glucoamylase and pro-sucrase-isomaltase. Incubation of microvillar membranes in vitro with either trypsin or canine pancreatic juice resulted in degradation of this high molecular mass band and a corresponding increase in the amount of protein in three bands representing molecular masses of 150, 133 and 106 kDa. In samples from control dogs aminopeptidase N was identified in the 133 kDa band by Western blotting and incubation with monospecific antiserum. These findings suggest that pancreatic enzymes play a major role in the normal post-translational processing of intestinal microvillar membrane proteins in the dog.
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PMID:Investigation of the physical properties of dog intestinal microvillar membrane proteins by polyacrylamide gel electrophoresis: a comparison between normal dogs and dogs with exocrine pancreatic insufficiency. 340 88

Acid alpha-glucosidase [EC 3.2.1.3] was purified from pig liver by a procedure including Sephadex G-100 affinity chromatography. Electrophoresis on SDS-polyacrylamide gel of the purified enzyme indicated the presence of two components with molecular weights of 73K and 64K. The two components of the enzyme were completely separated, in reasonable yield, by chromatography on a DEAE-5PW column. Both components catalyzed the hydrolysis of the alpha-1,4 and alpha-1,6 linkages of glycogen, maltose, isomaltose, dextrin, and a synthetic glucoside at acid pH. The pH optima of both components were 4.3 for maltase and glucoamylase, and 4.8 for isomaltase and dextrinase. But as to the activity on 4MU-alpha-Glc, the pH optimum of the larger component was 4.8 and that of the smaller component 5.3. The Km values of both components for 4MU-alpha-Glc, maltose, glycogen, isomaltose, and dextrin were 1.0 X 10(-4) M, 9.1 X 10(-3) M, 16.7 mg/ml, 6.7 X 10(-2) M, and 12.5 mg/ml, respectively. Erythritol, Tris, and turanose inhibited the two components competitively. The Ki values of the larger component were 5.0 X 10(-2) M, 13.3 X 10(-3) M, and 3.2 X 10(-3) M, and those of the smaller component were 2.5 X 10(-2) M, 6.1 X 10(-3) M, and 4.7 X 10(-3) M, for erythritol, Tris, and turanose, respectively.
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PMID:Purification and characterization of two components of acid alpha-glucosidase from pig liver. 351 91

A panel of monoclonal antibodies was produced against purified microvillus membranes of human small intestinal enterocytes. By means of these probes three disaccharidases (sucrase-isomaltase, lactase-phlorizin hydrolase, and maltase-glucoamylase) and four peptidases (aminopeptidase N, dipeptidylpeptidase IV, angiotension I-converting enzyme, and p-aminobenzoic acid peptide hydrolase) were successfully identified as individual entities by SDS PAGE and localized in the microvillus border of the enterocytes by immunofluorescence microscopy. The antibodies were used to study the expression of small intestinal hydrolases in the colonic adenocarcinoma cell line Caco 2. This cell line was found to express sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV, but not the other three enzymes. Pulse-chase studies with [35S]methionine and analysis by subunit-specific monoclonal antibodies revealed that sucrase-isomaltase was synthesized and persisted as a single-chain protein comprising both subunits. Similarly, lactase-phlorizin hydrolase was synthesized as a large precursor about twice the size of the lactase subunits found in the human intestine. Aminopeptidase N and dipeptidylpeptidase IV, known to be dimeric enzymes in most mammals, were synthesized as monomers. Transport from the rough endoplasmic reticulum to the trans-Golgi apparatus was considerably faster for the peptidases than for the disaccharidases, as probed by endoglycosidase H sensitivity. These results suggest that the major disaccharidases share a common biosynthetic mechanism that differs from that for peptidases. Furthermore, the data indicate that the transport of microvillus membrane proteins to and through the Golgi apparatus is a selective process that may be mediated by transport receptors.
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PMID:Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells. 389 50

Acid alpha-glucosidase was purified from the lysosomal/mitochondrial fraction of rat liver by acid precipitation, ammonium sulphate precipitation and affinity chromatography on Sephadex G 100, resulting in 17000-fold enrichment from that of the liver homogenate. The molecular weight of the enzyme was estimated to be 125000 from analytical gel filtration experiments. On SDS-polyacrylamide gel electrophoresis the purified enzyme showed only a single band having an apparent molecular weight of about 64000. Based on these results, it is concluded that lysosomal liver alpha-glucosidase consists of two subunits. The discontinuous system of SDS-polyacrylamide gel electrophoresis, however, revealed two closely migrating protein bands suggesting heterogeneity of the enzyme subunits.
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PMID:Purification and some properties of lysosomal alpha-glucosidase of rat liver. 391 37

Several approaches were adopted for the disruption and removal of the tegumental surface from protoscoleces of the horse strain of the hydatid organism, Echinococcus granulosus. The effectiveness of each method and the purity of subsequent microthrix-enriched fractions obtained by differential centrifugation were evaluated by electron microscopy, by the amount of protein released and by the degree of enrichment of surface plasma membrane marker enzymes. Incubation in saponin for 10 min produced the purest microtriche preparation, but in low yield; freeze/thawing, incubation in Triton X-100 for 10 min or in saponin for 20 min produced fractions containing significant amounts of relatively pure microtriches, but mild homogenization was a poor method for surface disruption and subsequent isolation of microtriches. Phosphodiesterase, adenosine triphosphatase (total and ouabain-inhibited), leucine aminopeptidase and glutamyltransferase were active in the protoscoleces but none were enriched in any of the microthrix fractions. In contrast, alkaline phosphatase, acid phosphatase, 5' nucleotidase and maltase were enriched significantly in all of the isolated microtriche preparations, which suggests that these enzymes are predominantly surface membrane bound. The protein profiles of the microthrix-enriched fractions, following SDS-PAGE, were basically similar, although there were some qualitative and quantitative differences in the proteins released by each isolation procedure. Three major PAS-staining components were present in all the preparations and these probably originated from the glycocalyx. One of these PAS-positive components, with an approximate molecular weight of 110 kDa, may be a glycoprotein specific to the horse strain of E. granulosus.
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PMID:Isolation, fractionation and partial characterization of the tegumental surface from protoscoleces of the hydatid organism, Echinococcus granulosus. 398 50

1. Cortisone administration to suckling rats leads prematurely to induction of enzymes of the intestinal microvillus plasma membrane and lengthening of the intestinal microvilli. To investigate the membrane changes that might be involved, a method for the isolation of a fraction enriched with microvillus plasma membrane was developed in suckling rats. Plasma-membrane fractions were compared from 13-day-old control rats and from 13-day-old rats given cortisol acetate by subcutaneous injection for 3 days. 2. After cortisol injection, the activity of maltase, trehalase, sucrase and leucyl beta-naphthylamidase increased markedly, and to the same extent, in intestinal homogenates and plasma-membrane preparations. Purification, and recovery of five marker enzymes with respect to homogenate activity, and recovery of protein, were similar for both membrane preparations, particularly after correction for non-membrane activity, which was high in suckling rats and affected by cortisol. 3. In material released from the plasma membrane by digestion with papain, maltase protein was increased after cortisol injection at least as much as maltase activity. Sucrase activity increased at least 200-fold, and this increase was associated with the appearance of a new sucrase band on polyacrylamide-gel electrophoresis. 4. Sodium dodecyl sulphate electrophoresis of plasma-membrane proteins revealed at least four additional macromolecules after cortisol injection. Concurrently several proteins disappeared from the plasma membrane. The added proteins appeared in the main to be removed from the plasma membrane by papain, whereas the deleted proteins were in the papain-resistant fraction. 5. Enzymic stimulation induced by cortisol acetate in the suckling-rat plasma membrane therefore appears to involve the addition of new proteins, rather than activation of proteins in situ. Deletion of proteins from the membrane during induction of hydrolytic enzymes may reflect other phenomena such as protein reorganization associated with the change in microvillus shape.
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PMID:Isolation of microvillus plasma membranes from suckling-rat intestine. The influence of premature induction of digestive enzymes by injection of cortisol acetate. 446 84

Antisera against purified pigeon small intestinal sucrase-isomaltase (S-I) and maltase-glucoamylase (M-G) were prepared from rabbits. Both sera showed cross-reactivity. It was demonstrated that the sucrase . isomaltase was purified to homogeneity, supporting our earlier results of SDS-PAGE of pigeon intestinal disaccharidases. Both the sucrase- isomaltase and maltase-glucoamylase activities were not inhibited by either specific or cross-reacting antibodies even when a several fold of either antibody was present. It is inferred from these immunochemical results that the two complexes in the pigeon intestine share many structural identities, and that their catalytic site(s) may not be involved in their antigenic domains.
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PMID:Studies on the intestinal disaccharidases of the pigeon IV. Immunochemical properties of sucrase . isomaltase and maltase . glucoamylase. 620 7

Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific alpha-glucosidase(s) were inhibited by 1-deoxynojirimycin with median inhibitory concentration of 3 microM, 20 microM, 12 microM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of 1-deoxynojirimycin and formed the basis for the design of the affinity ligand. Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was approximately equal to 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 degrees C in the presence of detergent (t 1/2 approximately equal to 20 days) and showed high specificity for the hydrolysis of the terminal (alpha 1,2)-linked glucose residue in the natural substrate Glc3-Man9-(GlcNAc)2.
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PMID:Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine-linked oligosaccharides. 623 11

Two membrane proteins, maltase and gp330 (the pathogenic antigen of Heymann nephritis), present in the proximal tubule brush border have recently been independently purified and found to be large glycoproteins of similar molecular weight (Mr = approximately 300,000) by SDS PAGE. To determine the relationship between the two, monoclonal antibodies raised against the purified proteins were used for comparative immunochemical analyses and immunocytochemical localization. When a detergent extract of [35S]methionine-labeled rat renal cortex was used for immunoprecipitation with monoclonal antimaltase IgG, a single band of approximately 300 kdaltons was precipitated, whereas a single 330-kdalton band was precipitated with monoclonal anti-gp330 IgG. Monoclonal antimaltase (gp300) IgG also immunoprecipitated maltase activity from solubilized renal maltase preparations, whereas monoclonal anti-gp330 IgG failed to do so. When cyanogen bromide-generated peptide maps of the two proteins were compared, there were many similar peptides, but some differences. When maltase and gp330 were localized by indirect immunofluorescence and by indirect immunoperoxidase and immunogold techniques at the electron microscope level, they were found to be differently distributed in the brush border of the initial (S1 and S2) segments of the proximal tubule: maltase was concentrated (approximately 90%) on the microvilli, and gp330 was concentrated (approximately 90%) in the clathrin-coated apical invaginations located at the base of the microvilli. We conclude that maltase (gp300) and the Heymann nephritis antigen (gp330) are structurally related membrane glycoproteins with a distinctive distribution in the proximal tubule brush border which may serve as markers for the microvillar and coated microdomains, respectively, of the apical plasmalemma.
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PMID:Microdomains of distinctive glycoprotein composition in the kidney proximal tubule brush border. 637 Oct 23


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