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)

The isolation of Saccharomyces cerevisiae plasma membrane was carried out after hypotonic lysis of yeast protoplasts treated with concanavalin A by two independent methods: a, at low speed centrifugation and b, at high speed centrifugation in a density gradient. Several techniques (electron microscopic, enzymic, tagging, etc.) were used to ascertain the degree of purification of the plasma membranes obtained. The low speed centrifugation technique as compared with the other method gave a higher yield of plasma membranes with a similar degree of purification. Analysis of the yeast plasma membrane of normally growing cells by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed at least 25 polypeptide bands. Twelve glycoprotein bands were also found, and their apparent molecular weights were determined. Treatment of the protoplasts with cycloheximide resulted in a significant decrease in the carbohydrate and protein content of the plasma membrane. The electrophoretic pattern of the plasma membrane of cycloheximide-treated cells showed a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff-positive bands. The isoelectric point of the most abundant proteins was low (pI 4) or lower than expected from previous data. A large part of the mannosyl transferase activity found in the cell (80%) was associated with the internal membranes, the remaining activity (20%) was located in the plasma membrane preparation. Part of the mannosyl transferase activity of the cells is located at the plasma membrane surface. Invertase (an external mannoprotein) is found in both the plasma and internal membranes, and as the specific activity dropped significantly following cycloheximide treatment of the cells, it is suggested that these membranes systems are the structures for the glycosylation of a precursor invertase and its subsequent release into the periplasmic space. Other transferase found in the plasma membrane preparation transfers glucose residues from UDPglucose to a poly(alpha(1 leads to 4) polymer identified as glycogen.
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PMID:The plasma membrane of Saccharomyces cerevisiae. Isolation and some properties. 34 15

Intracellular invertase from Streptococcus mutans GS5 was purified to near homogeneity by gel filtration and ion-exchange chromatography followed by preparative polyacrylamide gel electrophoresis. The invertase appeared to be composed of a single polypeptide chain with a molecular weight of 48,000. Extracellular invertase was identified in strain GS5 and was determined to have a molecular weight of 500,000. No antigenic relationship between these two forms of invertase was observed since antibody prepared against purified intracellular invertase neither affected extracellular invertase activity nor precipitated that enzyme on immunodiffusion. No antigenic relatedness between intracellular invertase and glucosyl- and fructosyl- transferases was detected since cross-reactivity with antibody prepared against either enzyme fraction was not observed after immunodiffusion. Using immunodiffusion and quantitative precipitin data, we examined the relationships of other S. mutans intracellular invertases to the serotype c enzyme. It appeared that the intracellular invertases from serotypes e, f, and g were structurally similar to the enzyme from serotype c, whereas the structure of invertases from serotypes a, b, and d appeared less similar to that of enzyme from serotype c.
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PMID:Purification and antigenic properties of intracellular invertase from Streptococcus mutans. 45 62

The [3H] phlorizin-binding component of brush border vesicles was enriched in situ by negative purification. Several procedures, known to effect selective solubilization of membrane components, were used separately or in combination to remove proteins unrelated to the binding. Deoxycholate ruptured the vesicles and released 67% of their protein, thereby increasing the specific [3H] phlorizin-binding activity of the pellet three-to fourfold. Extracting the deoxycholate-pellets with either NaI or alkaline solutions released up to 38% of the deoxycholate-insoluble protein without significantly affecting phlorizin binding. The polypeptide composition of the membranes at the different stages was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. A number of polypeptides present in the original vesicles could be ruled out as essential components of the [3H] phlorizin binding entity. Intact and deoxycholate-treated vesicles were subjected to proteolytic attack. Papain liberated sucrase and isomaltase from intact vesicles, but affected neither other Coomassie-stained bands nor phlorizin binding. Neither the protein composition nor the binding properties of sealed vesicles were influenced by trypsin or chymotrypsin. However, all the proteolytic enzymes tested on deoxycholate-treated membranes substantially reduced [3H] phlorizin binding and produced concomitantly the disappearance of several bands from the electrophoretic profile. Pretreatment of vesicles with papain, followed by deoxycholate extraction and incubation in alkaline media, increased the specific binding activity of the membranes up to ninefold by removing close to 90% of the protein. A limited number of polypeptides are suggested as possible candidates for the glycoside-binding site of intestinal brush borders.
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PMID:Partial purification of the sugar carrier of intestinal brush border membranes. Enrichment of the phlorizin-binding component by selective extractions. 52 29

Carboxypeptidase Y from Saccharomyces cerevisiae contains 14% mannose, the only neutral sugar present. An antiserum can be raised in rabbits which reacts with both the protein and the sugar moieties of the enzyme. This antiserum also precipitates yeast invertase and yeast cell wall mannan. Thus carboxypeptidase Y, which is known to be localized in yeast vacuoles, is very probably a mannoprotein. Tunicamycin inhibits the apparent formation of carboxypeptidase Y to a similar extent as that of the externally localized mannoprotein, invertase. No accumulation of an inactive nonglycosylated or partly glycosylated carboxypeptidase Y occurs as determined by the immunoprecipitation technique. Tunicamycin also inhibits the apparent formation of proteinase A, whereas it does not affect the increase in the activities of a number of other enzymes. It is suggested that in the synthesis of glycoproteins there exists a regulatory link between the synthesis of their polypeptide chains and the reactions involved in their glycosylation.
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PMID:Inhibition of the apparent rate of synthesis on the vacuolar glycoprotein carboxypeptidase Y and its protein antigen by turicamycin in Saccharomyces cerevisiae. 79 Oct 99

Sucrase-isomaltase complex and its functional subunits have been identified in homogenates of human small intestinal mucosa by use of Sephadex G-200 (superfine) chromatography aided by affinity of the isomaltase moiety for the dextran gel. The isomaltase subunit binds strongly to the gel at 4 degrees, and is eluted only after 2 column volumes; earlier recovery as a sharp peak can be achieved by raising column temperature to 37 degrees after elution of other proteins. Bio-Gel P-300 chromatography, density gradient, and equilibrium centrifugation demonstrated that the sucrase subunit (Stokes radius = 45 A, frictional ratio = 1.32, s20,w = 6.9, MW = 130,000) and the isomaltase subunit (Stokes radius = 45 A, frictional ratio = 1.30, s20,w = 6.6, MW = 120,000) are similar but unequal in size. The sucrase-isomaltase complex (Stokes radius = 70 A, frictional ratio = 1.61, s20,w = 9.8, MW = 280,000), appears to be an elongated hybrid molecule that is less symmetrical than either of itt subunits. Apparent Km and pH activity curves were indistinguishable for each enzyme whether present in the hybrid or in the free state. The sucrase-isomaltase complex, accounting for approximately 90 percent of native intestinal sucrase and isomaltase activities, was isolated and cleaved by 0.01 M beta-mercaptoethanol/6 M urea treatment into active sucrase and isomaltase subunits having biochemical characteristics identical with those of the free native moieties. Sodium dodecyl sulfate acrylamide gell electrophoresis of the complex also produced subunits having molecular weights very close to those for the active free sucrase and isomaltase moieties, indicating that each alpha-glucosidase appears to consist of a single polypeptide chain. Immunization of rabbits with pure sucrase-isomaltase complex yielded a monospecific precipitating antibody that reacted with the hybrid and the sucrase subunit, but had minimal affinity for the isomaltase subunit, providing further evidence that the sucrase-isomaltase molecule is a hybrid consisting of two distinct alpha-glucosidases.
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PMID:Human intestinal sucrase-isomaltase. Identification of free sucrase and isomaltase and cleavage of the hybrid into active distinct subunits. 80 75

Nine sucrose nonfermenting mutants have been isolated from yeast strain EK-6B, carrying the tightly linked SUC3 and MAL3 genes. These mutants are allelic to the SUC3 gene recessive in nature and none of them has detectable levels of either internal or external invertase. A single point mutation leading to the loss of both invertases suggests that either SUC3 is a control gene or codes for a polypeptide which is shared by both invertases.
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PMID:Genetic control of invertase formation in Saccharomyces cerevisiae. I. Isolation and characterization of mutants affecting sucrose utilization. 110 7

Production of heterologous proteins by yeast secretion imposes additional factors that need to be considered, which do not appear with production by direct expression. These include additional intracellular polypeptide processing dynamics through the secretory organelles and the protein concentration in the culture medium, which is the usual final destination of the product. Optimal control theory is applied to optimize fed-batch production of secreted protein. We maximize an objective function that includes both total production rate and product concentration. A mutant invertase is chosen as the model heterologous secretory protein. Optimal control control strategies have been obtained for the use of two different promoters for the gene transcription, a dere-pressible SUC2 promoter and a strong glycolytic GPD promoter. With the use of the strong GPD promoter, achieving maximum production occurs on the singular arc of maximum specific growth rate. As the object switches to maximum product concentration, operation occurs for longer periods of time at a slow glucose singular arc condition. The optimal control for maximizing protein production with the weak SUC2 promoter requires transitions between high and low glucose concentrations associated with multiple distinct singular arc conditions. For maximum product concentration, the high concentration branches of the singular arc supporting maximum growth rate and maximum secretion rate disappear. Operation stays essentially on the low glucose concentration branch of the singular arc, which maximizes the protein production rate and minimizes the dilution of the broth product concentration.
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PMID:Effect of transcription promoters on the optimal production of secreted protein in fed-batch reactors. 136 71

Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.
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PMID:Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota. 154 2

Four yeast secretion signals, the 19-amino-acid invertase signal sequence, the 17-amino-acid acid-phosphatase signal sequence, and the pre-sequence and prepro-sequence of prepro-alpha-factor have been used to look for the secretion of recombinant human insulin-like growth factor 1 (IGF1) from Saccharomyces cerevisiae. Only the prepro-sequence, often referred to as the alpha-factor leader and consisting of an N-terminal 19-amino-acid pre-sequence or signal sequence attached to a 66-amino-acid pro-region, permits secretion of IGF1. The signal sequences alone do not allow the translocation of IGF1 into the endoplasmic reticulum. This is evident from the fact that IGF1-like molecules, to which the signal sequences are still attached, accumulate intracellularly in the cytosol. Fusion of the pro-region of the alpha-factor leader to the C-terminus of the acid-phosphatase and invertase signal sequences allows IGF1 to be secreted once again. These results reveal the essential role of the pro-region of the alpha-factor leader in the secretion of IGF1 and indicate that it may have a function in guiding a nascent IGF1 polypeptide to a state in which translocation can occur.
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PMID:The pro-region of the yeast prepro-alpha-factor is essential for membrane translocation of human insulin-like growth factor 1 in vivo. 160 61

Mutations in the sucrase-isomaltase gene can lead to the synthesis of transport-incompetent or functionally altered enzyme in congenital sucrase-isomaltase deficiency (CSID) (Naim, H. Y., J. Roth, E. Sterchi, M. Lentze, P. Milla, J. Schmitz, and H. P. Hauri. J. Clin. Invest. 82:667-679). In this paper we have characterized two novel mutant phenotypes of CSID at the subcellular and protein levels. The first phenotype revealed a sucrase-isomaltase protein that is synthesized as a single chain, mannose-rich polypeptide precursor (pro-SI) and is electrophoretically indistinguishable from pro-SI in normal controls. By contrast to normal controls, however, pro-SI does not undergo terminal glycosylation in the Golgi apparatus. Subcellular localization of pro-SI by immunoelectron microscopy revealed unusual labeling of the molecule in the basolateral membrane and no labeling in the brush border membrane thus indicating that pro-SI is missorted to the basolateral membrane. Mapping of biosynthetically labeled pro-SI with four epitope- and conformation-specific monoclonal antibodies suggested that conformational and/or structural alterations in the pro-SI protein have prevented posttranslational processing of the carbohydrate chains of the mannose-rich precursor and have lead to its missorting to the basolateral membrane. The second phenotype revealed two variants of pro-SI precursors that differ in their content of mannose-rich oligosaccharides. Conversion of these forms to a complex glycosylated polypeptide occurs at a slow rate and is incomplete. Unlike its counterpart in normal controls, pro-SI in this phenotype is intracellularly cleaved. This cleavage produces an isomaltase-like subunit that is transport competent and is correctly sorted to the brush border membrane since it could be localized in the brush border membrane by anti-isomaltase mAb. The sucrase subunit is not transported to the cell surface and is most likely degraded intracellularly. We conclude that structural features in the isomaltase region of pro-SI are required for transport and sorting of the sucrase-isomaltase complex.
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PMID:Naturally occurring mutations in intestinal sucrase-isomaltase provide evidence for the existence of an intracellular sorting signal in the isomaltase subunit. 171 81


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