Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.26 (invertase)
 4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Saccharomyces cerevisiae Man9-alpha-mannosidase, responsible for trimming Man9GlcNAc2 in the endoplasmic reticulum to Man8GlcNAc2, the substrate for oligosaccharide elongation, has been purified to homogeneity from stabilized microsomal membranes without employing autolytic digestion. The activity was solubilized by the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulphonate (CHAPS), whose presence was necessary for maximal activity. Purification included Q-Sepharose ion-exchange chromatography, preparative isoelectric focusing and HPLC gel filtration on TSK 3000 matrix. Overall purification from post-nuclear supernatants was estimated to be 110,000-fold with a 50% recovery of activity. The purified enzyme hydrolysed Man9GlcNAc1,2 from thyroglobulin or oligosaccharide-lipid, but not invertase Man9GlcNAc, Man1 alpha 2Man1 alpha OCH3 or p-nitrophenyl-alpha-D-mannopyranoside. Conversion of thyroglobulin Man9GlcNAc to Man8GlcNAc was linear with time and enzyme concentration, with an apparent Km of 0.2 mM and a specific activity of 220 IU/mg. Glc3Man9GlcNAc2 from oligosaccharide-lipid was as good a substrate as Man9GlcNAc, but the lipid-linked Man7GlcNAc2 isomer was hydrolysed at only 10% of this rate. Hydrolysis of defined isomers of IgM and bovine thyroglobulin Man6,7,8GlcNAc indicated that, for maximal alpha 1,2-mannosidase activity, only the alpha 1,2-linked terminal mannoses on the alpha 3 branch of the Man9GlcNAc precursor were dispensable. Isomers lacking the terminal alpha 1,2-linked mannose on the alpha 6 branch were hydrolysed at only approximately 10% of the maximal rate. The enzyme exhibited a pI of 5.3 and a pH optimum at 6.5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the absence of reducing agents gave a single sharp band at 66 kDa, while in the presence of beta-mercaptoethanol equimolar amounts of two peptides, one of 44 kDa and one of 23 kDa, were obtained. Sizing on Sephacryl SF300, Superose 12 and TSK 3000 provided a holoenzyme mol. wt of 60-68 kDa, indicating that the isolated active form of the Man9-alpha-mannosidase was composed of one each of the sulphydryl-bonded dissimilar peptides. The enzyme bound to concanavalin A (ConA)-Sepharose and was eluted with alpha-methylmannoside, indicating the presence of high-mannose oligosaccharides. The Man9-alpha-mannosidase required low levels of Ca2+, which could be removed by EGTA. Activity was restored by Ca2+ or Zn2+, but not by Mg2+ or Mn2+.
Glycobiology 1991 Dec
PMID:Glycoprotein biosynthesis in yeast: purification and characterization of the endoplasmic reticulum Man9 processing alpha-mannosidase. 182 40

The TUP1 and CYC8 (= SSN6) genes of Saccharomyces cerevisiae play a major role in glucose repression. Mutations in either TUP1 or CYC8 eliminate or reduce glucose repression of many repressible genes and induce other phenotypes, including flocculence, failure to sporulate, and sterility of MAT alpha cells. The TUP1 gene was isolated in a screen for genes that regulate mating type (V.L. MacKay, Methods Enzymol. 101:325-343, 1983). We found that a 3.5-kb restriction fragment was sufficient for complete complementation of tup1-100. The gene was further localized by insertional mutagenesis and RNA mapping. Sequence analysis of 2.9 kb of DNA including TUP1 revealed only one long open reading frame which predicts a protein of molecular weight 78,221. The predicted protein is rich in serine, threonine, and glutamine. In the carboxyl region there are six repeats of a pattern of about 43 amino acids. This same pattern of conserved residues is seen in the beta subunit of transducin and the yeast CDC4 gene product. Insertion and deletion mutants are viable, with the same range of phenotypes as for point mutants. Deletions of the 3' end of the coding region produced the same mutant phenotypes as did total deletions, suggesting that the C terminus is critical for TUP1 function. Strains with deletions in both the CYC8 and TUP1 genes are viable, with phenotypes similar to those of strains with a single deletion. A deletion mutation of TUP1 was able to suppress the snf1 mutation block on expression of the SUC2 gene encoding invertase.
Mol Cell Biol 1990 Dec
PMID:Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae. 224 69

The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined. Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E. coli and Z. mobilis consensus sequences. Extracts from E. coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity. However, no transfructosylation activity (exchange reaction or levan formation) could be detected. This sucrase activity was different from that observed with the purified extracellular protein B46 from Z. mobilis. These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity. Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z. mobilis. The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus.
J Bacteriol 1990 Dec
PMID:Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis. 225 50

The SNF1 protein kinase is required for expression of the invertase gene in response to glucose deprivation in Saccharomyces cerevisiae. We selected for genes that in multicopy suppress the invertase defect of temperature-sensitive snf1 mutants. Increased dosage of the MSN1 gene restores high-level, regulated invertase expression in snf1-ts mutants, and disruption of MSN1 in the wild type reduces invertase expression a fewfold. MSN1 gene dosage does not affect SNF1 protein kinase activity in vitro. MSN1 encodes a 43-kilodalton protein, and a MSN1-beta-galactosidase fusion protein was localized in the nucleus. A LexA-MSN1 fusion protein, when bound to a lexA operator, activates transcription of an adjacent promoter. In vitro synthesized MSN1 protein exhibits weak, nonspecific DNA-binding activity.
Nucleic Acids Res 1990 Dec 11
PMID:Increased dosage of the MSN1 gene restores invertase expression in yeast mutants defective in the SNF1 protein kinase. 226 57

We have investigated the vacuolar delivery of alpha-mannosidase, a marker enzyme of the vacuolar membrane in the yeast Saccharomyces cerevisiae, and found that the enzyme has several unique characteristics in its biosynthesis and vacuolar delivery. alpha-Mannosidase has no typical signal sequence (Yoshihisa, T., and Anraku, Y. (1989) Biochem. Biophys. Res. Commun. 163, 908-915) but is located on the inner surface of the vacuolar membrane. The enzyme is synthesized as a 107-kDa polypeptide and converted to a 73-kDa polypeptide. Although the conversion depends on a vacuolar processing protease, proteinase A, it is much slower (t1/2 = 10 h) than the proteinase A-dependent processing of other vacuolar proteins. None of Asn-X-Thr/Ser sites on the 107-kDa alpha-mannosidase or on two alpha-mannosidase-invertase fusion proteins that are localized inside the vacuole receives N-linked oligosaccharide, whereas those sites on a carboxypeptidase Y-alpha-mannosidase fusion protein are N-glycosylated. The newly synthesized alpha-mannosidase is normally delivered to the vacuole and converted to the 73-kDa polypeptide even when the secretory pathway is blocked by a subset of sec mutations. These characteristics are different from those of other vacuolar proteins targeted to the vacuole via the secretory pathway. We conclude that alpha-mannosidase is delivered to the vacuole in a novel pathway separate from the secretory pathway.
J Biol Chem 1990 Dec 25
PMID:A novel pathway of import of alpha-mannosidase, a marker enzyme of vacuolar membrane, in Saccharomyces cerevisiae. 226 33

The effect of epidermal growth factor (EGF) on the ontogeny of the gastrointestinal tract was examined in New Zealand White rabbits. EGF, 40 micrograms X kg-1 X day-1, was administered to suckling animals from 3-18 days of age either intraperitoneally or orogastrically. Controls received saline. Animals were killed at 17-18 days of age. Body weight and wet weight of stomach, pancreas, and 10-cm segments of proximal, mid, and distal small intestine were measured. The total pancreas was homogenized for determination of protein, DNA, and amylase, and the intestinal mucosa was scraped, weighed, and homogenized for estimation of protein, DNA, sucrase, and lactase. While body weights were similar wet weight of stomach and pancreas were increased by intraperitoneal and orogastric EGF. Small intestinal wet weights were increased in all segments by intraperitoneal but not orogastric EGF, and both routes significantly increased mucosal DNA in the distal segment. EGF administered orogastrically induced precocious maturation of intestinal brush-border disaccharidase activities but had no effect on pancreatic amylase, whereas EGF administered intraperitoneally induced precocious maturation of pancreatic amylase but had no effect on brush-border disaccharidase activities. These findings suggest that both systemic and oral EGF play a role in regulating growth and postnatal maturation of the gastrointestinal tract.
Am J Physiol 1985 Dec
PMID:Effect of epidermal growth factor on ontogeny of the gastrointestinal tract. 241 92

We have used cell fusion to address the question of whether macromolecules are rapidly exchanged between lysosomes. Donor cell lysosomes were labeled by the long-term internalization of the fluid-phase pinocytic markers, invertase (sucrase), Lucifer Yellow, FITC-conjugated dextran, or Texas red-conjugated dextran. Recipient cells contained lysosomes swollen by long-term internalization of dilute sucrose or marked by an overnight FITC-dextran uptake. Cells were incubated for 1 or 2 h in marker-free media before cell fusion to clear any marker from an endosomal compartment. Recipient cells were infected with vesicular stomatitis virus as a fusogen. Donor and recipient cells were co-cultured for 1 or 2 h and then fused by a brief exposure to pH 5. In all cases, extensive exchange of content between donor and recipient cell lysosomes was observed at 37 degrees C. Incubation of cell syncytia at 17 degrees C blocked lysosome/lysosome exchange, although a "priming" process(es) appeared to occur at 17 degrees C. The kinetics of lysosome/lysosome exchange in fusions between cells containing invertase-positive lysosomes and sucrose-positive lysosomes indicated that lysosome/lysosome exchange was as rapid, if not more rapid, than endosome/lysosome exchange. These experiments suggest that in vivo the lysosome is a rapidly intermixing organellar compartment.
J Cell Biol 1987 Dec
PMID:Chinese hamster ovary cell lysosomes rapidly exchange contents. 244 96

Previous studies have demonstrated that external abdominal irradiation alters intestinal nutrient transport and brush border membrane (BBM) phospholipid content. This study was undertaken to assess the possible protective effect of oral dosing of enprostil, a synthetic E2 prostaglandin, on the BBM marker enzyme and lipid composition of non-irradiated control (CONT) animals and of rats exposed seven days perviously to a single dose of 600 cGy external abdominal irradiation (RAD). Half the CONT and RAD animals were orally dosed with enprostil 5 mcg/kg body weight, two and one mornings before the day of irradiation, and one hour before 600 cGy; the remaining half were dosed with placebo according to the same schedule. BBM were isolated and purified from the animals seven days post-irradiation for analysis of marker enzymes and lipid composition. Radiation was associated with a decline in jejunal and ileal BBM activity of sucrase. Enprostil was associated with a decline in the ratio of alkaline phosphatase/sucrase in jejunal BBM from irradiated rats, despite its preventing a radiation-associated decline in BBM sucrase activity. Radiation was associated with changes in ileal BBM phospholipids, and these alterations were not prevented with enprostil. Furthermore, enprostil given to non-radiated control rats altered BBM composition, such as increased jejunal and ileal lysophosphatidylethanolamine, and lowered ileal BBM sphingomyelin and phosphatidylethanolamine. Thus, enprostil failed to protect the intestine from the effects of abdominal irradiation on BBM phospholipids, but did partially prevent the effect of abdominal irradiation on jejunal sucrase activity.
Clin Invest Med 1989 Dec
PMID:Effect of oral enprostil, a synthetic prostaglandin E2, on intestinal brush border membrane lipid composition following abdominal irradiation in the rat. 251 20

The invertase (beta-fructofuranosidase, EC 3.2.1.26) of the rumen holotrich ciliate Isotricha prostoma has been purified. This is the first report of an enzyme purification from a known species of rumen protozoon. Cells were disrupted by ultrasonic treatment and the enzyme was purified from the cell-free extract by three successive liquid column chromatographies (Sepharose CL4B/octyl-Sepharose CL4B, DE52 DEAE-cellulose and concanavalin A-Sepharose 4B). This resulted in a 160-fold purification and a 15% yield. The major form of the purified enzyme was a tetramer with Mr about 350,000 that was readily dissociated by electrophoresis. The invertase was heterogeneous, as five types of monomers were shown by SDS/polyacrylamide-gel electrophoresis after denaturation. Part of this heterogeneity was due to different glycosylated forms of one of the polypeptides present in the purified enzyme. Isotricha prostoma invertase exhibited maximum activity at pH 5.5-6.0 and 50 degrees C. The kinetic properties of the purified enzyme were very similar to those of invertases from other sources such as yeast or plants (substrate and product inhibition, transferase activity).
Biochem J 1989 Dec 15
PMID:Purification and characterization of a heterogeneous glycosylated invertase from the rumen holotrich ciliate Isotricha prostoma. 251 52

1. Sugar-containing diets chosen not to affect intestinal structure or enterocyte turnover have been fed to mice previously maintained on a low carbohydrate diet in order to determine their ability to induce disaccharidase enzymes in the small intestine. 2. Glucose-, fructose- and 3-O-methyl-glucose-containing diets increased sucrase and maltase but not lactase activities in mouse jejunal homogenates. These effects were either absent or negligible in more distal regions of the small intestine. 3. Placing mice on glucose-, fructose- or 3-O-methyl-glucose-containing diets was further shown, by quantitative cytochemistry, to cause a 1.6-, 2.6- and 3.2-fold increase in the initial rate at which alpha-glucosidase activity (sucrase + maltase) appeared in the brush-border membrane of developing enterocytes. 4. The time during which alpha-glucosidase activity increased in enterocyte brush-border membranes fell from 30 h for low carbohydrate fed mice to 21, 19 and 17 h in mice fed glucose, fructose and 3-O-methyl-glucose respectively. Change of diet had no effect on the kinetics of lactase expression by developing enterocytes. 5. Maximal alpha-glucosidase activity detected in enterocyte brush-border membranes is equal to RT, where R is the initial rate of enzyme appearance and T is the time during which this rate operates. The ability of sugars to increase R selectively, but only at the expense of T, defines unexpected limits to the capacity of enterocytes to adapt to changes in luminal nutrition. 6. The above results are discussed in relation to other aspects of enterocyte differentiation recently subjected to quantitative analysis. The need to standardize other aspects of intestinal physiology and redefine the energy content of diets containing non-metabolizable substrates in this type of work is also emphasized.
J Physiol 1989 Dec
PMID:Sugar-dependent selective induction of mouse jejunal disaccharidase activities. 251 26


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>