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:3.2.1.26 (invertase)
4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank of E. coli harboring recombinant cosmids representative of the B. subtilis genome. It was shown that the sacA gene is located in a 2kb EcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for in B. subtilis and E. coli. Complementation of a sacA mutation was observed in Rec+ and REc- strains of B. subtilis. Expression of sucrase was also demonstrated in E. coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic in E. coli since the presence of the recombinant plasmids did not allow the transport of [U14C] sucrose and the growth of the cells. It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport of B. subtilis.
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PMID:Cloning and expression in Escherichia coli of the sucrase gene from Bacillus subtilis. 681 36

Temperature-sensitive secretory mutants (sec) of S. cerevisiae have been used to evaluate the stages and localization of glycoprotein oligosaccharide synthesis. At the nonpermissive growth temperature (37 degrees C), the sec mutants accumulate secretory organelles and glycoproteins. Histochemical staining and thin-section electron microscopy reveal that the secreted glycoprotein, acid phosphatase, is contained within one of three distinct organelles that accumulates in different mutants: ER; Golgi-like structures called Berkeley bodies; and 80--100 nm vesicles. When produced at 37 degrees C, invertase and acid phosphatase have less carbohydrate in the mutants that accumulate ER than in other mutants, or than in the wild-type strain. External invertase migrates on SDS-polyacrylamide gels as a heterogeneous species with an apparent molecular weight of 100 to 140 kd. Radiolabeled invertase, immunoprecipitated from extracts of ER-accumulating mutant cells, migrates as a set of three discrete protein species with apparent molecular weights of 79, 81, and 83 kd; the other mutants produce a form more like the secreted enzyme. In each case, removal of N-glycosidically linked oligosaccharides by treatment with endoglycosidase H produces a discrete species that migrates as a protein of 61 kd. Immunochemical analysis of bulk glycoprotein accumulated in the mutants suggests that a major portion of the N-linked oligosaccharide, the outer chain, is added after material passes from the ER.
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PMID:Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast. 702 44

Yeast plasma membranes were isolated from homogenized cells and analyzed by SDS-PAGE. Two glycoproteins of 160 000 and 240 000 molecular weight were found, both of which exhibited invertase activity (EC 3.2.1.26). By density gradient centrifugation a heavy membrane fraction which consisted of the glycoproteins and two hydrophobic proteins was isolated. Antibody labeling of protoplasts revealed a good correlation between the distribution of binding sites of the antibodies against the heavy fraction and the distribution of the intramembranous particles. The cytoplasmic surface of the yeast plasma membrane was visualized by freeze drying and subsequent platinum/carbon shadowing of membrane vesicles adsorbed to cationized glass and squirted with a hypotonic buffer stream. In contrast to the smooth exoplasmic surface the cytoplasmic surface showed paracrystalline arrays of particles which resembled in size, number and lattice constant the intramembranous particles. Removal of the adsorbed paracrystalline arrays and subsequent SDS-PAGE revealed the same protein pattern as the heavy membrane fraction. It can therefore be concluded that the glycoproteins which show invertase activity and the two hydrophobic proteins are the major components of the paracrystalline arrays. It is proposed that the glucose level of the nutrient medium influences the appearance and disappearance of the paracrystalline arrays, which consist mainly of invertase, because synthesis of invertase is inhibited by glucose levels higher than 1%.
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PMID:Isolation and characterization of paracrystalline arrays of the plasma membrane of baker's yeast Saccharomyces cerevisiae. 702 43

Brush borders free of nuclei were isolated by repeated homogenization and centrifugation in iso-osmotic medium. They showed typical morphology under electron microscopy. The mean recovery and enrichment of alkaline phosphatase activity in the brush-border fraction were 50% and 17.5-fold respectively. gamma-Glutamyl transpeptidase showed a close parallelism with alkaline phosphatase and sucrase in subcellular distribution. Microvillar membranes were purified from isolated brush borders; they showed a further enrichment for alkaline phosphatase and were composed of homogeneous vesicles. Both brush-border and microvillar-membrane preparations were analysed for contamination by basolateral and endoplasmic-reticular membranes. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the microvillar-membrane preparation in six different systems revealed approx. 40 components in the mol.wt. range 15 000-232 000. They were grouped into seven major classes on the basis of molecular weight and electrophoretic patterns.
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PMID:Characterization of brush borders purified in iso-osmotic medium and microvillar membranes subfractionated from mouse small intestine. 731 8

Intestinal brush border membrane hydrolases and HLA lymphocyte antigens have been examined in three siblings with sucrose intolerance and alcaptonuria, and their consanguineous parents. Sucrase-isomaltase activity was absent in the three patients, and corresponded with the gel electrophoresis of SDS-solubilized brush border membranes, which failed to demonstrate the protein band normally associated with sucrase-isomaltase complex. The activities of all brush border membrane enzymes in the mother were normal, while those of the father were generally low. The use of hydrolytic capacity ratios, however, permitted the designation of both parents as heterozygotes. Significant homogentisic aciduria was found only in the three propositi, and no effect of homogentisic acid on the sucrase activities of two normal, unrelated children could be demonstrated in vitro. The HLA lymphocyte antigen profiles of all seven family members demonstrated remarkable histocompatibility in five of them.
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PMID:Alcaptonuria and sucrase-isomaltase deficiency in three offspring of a consanguineous marriage. 734 88

A method for analysing microgram amounts of microvillar membranes by two-dimensional electrophoresis (protein mapping) is described, and has been used to characterize the microvillar proteins of the small intestine of German shepherd, corgi, and beagle dogs. Detergent-solubilized microvillar membranes were radiolabelled with 14C and separated by isoelectric focussing followed by SDS-PAGE. Proteins were detected fluorographically and glycoproteins by lectin-affinity staining. The microvillar hydrolases alkaline phosphatase and dipeptidyl aminopeptidase IV were identified by active-site labelling and aminopeptidase N by immunoprecipitation. Changes following pancreatic duct diversion were consistent with accumulation of pro-sucrase-isomaltase and diminished expression of the sucrase and isomaltase subunits. Cytoskeletal proteins were concentrated in the core fraction remaining after extraction of microvillar membranes with Triton X-100. There were no consistent differences between dogs of different breed, and the canine protein maps were similar to the human.
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PMID:Characterization of microvillar membrane proteins of dog small intestine by two-dimensional electrophoresis. 758 24

An intracellular beta-D-fructofuranosidase produced by Aspergillus sydowi IAM 2544 was purified by Q-Sepharose and Alkyl-Sepharose chromatographies. The molecular mass was 50 kDa by SDS-PAGE analysis. The optimum pH and temperature of sucrose hydrolyzing activity of the enzyme were 5.5 and 75 degrees C, respectively, but those of fructosyl transferase activity were 5.2 and 55 degrees C, respectively. The enzyme efficiently transferred the fructose residue of sucrose as a donor to trehalose as an acceptor. And the amount of fructosyl and oligofructosyl trehaloses produced was changed by the molar ratio of trehalose as an acceptor to sucrose as a donor used. The most efficient production of the transferred products was achieved at the reaction conditions in the range of molar ratios of 1:1 to 3:1 (trehalose:sucrose). The chemical structures of these new kinds of resulting series of fructosyl and oligofructosyl trehaloses produced were identified as O-beta-D-Fru-(2-->6)-alpha-D-Glc-(1-->1)-alpha-D-glucopyranoside, O-beta-D-Fru-(2-->6)-alpha-D-Glc-(1-->1)-alpha-D-glucopyranoside, and O-beta-D-Fru-(2-->1)-O-beta-D-Fru-(2-->1)-O-beta-D-Fru-(2-->6)-alpha-D-G lc- (1-->1)-alpha-D-glucopyranoside. These results indicate that beta-fructofuranosidase from Aspergillus sydowi specifically transferred the fructose residue of sucrose to the C6-OH position of the glucose residue of trehalose at the early stage of the reaction, following the elongation of the fructose residue by the transfructosylation of the enzyme to form oligofructosyl trehalose of a longer fructose chain.
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PMID:Enzymatic synthesis of novel fructosyl and oligofructosyl trehaloses by Aspergillus sydowi beta-fructofuranosidase. 776 19

A secreted invertase was purified 23-fold by ultrafiltration, ion-exchange, and gel filtration chromatography from the culture supernatant of 18 h sucrose-grown cultures of Aspergillus niger. The purified enzyme hydrolysed sucrose and raffinose but there was no detectable hydrolysis of inulin, melezitose or PNPG. Invertase activity was optimal at pH 5.5 and 50 degrees C. The molecular mass of reduced invertase was 115 kDa, as determined by SDS gel electrophoresis. The native molecular weight of between 225 kDa and 250 kDa, estimated by electrophoresis under non-denaturing conditions, suggests that the protein is a dimer of identical subunits. The suc1 gene encoding this protein was completely-sequenced. The translated sequence yields a protein of 566 amino acids with a calculated molecular mass of 61 kDa, suggesting that carbohydrates represent about 50% of the mass of the protein.
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PMID:Purification and characterisation of an Aspergillus niger invertase and its DNA sequence. 835 33

The cDNA sequence encoding mature human C9 protein and its signal peptide was cloned into three expression vectors for expression in COS-7 (mammalian), Spodoptera frugiperda IPLB-SF-21AE (insect), and Saccharomyces cerevisiae (yeast) cells. In addition, C9 cDNA encoding only the mature protein was fused to the yeast invertase leader sequence (SUC2) and cloned for expression in yeast. Under optimal conditions COS-7 and IPLB-SF-21AE cells secreted recombinant C9 (rC9) at concentrations of about 111 and 700 ng C9/ml culture supernatant, respectively. By comparison S. cerevisiae, whether transformed with C9 cDNA containing its native or yeast invertase leader sequence, secreted only very small amounts of rC9 (5-10 ng/ml). However, upon lysis concentrations of up to 500 ng/mg dry wt were found in yeast cells transformed with C9 cDNA. SDS-PAGE followed by Western blot analysis revealed COS-7 cell and S. cerevisiae expressed rC9 to have a MW similar to that of native C9 purified from human serum, while rC9 from IPLB-SF-21AE cells was about 4 kDa smaller. No hemolytic activity of S. cerevisiae secreted rC9 could be detected and the specific hemolytic activity of S. cerevisiae intracellular rC9 was also very low. However, the specific hemolytic activities of COS-7 and IPLB-SF-21AE secreted rC9 were indistinguishable from that of purified native human C9. Thus, for future studies on the structure and function of C9 where the production of large quantities of mutant protein would be desirable, the baculovirus-insect cell expression system appears to offer considerable advantages.
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PMID:The expression of hemolytically active human complement protein C9 in mammalian, insect, and yeast cells. 847 47

Two isoenzymes of maltase (EC 3.2.1.20) were purified to homogeneity from Candida albicans. Isoenzymes I and II were found to have apparent molecular masses of 63 and 66 kDa on SDS/PAGE with isoelectric points of 5.0 and 4.6 respectively. Both isoenzymes resembled each other in similar N-terminal sequence, specificity for the alpha(1-->4) glycosidic linkage and immune cross-reactivity on Western blots using a maltase II antigen-purified rabbit antibody. Maltase was induced by growth on sucrose whereas beta-fructofuranosidase activity could not be detected under similar conditions. Maltase I and II were shown to be unglycosylated enzymes by neutral sugar assay, and more than 90% of alpha-glucosidase activity was recoverable from spheroplasts. These data, in combination with other results from this laboratory [Geber, Williamson, Rex, Sweeney and Bennett (1992) J. Bacteriol. 174, 6992-6996] showing lack of a plausible leader sequence in genomic or mRNA transcripts, suggest an intracellular localization of the enzyme. To establish further the mechanism of sucrose assimilation by maltase, the existence of a sucrose-inducible H+/sucrose syn-transporter was demonstrated by (1) the kinetics of sucrose-induced [14C]sucrose uptake, (2) recovery of intact [14C]sucrose from ground cells by t.l.c. and (3) transport of 0.83 mol of H+/mol of [14C]sucrose. In total, the above is consistent with a mechanism whereby sucrose is transported into C. albicans to be hydrolysed by an intracellular maltase.
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PMID:Role of maltase in the utilization of sucrose by Candida albicans. 848 4


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