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:3.2.1.20 (alpha-glucosidase)
4,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Polymerase chain reaction-based methodology was used to obtain a cDNA clone (MAL2) from potato (Solanum tuberosum L.) with the sequence characteristics of an alpha-glucosidase. Phylogenetic analysis of the deduced polypeptide encoded by this cDNA demonstrated that the most similar sequences were alpha-glucosidases and alpha-xylosidases of plant origin. The MAL2 cDNA was expressed in Escherichia coli and the recombinant MAL2 protein was affinity-purified. MAL2 catalysed the hydrolysis of a range of maltooligomers and p-nitrophenyl-alpha-D-glucopyranoside with a pH optimum of 5.5-5.7. The substrate with the lowest Km value was maltotetraose (3.7 mM). The MAL2 expression product did not catalyse the hydrolysis of xyloglucan oligosaccharides, p-nitrophenyl-alpha-D-xylopyranoside or gelatinised potato starch. MAL2 was down-regulated in transgenic potato plants using an antisense approach. In several independent transgenic antisense lines, MAL2 expression was severely down-regulated. Despite this, no decrease in total extractable alpha-glucosidase and alpha-xylosidase activity could be detected in tissues from the transgenic plants. In glasshouse trials, no visible phenotype, change in tuber yield or carbohydrate content was associated with MAL2 down-regulation. The implications of these results are discussed.
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PMID:Copy-DNA cloning and characterisation of a potato alpha-glucosidase: expression in Escherichia coli and effects of down-regulation in transgenic potato. 1146 91

Aspergillus nidulans possessed an alpha-glucosidase with strong transglycosylation activity. The enzyme, designated alpha-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to alpha-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other alpha-glucosidases. We propose here that AgdB is a novel alpha-glucosidase with unusually strong transglycosylation activity.
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PMID:Novel alpha-glucosidase from Aspergillus nidulans with strong transglycosylation activity. 1187 75

We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity. Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa. Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites. Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions. Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits. The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity.
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PMID:cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea. 1288 65

Processing alpha-glucosidase I, which is encoded by CWH41, regulates one of the key steps in asparagine-linked glycoprotein biosynthesis by cleaving the terminal alpha-1,2-linked glucose from Glc(3)Man(9)GlcNAc(2), the common oligosaccharide precursor. This cleavage is essential for further processing of the oligosaccharide to the complex, hybrid, and high mannose type carbohydrate structures found in eukaryotes. A method is described for the purification of the soluble form of the alpha-glucosidase I, from recombinant Saccharomyces cerevisiae overexpressing CWH41. A homogeneous enzyme preparation was obtained in higher yield than previously reported. Cultivation of recombinant S. cerevisiae in a fermenter increased the biomass 1.7 times per liter and enzyme production 2 times per liter compared to cultivation in shake flasks. Ammonium sulfate precipitation with three chromatography steps, including chromatography on an N-(5'-carboxypentyl)-1-deoxynojirimycin column, resulted in highly purified enzyme with no detectable contamination by other alpha- and beta-aryl-glycosidases. The purification procedure reproducibly yielded 40 microg of pure enzyme per gram wet biomass. Enzyme that was purified using an alternative procedure contained minor impurities and was hydrolyzed by an endogenous proteolytic activity to peptides that retained full catalytic activity. Controlled trypsin hydrolysis of the highly purified enzyme released polypeptide(s) containing the alpha-glucosidase I catalytic domain, with no loss of catalytic activity. This suggests that the catalytic domain of yeast alpha-glucosidase I is resistant to trypsin hydrolysis and remains fully functional after cleavage.
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PMID:An improved purification procedure for soluble processing alpha-glucosidase I from Saccharomyces cerevisiae overexpressing CWH41. 1468 Sep 56

An alpha-glucosidase was solubilised from a mixed membrane fraction of Entamoeba histolytica and purified to homogeneity by a two-step procedure consisting of ion exchange chromatography in a Mono Q column and affinity chromatography in concanavalin A-sepharose. Although the enzyme failed to bind the lectin, this step rendered a homogenous and more stable enzyme preparation that resolved into a single polypeptide of 55 kDa after SDS-PAGE. As measured with 4-methylumbelliferyl-alpha-D-glucopyranoside (MUalphaGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 degrees C. Although the enzyme preferentially hydrolysed nigerose (alpha1,3-linked), it also cleaved kojibiose (alpha1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (alpha1,4), trehalose (alpha1,1) and isomaltose (alpha1,6). Activity on alpha1,3- and alpha1,2-linked disaccharides was strongly inhibited by the glycoprotein processing inhibitors 1-deoxynojirimycin and castanospermine but was unaffected by australine. Glucose and particularly 3-deoxy-D-glucose and 6-deoxy-D-glucose were strong inhibitors of activity, whereas 2-deoxy-D-glucose and other monosaccharides had no effect. Enzyme activity on MUalphaGlc was very sensitive to inhibition by diethylpyrocarbonate suggesting a critical role of histidine residues in enzyme catalysis. Other amino acid modifying reagents such as N-ethylmaleimide and N-(3-dimethylaminopropyl)-N'ethylcarbodiimide showed a moderate effect or none at all, respectively. Results are discussed in terms of the possible involvement of this glycosidase in N-glycan processing.
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PMID:Purification and biochemical characterisation of a membrane-bound alpha-glucosidase from the parasite Entamoeba histolytica. 1501 35

Alpha-glucosidase I initiates the trimming of newly assembled N-linked glycoproteins in the lumen of the endoplasmic reticulum (ER). Site-specific chemical modification of the soluble alpha-glucosidase I from yeast using diethylpyrocarbonate (DEPC) and tetranitromethane (TNM) revealed that histidine and tyrosine are involved in the catalytic activity of the enzyme, as these residues could be protected from modification using the inhibitor deoxynojirimycin. Deoxynojirimycin could not prevent inactivation of enzyme treated with N-bromosuccinimide (NBS) used to modify tryptophan residues. Therefore, the binding mechanism of yeast enzyme contains different amino acid residues compared to its mammalian counterpart. Catalytically active polypeptides were isolated from endogenous proteolysis and controlled trypsin hydrolysis of the enzyme. A 37-kDa nonglycosylated polypeptide was isolated as the smallest active fragment from both digests, using affinity chromatography with inhibitor-based resins (N-methyl-N-59-carboxypentyl- and N-59-carboxypentyl-deoxynojirimycin). N-terminal sequencing confirmed that the catalytic domain of the enzyme is located at the C-terminus. The hydrolysis sites were between Arg(521) and Thr(522) for endogenous proteolysis and residues Lys(524) and Phe(525) for the trypsin-generated peptide. This 37-kDa polypeptide is 1.9 times more active than the 98-kDa protein when assayed with the synthetic trisaccharide, alpha-D-Glc1,2alpha-D-Glc1,3alpha-D-Glc-O(CH2)(8)COOCH(3), and is not glycosylated. Identification of this relatively small fragment with catalytic activity will allow mechanistic studies to focus on this critical region and raises interesting questions about the relationship between the catalytic region and the remaining polypeptide.
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PMID:Binding residues and catalytic domain of soluble Saccharomyces cerevisiae processing alpha-glucosidase I. 1601 48

Inspection of the genome sequence of Lactobacillus casei ATCC 334 revealed two operons that might dissimilate the five isomers of sucrose. To test this hypothesis, cells of L. casei ATCC 334 were grown in a defined medium supplemented with various sugars, including each of the five isomeric disaccharides. Extracts prepared from cells grown on the sucrose isomers contained high levels of two polypeptides with M(r)s of approximately 50,000 and approximately 17,500. Neither protein was present in cells grown on glucose, maltose or sucrose. Proteomic, enzymatic, and Western blot analyses identified the approximately 50-kDa protein as an NAD(+)- and metal ion-dependent phospho-alpha-glucosidase. The oligomeric enzyme was purified, and a catalytic mechanism is proposed. The smaller polypeptide represented an EIIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Phospho-alpha-glucosidase and EIIA are encoded by genes at the LSEI_0369 (simA) and LSEI_0374 (simF) loci, respectively, in a block of seven genes comprising the sucrose isomer metabolism (sim) operon. Northern blot analyses provided evidence that three mRNA transcripts were up-regulated during logarithmic growth of L. casei ATCC 334 on sucrose isomers. Internal simA and simF gene probes hybridized to approximately 1.5- and approximately 1.3-kb transcripts, respectively. A 6.8-kb mRNA transcript was detected by both probes, which was indicative of cotranscription of the entire sim operon.
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PMID:The sim operon facilitates the transport and metabolism of sucrose isomers in Lactobacillus casei ATCC 334. 1831 Mar 37

N-glycosylation is a common protein modification. Joining of polypeptide and carbohydrate elements into hybrid molecules provides an opportunity to fine-tune protein properties. However, the role of N-glycosylation on the development of multicellular organisms remains elusive. Here we report a hypomorphic allele of KNOPF/GLUCOSIDASE 1, which allows us to describe the effects of impaired alpha-glucosidase I on post-embryonic development of plants for the first time. This knf-101 mutation alters cell shape but does not affect cell arrangements, except for the patterning of specialized epidermal cells, delineating the significance of N-glycan processing during epidermal development in Arabidopsis.
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PMID:A novel mutation in KNOPF uncovers the role of alpha-glucosidase I during post-embryonic development in Arabidopsis thaliana. 1850 69

alpha-glucosidase inhibitors (alphaGIs) increase active glucagon-like peptide-1 (GLP-1) and reduce the total glucosedependent insulinotropic polypeptide (GIP) levels, but their ability to prevent diabetes remains uncertain. Dipeptidyl peptidase-4 (DPP-4) inhibitors, such as sitagliptin, increase active GLP-1 and GIP levels and improve hyperglycemia in a glucose-dependent fashion. However, the effectiveness of their combination in subjects with normal glucose tolerance (NGT) or impaired glucose tolerance (IGT) is uncertain. The present study evaluated the effect of miglitol, sitagliptin, and their combination on glucose, insulin and incretin levels in non-diabetic men. Miglitol and sitagliptin were administered according to four different intake schedules (C: no drug, M: miglitol; S: sitagliptin, M+S: miglitol and sitagliptin). The plasma glucose levels were significantly lower for M, S and M+S than for the control. The areas under the curve (AUCs) of the plasma active GLP-1 level in the M, S, and M+S groups were significantly greater than that in the control group. The AUC of the plasma active GLP-1 level was significantly greater for M+S group than for the M and S groups. The AUC of the plasma total GIP level was significantly smaller for M+S group than for the control and M and S groups. The results of our study suggest that miglitol, sitagliptin, or their combination contributes to the prevention of type 2 diabetes.
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PMID:Effects of miglitol, sitagliptin or their combination on plasma glucose, insulin and incretin levels in non-diabetic men. 2051 6


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