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)

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.
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PMID:Purification and characterization of acid trehalase from the yeast suc2 mutant. 328 51

Yeast invertase exists in two different forms. The cytoplasmic enzyme is nonglycosylated, whereas the external invertase contains about 50% carbohydrate of the high mannose type. The protein moieties of both enzymes are identical. The two invertases have been used previously as a model system to investigate the influence of covalently linked carbohydrate chains on the stability of large glycoproteins, and controversial results were obtained. Here, we measured thermal and denaturant-induced unfolding by various probes, such as the loss of enzymatic activity, and by the changes in absorbance and fluorescence. The ranges of stability of the two invertases were found to be essentially identical, indicating that the presence of a high amount of carbohydrate does not significantly contribute to the stability of external invertase. Earlier findings that invertase is stabilized by glycosylation could not be confirmed. The stability of this glycoprotein is apparently determined by the specific interactions of the folded polypeptide chain. Unlike the glycosylated form, the carbohydrate-free invertase is prone to aggregation in the denatured state at high temperature and in a partially unfolded form in the presence of intermediate concentrations of guanidinium chloride.
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PMID:The stability of yeast invertase is not significantly influenced by glycosylation. 328 23

In order to gain information on the ability of the glycosylation system of Schizosaccharomyces pombe to process heterologous glycoproteins, the expression of Saccharomyces cerevisiae invertase in the former yeast was studied. Sc. pombe cells are able to produce enzymatically active invertase from the S. cerevisiae SUC2 gene introduced by transformation and the enzyme is glycosylated and secreted into the cell wall. However, Sc. pombe transformants do not glycosylate the heterologous enzyme as their own invertase since it is not bound by the lectin from Bandeiraea simplicifolia seeds, which indicates the absence of terminal galactose residues. Moreover, the electrophoretic mobility of the heterologous invertase is similar to that of the large enzyme from S. cerevisiae, both in its native form and after being deglycosylated with Endo H. These results suggest that the polypeptide chain of S. cerevisiae invertase is the primary factor for the glycosylation in Sc. pombe cells.
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PMID:Synthesis of Saccharomyces cerevisiae invertase by Schizosaccharomyces pombe. 329 86

Oligosaccharide processing is controlled by host- and protein-dependent factors. To increase our understanding of the relative contribution of those factors we studied the glycosylation of yeast invertase expressed in a heterologous system. Invertase synthesized in psi-2 cells (an NIH 3T3-derived packaging line) is secreted efficiently, enzymatically active, and heavily glycosylated. It was estimated that the protein contains 8 or 9 carbohydrate chains. Two classes can be observed, of an approximate size of 100-110 kDa and 115-130 kDa, respectively. The size differences are due to differences in glycosylation. The smaller class contains two high-mannose carbohydrate chains; the remainder is of the complex type, sialylated and most likely tri- or tetraantennary. This profile parallels the situation observed with invertase glycosylation in yeast, where 2 of 9 or 10 chains remain unprocessed. The larger size class of invertase expressed in mouse fibroblasts has a different profile, since it contains probably only complex-type glycans. There are no apparent differences, however, in the size of the protein backbone between the two size classes. When invertase is synthesized in the presence of the mannosidase inhibitor 1-deoxymannojirimycin, processing is blocked completely, since all glycans are susceptible to endo-beta-N-acetylglucosaminidase H. The glucosidase inhibitor 1-deoxynojirimycin does not inhibit processing completely. In both cases secretion of the protein is not affected. The glycosylation inhibitor tunicamycin prevents secretion of invertase completely when cells are cultured at 37 degrees C. At 26 degrees C, however, nonglycosylated invertase can be detected in the medium. These data suggest that glycosylation of invertase seems to be essential for the early steps of the secretory pathway but is less critical for later events.
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PMID:Expression of the Saccharomyces cerevisiae glycoprotein invertase in mouse fibroblasts: glycosylation, secretion, and enzymatic activity. 329 66

Human alpha-1-antitrypsin (alpha-AT) is a major serum protein with protease inhibitory activity. Three asparagine residues in alpha-AT are glycosylated with the mammalian 'complex' pattern of carbohydrate as the protein is secreted from cells in the liver. To examine the glycosylation and secretion of human alpha-AT by Saccharomyces cerevisiae, the yeast invertase secretion signal codons were substituted for the native secretion signal coding DNA of an alpha-AT cDNA, and the fusion gene was placed on an autonomously replicating yeast--Escherichia coli shuttle vector under control of the yeast triosephosphate isomerase (TPI) promoter. Yeast strains transformed with this plasmid produce human alpha-AT and secrete about one-fifth of it into the culture broth. Approximately 80% of the alpha-AT produced in yeast is not in the culture broth but is inside the cell within the secretory pathway. This internal alpha-AT is heterogeneous, consisting of molecules with core carbohydrate on either two or all three of the asparagine receptors. Human alpha-AT secreted into the culture broth contains, in addition to core carbohydrate, variable numbers of mannose outer chains, typical of secreted yeast proteins such as invertase. All carbohydrate is removed by endoglycosidase H treatment. Examination of alpha-AT secreted from an mnn9 mutant, which blocks addition of variable numbers of outer mannose chains, revealed a homogeneous alpha-AT product which, like alpha-AT isolated from human serum, bears carbohydrate on three asparagine residues per molecule.
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PMID:Glycosylation and secretion of human alpha-1-antitrypsin by yeast. 331 63

The yeast Saccharomyces cerevisiae X2180 strain with the mnn1 mnn2 mnn9 mutations, all of which affect mannoprotein glycosylation, synthesizes N-linked oligosaccharides having the following structure: (Formula: see text) whereas the mnn1 mnn2 mutant extends the alpha 1----6-linked backbone of some of the core oligosaccharides by adding 20-30 mannose units. Membrane fractions from the mnn1 mnn2 and mnn1 mnn2 mnn9 mutants are equally effective in catalyzing transfer from GDP-[3H]mannose to add mannose in both alpha 1----2 and alpha 1----6 linkages to an oligosaccharide having the following structure: (Formula: see text) but neither membrane preparation can utilize the homologous mnn1 mnn2 mnn9 oligosaccharide as an acceptor. Thus, addition of the alpha 1----2-linked mannose side chain to the terminal alpha 1----6-linked mannose in oligosaccharides of the mnn9 mutant inhibits the elongation reaction and may serve as an important structural control of mannoprotein glycosylation. The mnn9 mutation also increases the transit time for invertase secretion, meaning that this mutation could affect the processing machinery in the Golgi apparatus.
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PMID:Regulation of the protein glycosylation pathway in yeast: structural control of N-linked oligosaccharide elongation. 332 Oct 55

A two-step method for the selection of transformants of prototrophic industrial strains of the methylotrophic yeast Pichia pastoris has been developed. This method is based on our observation that P. pastoris cannot use sucrose as the sole carbon source (Suc-) and that introduction of the invertase gene (SUC2) of Saccharomyces cerevisiae renders P. pastoris Suc+. P. pastoris was transformed with a plasmid which contains the SUC2 gene of S. cerevisiae and an autonomously replicating sequence PARS1 from P. pastoris. The transformants were initially allowed to regenerate on medium containing dextrose and the regenerated cells were pooled and plated on sucrose medium to screen for Suc+ transformants. It was shown that the Suc+ transformants of P. pastoris with the autonomously replicating plasmid were highly unstable with respect to the plasmid maintenance, even when grown on sucrose as the sole carbon and energy source. This high instability was attributed to an efficient cross-feeding by Suc- segregants on glucose and fructose generated due to hydrolysis of sucrose by the invertase enzyme secreted by Suc+ cells. Spontaneous integration of the plasmid DNA resulting in a stable Suc+ phenotype was also observed. However, stable Suc+ transformants were obtained more readily by integration of SUC2 into P. pastoris genome following transformation with a linearized plasmid with the ends homologous to P. pastoris HIS4 locus. All such integrants were completely stable for Suc+ phenotype after 20 generations of growth in a nonselective medium.
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PMID:Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation of Pichia pastoris. 332 36

Two glycopeptide hydrolases, an endo-beta-N-acetylglucosaminidase and peptide:N-glycanase (amidase), have been isolated from defatted jack bean meal by standard procedures involving differential solubility and column chromatography. The purified products appear to be free of contaminating proteases and exoglycosidases, and their substrate specificity has been explored with regard to both glycan and peptide structure of the substrates. The endoglycosidase appears to be specific for high mannose glycans; no hydrolysis of either hybrid or complex glycans has been observed. It shows limited activity with two intact glycoproteins, ribonuclease B and yeast invertase, and gives optimal rate with glycopeptides. Free glycan-Asn derivatives are poor substrates in comparison with glycopeptides or glycan-Asn derivatives where the alpha-amino group has been dansylated. The amidase will liberate both high mannose, hybrid, and asialo-complex glycans from both proteins and peptides, but many glycans in intact proteins or in long peptides are resistant to the amidase and become active as substrates only after further proteolytic cleavage. The best substrates appear to be those with the glycosylated asparagine no more than 4-5 residues in from either the NH2- or COOH-terminal end of the peptide. Sialylated glycans do not appear to be released by the amidase.
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PMID:Purification and characterization of two glycopeptide hydrolases from jack beans. 333 94

Mutations in the SNF2 gene of Saccharomyces cerevisiae prevent derepression of the SUC2 (invertase) gene, and other glucose-repressible genes, in response to glucose deprivation. We have isolated 25 partial phenotypic revertants of a snf2 mutant that are able to derepress secreted invertase. These revertants all carried suppressor mutations at a single locus, designated SSN20 (suppressor of snf2). Alleles with dominant, partially dominant and recessive suppressor phenotypes were recovered, but all were only partial suppressors of snf2, reversing the defect in invertase synthesis but not other defects. All alleles also caused recessive, temperature-sensitive lethality and a recessive defect in galactose utilization, regardless of the SNF2 genotype. No significant effect on SUC2 expression was detected in a wild-type (SNF2) genetic background. The ssn20 mutations also suppressed the defects in invertase derepression caused by snf5 and snf6 mutations, and selection for invertase-producing revertants of snf5 mutants yielded only additional ssn20 alleles. These findings suggest that the roles of the SNF2, SNF5 and SNF6 genes in regulation of SUC2 are functionally related and that SSN20 plays a role in expression of a variety of yeast genes.
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PMID:Suppressors of SNF2 mutations restore invertase derepression and cause temperature-sensitive lethality in yeast. 351 73

A series of high mannose oligosaccharides with the size range Man8-14GlcNAc was purified from Saccharomyces cerevisiae invertase, and the composition of each was determined by chemical analysis. Purity and composition were verified by 1H NMR spectroscopy at 500 MHz, and structures were assigned on the basis of chemical shifts in C1-H and C2-H protons of similarly substituted compounds of known structure. Such analyses showed that these invertase oligosaccharides were a homologous series of homogeneous compounds, each related to the next member by addition of 1 mol of mannose in a specific alpha-linked configuration. Man8GlcNAc purified from the total glycoprotein fraction of disrupted yeast was the smallest species found and had the same homogeneous structure as that previously reported for the Man8GlcNAc from invertase (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Digestion of Man8-13GlcNAc species from invertase with Aspergillus satoi alpha 1,2-mannosidase provided products that were consistent with the structures assigned by 1H NMR as did fast atom bombardment-mass spectroscopy fragmentation analysis of the Man9,10GlcNAc oligosaccharides. These results lead to the proposal that Man8GlcNAc is the only trimming intermediate in Saccharomyces sp., and the remaining Man9-14GlcNAc oligosaccharides are biosynthetic intermediates which define the principal pathway of single-step mannose addition in the formation of the inner core of yeast mannan.
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PMID:Structure of yeast external invertase Man8-14GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy. 352 34


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