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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)

1. The distributions of several enzymes and other marker components were examined after zonal centrifugations of whole homogenates from glucose-repressed Saccharomyces cerevisiae on sucrose and iso-osmotic Ficoll, and the composition and morphology of the fractions were investigated. 2. After high-speed zonal centrifugation most of the protein, acid and alkaline phosphatases, alkaline pyrophosphatase, adenosine monophosphatase, beta-fructofuranosidase, alpha-mannosidase, NADPH-cytochrome c oxidoreductase and an appreciable amount of phospholipid and sterol were non-sedimentable, i.e. were at densities below 1.09 (g/cm3). Most of the RNA was at p=1.06-1.08 in Ficoll and at p=1.09-1.11 in sucrose. 3. The bulk of the Mg2+-dependent adenosine triphosphatase (Mg-ATPase) was coincident with the main peak of phospholipid and sterol, at median density 1.10, which was also rich in smooth-membrane vesicles. In Ficoll, a minor peak of phospholipid and sterol at p-1.12-1.15 contained a smaller part of the oligomycin-insensitive Mg-ATPase and heavy membrane fragments. In sucrose, several minor peaks of Mg-ATPase were in the mitochondrial density range, and a peak of oligomycin-insensitive Mg-ATPase coincident with a minor peak of phospholipid and sterol at around p-1.25 contained heavy membrane fragments of high carbohydrate content, especially mannose. 4. Further purification of the oligomycin-insensitive Mg-ATPase containing membrane preparations was performed on Urografin gradients. 5. It is argued that the oligomycin-insensitive Mg-ATPase containing membranes are fragments of the plasma membrane, but have different densities because they contain different amounts of glycoprotein particles.
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PMID:Distribution of membranes, especially of plasma-membrane fragments, during zonal centrifugations of homogenates from glucose-repressed Saccharomyces Cerevisiae. 13 74

Previously, Man8-14GlcNAc oligosaccharides were isolated from highly purified Saccharomyces cerevisiae invertase and shown by one-dimensional 1H NMR spectroscopy and alpha 1,2-linkage-specific mannosidase digestion to constitute a homologous series of nearly homogeneous compounds, which appeared to define the intermediates in oligosaccharide core synthesis in yeast (Trimble, R.B. and Atkinson, P.H. (1986) J. Biol. Chem., 261, 9815-9824). To evaluate whether invertase oligosaccharides reflected global core processing of yeast glycans, the soluble glycoprotein pool of disrupted log-phase cells was digested with endo-beta-N-acetyl-glucosaminidase H and Man8-13GlcNAc were isolated by Bio-Gel P-4 chromatography. Although analysis of each size class by one-dimensional 400 MHz and two-dimensional 500 MHz phase-sensitive COSY 1H NMR spectroscopy revealed considerable structural heterogeneity in all but Man8GlcNAc, the major positional isomer in Man9-13GlcNAc (approximately 50%) was identical to that previously elucidated on invertase. The heterogeneity resided in four families of oligosaccharides: (i) Glc3Man9GlcNAc----Man8 GlcNAc trimming intermediates; (ii) alpha-mannosidase degradation products of the principal isomers; (iii) mannan elongation intermediates; (iv) core structures with the alpha 1,2-linked mannose usually removed by the processing alpha-mannosidase. The potential for the vacuolar alpha-mannosidase (AMS1 gene product) to generate heterogeneity in vitro was confirmed by isolating oligosaccharides from AMS1 and ams1 yeast strains in the presence of a Man13GlcNAc[3H]-ol marker (where GlcNAc[3H]-ol is N-acetylglucosamin [1-3H]itol). Degradation of the Man13GlcNAc[3H]-ol to Man9-12GlcNAc[3H]-ol occurred in the former, but not in the latter. A role for the vacuolar alpha-mannosidase in generating at least some heterogeneity in vivo was inferred from the 1H NMR spectrum of the AMS1 Man11GlcNAc pool, which showed more structural isomerism than seen in the spectrum of a comparable ams1 Man11GlcNAc preparation. Thus, the principal biosynthetic pathway of inner core mannan in Saccharomyces is defined by the Man8-13GlcNAc oligosaccharides found on external invertase, while structural heterogeneity in these size classes results from precursor processing in the endoplasmic reticulum, core extension in the Golgi and metabolic degradation in the vacuole.
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PMID:Structural heterogeneity in the Man8-13GlcNAc oligosaccharides from log-phase Saccharomyces yeast: a one- and two-dimensional 1H NMR spectroscopic study. 155 Sep 91

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+.
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PMID:Glycoprotein biosynthesis in yeast: purification and characterization of the endoplasmic reticulum Man9 processing alpha-mannosidase. 182 40

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.
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PMID:A novel pathway of import of alpha-mannosidase, a marker enzyme of vacuolar membrane, in Saccharomyces cerevisiae. 226 33

Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth. This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case. Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II. Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes. Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.
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PMID:Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. 306 74

The immature sugar cane stalks studied contained less than 7% sucrose, and showed the activities of enzymes such as invertase, alpha-galactosidase, alpha-mannosidase, beta-N-acetylglucosaminidase, beta-glucosidase, beta-xylosidase, and beta-galactosidase. The alpha-galactosidase was highly purified by ammonium sulfate fractionation, gel filtration on a Sephadex G-100 column, ionexchange chromatography on DEAE-cellulose, and CM-cellulose columns, and heat treatment (60 degrees C, 15 min) in the presence of 0.2 m D-galactose. In polyacrylamide gel electrophoresis, the purified enzyme was homogeneous, having a molecular weight of approximately 46,000. In gelfiltration, it was approximately 47,000. The activity was optimum at pH 4.5 and at 60 degrees C. The purified enzyme hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km, 0.83 mM; Vmax, 25.0 mumol/mg/min), raffinose (Km, 25.9 mM; Vmax, 15.4 mumol/mg/min), and stachyose (Km, 13.0 mM; Vmax 2.7 mumol/mg/min), in addition to melibiose, guar gum, and locust bean gum. The hydrolysis of p-nitrophenyl-alpha-D-galactopyranoside was markedly inhibited by HgCl2, AgNO3, p-chloromercuribenzoate (PCMB), L-ascorbic acid, melibiose, stachyose, and D-galactose. Also the purified enzyme showed a lectin activity with trypsinized erythrocytes.
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PMID:Purification and properties of alpha-galactosidase from immature stalks of Saccharum officinarum (sugar cane). 627 79

The addition of 88 mM sucrose to the culture medium of human skin fibroblasts from normal subjects caused remarkable increase in the intracellular lysosomal hydrolase activities. The mechanism of this induction by sucrose loading was carefully studied with several fibroblast strains of different inherited lysosomal storage disorders. In single lysosomal hydrolase defect such as GM1-gangliosidosis, mannosidosis and Sandhoff disease, no induction of the deficient hydrolase was found with 88 mM sucrose loading. In contrast, sucrose loading caused normalization of intracellular lysosomal hydrolase activities in I-cell disease fibroblasts and cytoplasmic inclusion materials disappeared. Subsequent investigations reveal that I-cell disease cells are classified into three subgroups by the degree of hydrolase induction by sucrose loading; a high responding, an intermediate responding and a no-response group. The heterogeneity may be based upon different induction by sucrose loading of the enzyme, probably the residual phosphotransferase which is involved in the processing steps of lysosomal enzyme molecules. With the addition of mannose-6-phosphate and 10 mM NH4Cl to cultured skin fibroblasts, it was shown that sucrose loading caused increased synthesis of lysosomal enzyme proteins. The result of the test with 2,4-dinitrophenol suggests that sucrose is indeed pinocytosed by cultured human skin fibroblasts and localized in lysosomes and that this event is the essential factor to trigger the induction of lysosomal hydrolases. Simultaneous loading of both invertase and sucrose in cultured cells caused no induction of alpha-mannosidase activity. This result indicates that invertase is also pinocytosed, reaches the lysosomes and hydrolyzes sucrose in the lysosomes. Lysosomal overloading with sucrose resulted in induction of lysosomal hydrolases and invertase blocked the induction of alpha-mannosidase activity. However, some induction still exists in beta-galactosidase and alpha-fucosidase activity. Thus it is very likely that the induction of lysosomal hydrolases demands a complicated process. In this article, we investigated the effects of sucrose on the lysosomal hydrolases in cultured human skin fibroblasts of several inherited lysosomal storage disorders and normal subjects and discuss the possible mechanism of the induction of lysosomal hydrolase activities by sucrose loading.
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PMID:The effects of sucrose loading on lysosomal hydrolases. 670 43

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).
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PMID:Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing. 675 47

Glycosidases and glycosyltransferases were electrophoresed in the presence of sodium dodecyl sulfate (SDS) in a thin-layer gel supported by a glass plate, treated with the nonionic detergent Triton X-100, and specifically stained for the sugar-releasing activity of these enzymes. Staining is based on conversion of monosugars or a sugar phosphate to glucose-6-phosphate by the appropriate intermediary enzymes, reduction of NADP+ to NADPH, and accumulation of reduced Nitroblue Tetrazolium in the gel. Among the enzymes tested, alpha-glucosidase, beta-glucosidase and beta-mannosidase could not be renatured, whereas beta-fructofuranosidase and alpha-mannosidase could be renatured unless heated before electrophoresis. Sucrose phosphorylase, glucosyltransferase and fructosyltransferase, which are single-peptide proteins with no cystine bond, could be renatured even after pretreatment with SDS and/or mercaptoethanol at 100 degrees C for 10 min. However, exclusive heating remarkably decreased the activities of these enzymes. Two-dimensional separation of the five renaturable enzymes was done in a single thin-layer gel, using SDS-electrophoresis in the first dimension and isoelectric focusing in the second dimension.
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PMID:Renaturation and activity staining of glycosidases and glycosyltransferases in gels after sodium dodecyl sulfate-electrophoresis. 752 70

Processing of N-linked oligosaccharides in Saccharomyces cerevisiae begins with the removal of glucose and mannose residues from Glc3Man9GlcNAc2 to form a single isomer of Man8GlcNAc2. The importance of mannose removal for subsequent outer chain synthesis was examined in strains of S. cerevisiae disrupted in the MNS1 gene encoding a specific alpha 1,2-mannosidase responsible for Man8GlcNAc2 synthesis [Camirand, Heysen, Grondin and Herscovics (1991) J. Biol. Chem. 266, 15120-15127]. Both MNS1 transcripts of 1.85 kb and 1.7 kb were not observed in Northern blots of mns1 cells (i.e. cells containing the disrupted gene). Analysis on Bio-Gel P-6 of endo-beta-N-acetylglucosaminidase-H-sensitive oligosaccharides following a 10 min pulse with [2-3H]mannose revealed similar amounts of labelled outer chains excluded from the gel in both control and mns1 cells. H.p.l.c. of the included oligosaccharides showed that a Man9GlcNAc, rather than a Man8GlcNAc, intermediate was formed in mns1 cells. Analysis of [3H]mannose-labelled core oligosaccharides from immunoprecipitated CPY and invertase by h.p.l.c. showed a similar size distribution in mns1 and control cells. Invertase immunoprecipitated from [35S]methionine-labelled mns1 cells was highly glycosylated, but migrated slightly faster than that from control cells on denaturing PAGE, indicating a small difference in glycosylation. A similar difference in mobility was observed for invertase activity stain following non-denaturing gel electrophoresis. It is concluded that the alpha-mannosidase encoded by MNS1 is the only enzyme responsible for mannose removal in vivo, and that this processing step is not essential for outer chain synthesis.
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PMID:Disruption of the processing alpha-mannosidase gene does not prevent outer chain synthesis in Saccharomyces cerevisiae. 843 91


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