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)

The archaeon Methanopyrus kandleri is the most thermophilic methanogen presently known. It contains a chaperonin (thermosome) which represents a 951 kDa homo-hexadecameric protein complex with NH4+-dependent ATPase activity. Since its synthesis is not increased upon heat shock, we set out to test its chaperone function. In order to obtain the chaperonin in amounts sufficient for functional investigations, the gene encoding the 60 kDa subunit was expressed in E. coili BL21 (DE3) cells. Purification yielded soluble, high-molecular-mass double-ring complexes, indistinguishable from the natural thermosome. In order to study the functional properties of the recombinant protein complex, pig citrate synthase, yeast alcohol dehydrogenase, yeast alpha-glucosidase, bovine insulin, and Thermotoga phosphoglycerate kinase were used as model substrates. The results demonstrate that the recombinant M. kandleri thermosome possesses a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins after chemical denaturation. However, the chaperonin only forms dead-end complexes with its non-native substrates, no release is detectable at temperatures between 25 and 60 degrees C.
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PMID:The recombinant thermosome from the hyperthermophilic archaeon Methanopyrus kandleri: in vitro analysis of its chaperone activity. 1006 37

The scaled down system developed in Part I of this series was further validated by using a 1-cm diameter column for method development studies for the separation of two model proteins, alcohol dehydrogenase and alpha-glucosidase, from unclarified yeast homogenate by hydrophobic interaction expanded bed chromatography based on the STREAMLINE matrix. The efficacy of solids removal and establishment of optimal binding and separation condition by stepwise elution were investigated. Equilibration of the EBA column and loading at high salt strengths affected the subsequent recovery of the two target proteins. Although good resolution between the target proteins could be achieved, peak tailing was found to be a consistent problem. The optimised separation protocol was scaled up 25-fold to a column diameter of 5.0 cm. The results were in good agreement with the run conducted in the 1-cm column, indicating the potential of using the small columns as an viable approach for method scouting and development studies.
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PMID:Expanded bed chromatography of proteins in small-diameter columns. II. Methods development and scale up. 1084 May 98

A new type of microfiltration (MF) bioreactor, developed in our laboratory, was investigated for use in improving efficiency of the production of extremophilic enzymes. In spite of the difficulties in cultivating hyperthermophiles, we achieved, in 300 h fermentation, more than 38 g/l dry weight of Sulfolobus solfataricus using a MF technique, and we demonstrated that the activity of alcohol dehydrogenase (ADH), as the reporter enzyme, was not affected by cell density. However, hyperthermophile cultivation is difficult to scale up because of evaporation and the very low growth rate. Thus, to achieve high productivity we cultivated, in the MF bioreactor, recombinant mesophilic hosts engineered for the production of two thermophilic enzymes, namely, trehalosyldextrin-forming enzyme (SsTDFE) and trehalose-forming enzyme (SsTFE) from Sulfolobus solfataricus. The traditional Luria-Bertani broth used for recombinant Escherichia coli growth was replaced with a semidefined medium. The latter was used in both the batch and the MF experiments, and the ratio of complex components (e.g., yeast extract and tryptone) to a simple carbon source (glycerol) was decreased during the fed-batch phase to further decrease the medium cost in view of industrial applications. The bioprocess developed was able to improve productivity 500 fold for rSsTFE and 60 fold for rSsTDFE with respect to the wild type cultivated in MF mode. Comparisons with another recombinant enzyme, alpha-glucosidase (rSsalphagly), from Sulfolobus solfataricus produced in our MF bioreactor are reported.
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PMID:Innovative fermentation strategies for the production of extremophilic enzymes. 1145 63

The eukaryotic cytoskeletal protein tubulin is a heterodimer of two subunits, alpha and beta, and is a building block unit of microtubules. In a previous communication we demonstrated that tubulin possesses chaperone-like activities by preventing the stress-induced aggregation of various proteins (Guha, S., Manna, T. K., Das, K. P., and Bhattacharyya, B. (1998) J. Biol. Chem. 273, 30077-30080). As an extension of this observation, we explored whether tubulin, like other known chaperones, also protected biological activity of proteins against thermal stress or increased the yields of active proteins during refolding from a denatured state. We show here that tubulin not only prevents the thermal aggregation of alcohol dehydrogenase and malic dehydrogenase but also protects them from loss of activity. We also show that tubulin prevents the aggregation of substrates during their refolding from a denatured state and forms a stable complex with denatured substrate. The activity of malic dehydrogenase, alpha-glucosidase, and lactate dehydrogenase during their refolding from urea or guanidium hydrochloride denatured states increased significantly in presence of tubulin compared with that without tubulin. These results suggest that tubulin, in addition to its role in mitosis, cell motility, and other cellular events, might be implicated in protein folding and protection from stress.
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PMID:Chaperone-like activity of tubulin. binding and reactivation of unfolded substrate enzymes. 1150 63

Previously we demonstrated that a novel stress protein is induced in fish cells by the infection of a fish rhabdovirus (Cho W. J., Cha, S. J., Do, J. W., Choi, J. Y., Lee, J. Y., Jeong, C. S., Cho, K. J., Choi, W. S., Kang, H. S., Kim, H. D., and Park, J. W. (1997) Biochem. Biophys. Res. Commun. 233, 316-319). In this paper, we present the molecular cloning and characterization of a gene encoding this protein named virus-inducible stress protein (VISP). The VISP was purified partially by immunoprecipitation using a monoclonal antibody against the VISP and further purified by the electroelution from a SDS-PAGE gel. The protein was subjected to internal protein sequencing, and the sequence of three peptides was determined. Degenerate oligonucleotides based on the three peptide sequences were used to screen a cDNA library from rhabdovirus-infected CHSE-214 fish cells, and a cDNA of a 2193-bp open reading frame encoding the VISP with 730 amino acid residues (M(r) = 79.84) was identified. Whereas the nucleotide sequence of VISP shows no similarity with other genes in the GenBank(TM), the amino acid sequence of the VISP has similarity with the bacterial extracellular solute-binding protein family 5 (SBP_bac_5) that is proposed to have chaperone activity. Thus, we explored whether the VISP also had chaperone-like activity. Purified recombinant VISP expressed in Escherichia coli promoted the functional folding of alpha-glucosidase after urea denaturation and also prevented thermal aggregation of alcohol dehydrogenase. These results suggest that the VISP has amino acid sequence similarity with SBP_bac_5 and that it has chaperone activity that may play a role in virus infection.
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PMID:Molecular cloning of a novel chaperone-like protein induced by rhabdovirus infection with sequence similarity to the bacterial extracellular solute-binding protein family 5. 1218 66

Spectrin, the major constituent protein of the erythrocyte membrane skeleton, exhibits chaperone activity by preventing the irreversible aggregation of insulin at 25 degrees C and that of alcohol dehydrogenase at 50 degrees C. The dimeric spectrin and the two subunits, alpha-spectrin and beta-spectrin prevent such aggregation appreciably better, 70% in presence of dimeric spectrin at an insulin:spectrin ratio of 1:1, than that in presence of the tetramer of 25%. Our results also show that spectrin binds to denatured enzymes alpha-glucosidase and alkaline phosphatase during refolding and the reactivation yields are increased in the presence of the spectrin derivatives when compared with those refolded in their absence. The unique hydrophobic binding site on spectrin for the fluorescence probe, 6-propionyl-2-(dimethylamino)naphthalene (Prodan) has been established to localize at the self-associating domain with the binding stoichiometry of one Prodan/both dimeric and tetrameric spectrin. The other fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, does not show such specificity for spectrin, and the binding stoichiometry is between 3 and 5 1-anilinonaphthalene-8-sulfonic acid/dimeric and tetrameric spectrin, respectively. Regions in alpha- and beta-spectrins have been found to have sequence homology with known chaperone proteins. More than 50% similarities in alpha-spectrin near the N terminus with human Hsp90 and in beta-spectrin near the C terminus with human Hsp90 and Escherichia coli DnaJ have been found, indicating a potential chaperone-like sequence to be present near the self-associating domain that is formed by portions of alpha-spectrin near the N terminus and the beta-spectrin near the C terminus. There are other patches of sequences also in both the spectrin polypeptides, at the other termini as well as in the middle of the rod domain having significant homology with well known chaperone proteins.
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PMID:Chaperone activity and prodan binding at the self-associating domain of erythroid spectrin. 1549 10

Intracellular products, not secreted from the microbial cell, are released by breaking the cell envelope consisting of cytoplasmic membrane and an outer cell wall. Hydrodynamic cavitation has been reported to cause microbial cell disruption. By manipulating the operating variables involved, a wide range of intensity of cavitation can be achieved resulting in a varying extent of disruption. The effect of the process variables including cavitation number, initial cell concentration of the suspension and the number of passes across the cavitation zone on the release of enzymes from various locations of the Brewers' yeast was studied. The release profile of the enzymes studied include alpha-glucosidase (periplasmic), invertase (cell wall bound), alcohol dehydrogenase (ADH; cytoplasmic) and glucose-6-phosphate dehydrogenase (G6PDH; cytoplasmic). An optimum cavitation number Cv of 0.13 for maximum disruption was observed across the range Cv 0.09-0.99. The optimum cell concentration was found to be 0.5% (w/v, wet wt) when varying over the range 0.1%-5%. The sustained effect of cavitation on the yeast cell wall when re-circulating the suspension across the cavitation zone was found to release the cell wall bound enzyme invertase (86%) to a greater extent than the enzymes from other locations of the cell (e.g. periplasmic alpha-glucosidase at 17%). Localised damage to the cell wall could be observed using transmission electron microscopy (TEM) of cells subjected to less intense cavitation conditions. Absence of the release of cytoplasmic enzymes to a significant extent, absence of micronisation as observed by TEM and presence of a lower number of proteins bands in the culture supernatant on SDS-PAGE analysis following hydrodynamic cavitation compared to disruption by high-pressure homogenisation confirmed the selective release offered by hydrodynamic cavitation.
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PMID:Disruption of Brewers' yeast by hydrodynamic cavitation: Process variables and their influence on selective release. 1657 Mar 16

Azasugars, "nitrogen in the ring" analogues of monosaccharides, are known to be distributed in select plant, fungal. and bacterial species. We identify Chitinophaga pinensis DSM 2588 as the first bacterial source of the plant pyrrolidine azasugar 1,4-dideoxy-1,4-aminoarabinitol (DAB-1). Comparative sequence analyses identified C. pinensis as a putative azasugar producer, via observation of a three-gene cluster coding for putative aminotransferase, alcohol dehydrogenase, and sugar phosphatase enzymes, similar to the previously reported azasugar biosynthetic signature identified in Bacillus amyloliquefaciens FZB42. Multistep fractionation of C. pinensis culture media guided by a maltase inhibition assay yielded a component with a mass consistent with the structure of DAB-1. Heterologous expression of the three-gene cluster in E. coli, a non-azasugar producer, led to the isolation of nectrisine, a biosynthetic precursor to DAB-1, which displayed potent slow tight binding inhibition of maltase. Reduction of nectrisine with NaBH4 removed the slow tight binding inhibition kinetics, and MS analysis provided evidence for the production of a compound matching that of the isolated DAB-1 from C. pinensis. 1H NMR analysis of the nectrisine produced in E. coli after NaBD4 reduction produced a spectrum consistent with DAB-1 deuterated at C-1, primarily at the pro-S position. These results support the idea that the azasugar three-gene cluster represents a general biosynthetic path leading to several different compounds, which may prove useful for the identification of other azasugar-producing organisms.
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PMID:Functional Analysis of a Gene Cluster from Chitinophaga pinensis Involved in Biosynthesis of the Pyrrolidine Azasugar DAB-1. 3179 83


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