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)

Yeast secretory mutants sec53 and sec59 define a posttranslational stage in the penetration of glycoprotein precursors into the endoplasmic reticulum (ER). In the previous report we showed that at the restrictive temperature (37 degrees C) these mutants accumulate enzymatically inactive and incompletely glycosylated forms of the secretory enzyme invertase and the vacuolar enzyme carboxypeptidase Y. Cell fractionation experiments reveal that these precursor forms remain firmly bound to the ER membrane. However, upon return to the permissive temperature (24 degrees C), the invertase precursors are glycosylated, become partially active, and are secreted. Thermoreversible conversion does not require protein synthesis, but does require energy. In contrast to the effect of these mutations, inhibition of oligosaccharide synthesis with tunicamycin at 37 degrees C causes irreversible accumulation of unglycosylated invertase. The effect of the drug is exaggerated by high temperature since unglycosylated invertase synthesized in the presence of tunicamycin at 25 degrees C is secreted. A portion of the invertase polypeptide accumulated at 37 degrees C is preserved when membranes from sec53 and sec59 are treated with trypsin. In the presence of Triton X-100 or saponin, the invertase is degraded completely. The protected fragment appears to represent a portion of the invertase polypeptide that is embedded in or firmly associated with the ER membrane. This association may develop early during the synthesis of invertase, so that in the absence of translocation, some of the completed polypeptide chain remains exposed on the cytoplasmic surface of the ER.
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PMID:Genes required for completion of import of proteins into the endoplasmic reticulum in yeast. 636 72

Yeast cells secrete a variety of glycosylated proteins. At least two of these proteins, invertase and acid phosphatase, fail to be secreted in a new class of mutants that are temperature-sensitive for growth. Unlike the yeast secretory mutants previously described (class A sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell., 21:205-420), class B sec mutants (sec 53, sec 59) fail to produce active secretory enzymes at the restrictive temperature (37 degrees C). sec 53 and sec 59 appear to be defective in reactions associated with the endoplasmic reticulum. Although protein synthesis continues at a nearly normal rate for 2 h at 37 degrees C, incorporation of [3H]mannose into glycoprotein is reduced. Immunoreactive polypeptide forms of invertase accumulate within the cell which have mobilities on SDS PAGE consistent with incomplete glycosylation: sec 53 produces little or no glycosylated invertase, and sec 59 accumulates forms containing 0-3 of the 9-10 N-linked oligosaccharide chains that are normally added to the protein. In addition to secreted enzymes, maturation of the vacuolar glycoprotein carboxypeptidase Y, incorporation of the plasma membrane sulfate permease activity, and secretion of the major cell wall proteins are blocked at 37 degrees C.
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PMID:Yeast secretory mutants that block the formation of active cell surface enzymes. 636 71

Various amino acid insertions have been introduced into the proximal portion of the signal sequence of secreted yeast invertase. The altered invertase genes have been reintroduced into yeast and monitored for their ability to direct synthesis of secreted invertase in vivo. The insertions should alter the signal polypeptide local secondary structure as predicted by the Chou and Fasman rules (1978). Secretion of these altered invertase polypeptides is not blocked by the amino acid insertions.
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PMID:Conformational alterations in the proximal portion of the yeast invertase signal peptide do not block secretion. 639 90

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.
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PMID:Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae. 644 5

Microscopical studies showed that initial differentiation of the guinea-pig small intestine occurs between days 35 and 55 of foetal development. Changes observed at this time include formation of villi (by day 42), elaboration of submucosal duodenal Brunner's glands (by day 49) and the appearance of a well-developed microvillus membrane (by day 56). Different microvillus membrane-associated hydrolases appear at different stages of foetal and postnatal development. The 'early' enzymes such as aminopeptidase, alkaline phosphatase and sucrase show a sharp increase and reach their maximal levels between days 35 and 50, whereas the late enzymes such as dipeptidyl peptidase IV and lactase increase gradually between days 35 and 50, and reach maximal activity between days 50 and 60. A combination of techniques involving precipitation with Mg2+ followed by fractionation on sucrose density gradients has enabled us to prepare, for the first time, a 21-fold enriched microvillus membrane fraction from the foetal intestine. Polypeptide analysis of this membrane fraction by sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed the presence of developmentally specific polypeptides at different stages of foetal and postnatal development. Three polypeptides of molecular weights 205 000, 80 000 and 47 000 are major microvillus membrane components at the 40-day foetal stage. Two other polypeptides of molecular weights 60 000 and 131 000 are major microvillar components at 56-day and older foetal stages as well as at the 3-day neonatal stage. The adult microvillus membrane contained 112 000 and 122 000 Mr polypeptides as major components. The above results were confirmed using two-dimensional isoelectric focussing-sodium dodecyl sulphate/polyacrylamide gel electrophoretic techniques.
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PMID:Structural and biochemical differentiation of the mammalian small intestine during foetal development. 653 51

The enzyme responsible for all of the isomaltase activity and much of the maltase activity in the small intestine of the Californian sea lion (Zalophus californianus) was isolated by detergent solubilization of the brush-border membrane, followed by immunoadsorption chromatography using antibodies directed against rabbit sucrase-isomaltase. In 0.1% Triton X-100, sea lion isomaltase occurs as a monomer of Mr = 245,000 and is composed of a single polypeptide chain. As judged from the stoichiometry of the covalent binding of the affinity label, conduritol-B-epoxide, this polypeptide chain carries two enzymatically active sites; they are apparently identical and do not show either positive or negative cooperativity. In addition to cross-reacting immunologically with rabbit sucrase-isomaltase, sea lion isomaltase has similar overall enzymatic properties, with the exception of not hydrolyzing sucrose. The Alaskan fur seal (Collarhinus ursinus) has a two-active site isomaltase; however, in contrast to the sea lion, this animal is endowed with a small but significant sucrase activity. Along with (fully active) pro-sucrase-isomaltase, sea lion isomaltase is one of the very few examples of enzymes with more than one active site on a single polypeptide chain acting "in parallel" (rather than "in series"). Furthermore, this enzyme triggers some interesting questions on the phylogenetical pedigree of small intestinal sucrase-isomaltase.
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PMID:A two-active site one-polypeptide enzyme: the isomaltase from sea lion small intestinal brush-border membrane. Its possible phylogenetic relationship with sucrase-isomaltase. 671 26

The radiation target size for invertase activity has been determined for the Saccharomyces cerevisiae glycoprotein which contains 50% carbohydrate. Identical inactivation curves were observed for the native enzyme as well as samples depleted of carbohydrate by incubation with Endo-beta-N-acetylglucosaminidase H. The functional unit of 120,000 daltons was unaltered by the per cent of oligosaccharide cleaved by the enzyme, or by the presence or absence of the released sugars. The irradiated samples showed no change in hexose content even after radiation exposures which grossly destroyed enzymatic activity. Reducing sugars appeared in the irradiated samples, indicating radiation damage to the oligosaccharides. These results unequivocally identify the enzymatically functional portion of the invertase molecule as the polypeptide homodimer, independent of the covalently-bound carbohydrates, and indicate that transfer of radiation energy from protein to oligosaccharide or vice versa is inefficient.
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PMID:Radiation inactivation of the glycoprotein, invertase. 675 36

Poly(A)-containing RNA from Saccharomyces cerevisiae was translated in the wheat germ protein-synthesizing system and yielded as one of its products a polypeptide which was identified as invertase. This characterization was based on the presence of a protein band at 60,000 daltons following immunoprecipitation with a specific invertase antibody, and the formation from this protein of staphylococcal V8 protease and cyanogen bromide peptides, which were similar in size to those from carbohydrate-depleted invertase. The observed increase in invertase activity from the early to mid logarithmic stage of cell growth was synchronous with the appearance of translatable invertase mRN in these cells. No translatable invertase mRNA, however, was detected in late logarithmic cells, although a constant level of invertase activity was present in the culture medium.
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PMID:Growth phase dependence of invertase mRNA levels in yeast. 699 70

Detergent-solubilized pig intestinal sucrase . isomaltase (EC 3.2.1.48-EC 3.2.1.10) was purified 40 to 100 times with a yield of 10 to 20% by a rapid immunoadsorbent technique. The purified enzyme was shown to be homogeneous by immunoelectrophoresis and was essentially free of other known brush border peptidases and disaccharidases. Intestinal sucrase . isomaltase isolated from pigs with intact pancreatic ducts consisted of two polypeptide chains with apparent molecular weights of 140,000 and 150,000, respectively. In contrast, the enzyme isolated from pigs in which the pancreas was completely disconnected from the duodenum 3 days before killing migrated in polyacrylamide gel electrophoresis in dodecyl sulfate as a single polypeptide chain with an apparent molecular weight of 260,000. Treatment with pancreatic proteases in vitro converted the large polypeptide chain into bands with molecular weights equal to or somewhat larger than those of sucrase . isomaltase purified from normal pigs. No increase of enzymatic activity could be detected during this transformation. It is suggested that the single-chain sucrase . isomaltase represents a precursor, which is converted to the final sucrase . isomaltase in vivo by pancreatic proteolytic enzymes. This is one of the few examples in vertebrates of a single polypeptide chain carrying two enzymatically active sites. The significance of the result for the mechanism of the biosynthesis of sucrase . isomaltase is discussed.
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PMID:A fully active, two-active-site, single-chain sucrase.isomaltase from pig small intestine. Implications for the biosynthesis of a mammalian integral stalked membrane protein. 700 20

We have studied regulation of invertase putative structural genes (SUC) in S. cerevisiae and the synthetic relationship between secreted, glycosylated invertase (E.C.3.2.1.26) and the cytoplasmic, nonglycosylated form of the enzyme. Using immunoprecipitation and gel electrophoresis, we have analyzed invertase polypeptides and glycopeptides synthesized in vitro and in vivo. Analysis of size-fractionated mRNA from a SUC2 strain has shown that three mature, catabolite-repressible mRNA species direct the in vitro synthesis of three invertase polypeptides that have differing molecular weights. Two of these polypeptides, P63 and P62 (63 and 62 kd), are larger than the polypeptides of the secreted enzyme and are cotranslationally processed by microsomal membranes in vitro to yield secreted invertase glycopeptides (GP90 and GP87). The smallest polypeptide, P60 (60 kd), which comigrates electrophoretically with cytoplasmic invertase, is not processed. Posttranslationally, a microsomal-membrane detergent extract removes approximately 20 aminoacids from P62 but not from P60. In vitro translations of mRNAs from a genetically confirmed suc3 mutant strain, from the parental SUC3 strain and from derivative meiotic segregants have shown that the three polypeptides (and therefore three mRNA species) are encoded by one gene. Analysis of in vivo radiolabeled invertase from the same SUC3 and suc3 strains has verified that the SUC3 locus contains the structural gene for secreted and cytoplasmic invertase. Through the derepressed synthesis of multiple primary or processed transcripts, the SUC2 and SUC3 genes are regulated to produce multiple invertase polypeptides. The larger two polypeptides appear to be processed and secreted to yield glycosylated invertase, while the smallest remains in the cytoplasm.
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PMID:Distinct repressible mRNAs for cytoplasmic and secreted yeast invertase are encoded by a single gene. 702 48


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