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)

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

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

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

The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles. When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinity-purified anticlathrin IgGs--one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. ( Louvard , D., C. Morris, G. Warren, K. Stanley, F. Winkler , and H. Reggio , 1983, EMBO [Eur. Mol. Biol. Organ.] J., 2:1655-1664), which detects both the heavy and light chains. As viewed by light microscopy (immunofluorescence or immunoperoxidase), staining with both anticlathrins was concentrated at the base of the proximal tubule microvilli. Immunoelectron microscopic localizations carried out on brush border fractions (using peroxidase and gold conjugates) demonstrated specific binding of anticlathrin IgGs to the lattice-like cytoplasmic coat. When brush border fractions were reacted with monoclonal antibodies prepared against gp330 and maltase, proteins that serve as markers for the membrane of the apical invaginations and microvilli, respectively ( Kerjaschki , D., L. Noronha - Blob , B. Sacktor , and M. G. Farquhar , 1984, J. Cell Biol., 98:1505-1513), the two proteins retained their restrictive distribution in the brush border. The findings demonstrate (a) that the cytoplasmic coat of the proximal tubule intermicrovillar apical invaginations is composed of clathrin heavy and light chains, and (b) that the differential distribution of proteins in these two brush border microdomains is maintained in appropriately prepared brush border fractions.
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PMID:Presence of an extensive clathrin coat on the apical plasmalemma of the rat kidney proximal tubule cell. 637 81

We have studied some characteristics of alpha-1,4-glucosidases in human male reproductive organs in order to obtain information on the origin of the enzyme in seminal plasma. Acid and neutral enzymes could be distinguished on the basis of their selective inhibition either by SDS (acid enzyme) or MTT (neutral enzyme). Only the epididymis contained a significant amount of SDS resistant neutral alpha-1,4-glucosidase which was comparable to what has been isolated in seminal plasma. The similarity of epididymal and seminal plasma neutral enzymes was further confirmed by ultracentrifugation on sucrose density gradients, which permitted a complete separation of neutral (11S) and acid (4S) iso-enzymes. The 11S form was present in epididymis and in seminal plasma, but was totally absent in seminal vesicles, prostates and testis. The epididymal enzyme also had some of the unique characteristics found in the seminal plasma enzyme: it precipitated upon dialysis against distilled water, and its mobility on SDS polyacrylamide gel electrophoresis was identical to that of form 1 in seminal plasma. These results, although they do not constitute absolute proof of the identity of epididymal and seminal plasma alpha-glucosidase, certainly provide strong support for this hypothesis.
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PMID:Similar biochemical properties of human seminal plasma and epididymal alpha-1,4-glucosidase. 638 46

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

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

These studies examined the potential for reorganization and differentiation of dissociated 18-day fetal rat intestine. Cultures of trypsin-dissociated fetal intestine were maintained in vitro for 1 week on a three-dimensional matrix, then transplanted into syngeneic hosts. When harvested after 4 weeks, these transplants consistently demonstrated organotypic differentiation. Spherical structures containing crypts with frequent mitotic figures and villi lined with columnar epithelium had formed. PAS staining demonstrated positive epithelial cell brush borders, goblet cells, and luminal contents. Significant levels of the microvillus membrane enzymes lactase, sucrase, maltase, and alkaline phosphatase were present in the luminal contents. Sucrase-isomaltase, an enzyme characteristic of postweaning small intestine, was demonstrated by immunoprecipitation and SDS-PAGE. Thus, both morphological and biochemical maturation occurred in the transplants.
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PMID:Organotypic differentiation of trypsin-dissociated fetal rat intestine. 661 90


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