Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

6-Hydroxy-D-nicotine oxidase (6-HDNO) was expressed in Escherichia coli JM109 cells from the recombinant plasmid pAX-6-HDNO as a beta-galactosidase-6-HDNO fusion protein. Affinity chromatography of the fusion protein on p-aminobenzyl-1-thio-beta-galactopyranoside-agarose and subsequent digestion with protease Xa yielded highly purified apo6-HDNO. Incubation of the purified protein with [14C]FAD demonstrated that flavinylation of apo6-HDNO proceeds autocatalytically. Phosphorylated three-carbon compounds such as glycerol-3-P, which are known to stimulate the formation of the histidyl (N3)-(8 alpha) FAD between apo6-HDNO and FAD (Brandsch, R., and Bichler, V. (1989) Eur. J. Biochem. 182, 125-128), could be replaced in their action by high concentrations of glycerol (45%) or sucrose (20%). These substances apparently induced and stabilized a conformational state of the apoenzyme compatible with covalent attachment of FAD. In the absence of glycerol the apoenzyme readily lost the ability to form holoenzyme at temperatures above 30 degrees C. Holoenzyme formation protected the 6-HDNO polypeptide from this thermal denaturation. Autoflavinylation of 6-HDNO was inhibited by the sulfhydryl reagents dithionitrobenzoate or N-ethylmaleimide. Inhibition was prevented by mercaptoethanol or FAD, but not 6-hydroxy-D-nicotine, the substrate of the holoenzyme. A cysteine-thiol group may therefore be involved in reactions leading to the covalent attachment of FAD to apo6-HDNO. When flavinylation of apo6-HDNO proceeded under anaerobic conditions, the amount of incorporation of [14C]FAD into the polypeptide was indistinguishable from reactions performed in the presence of O2. None of the FAD-derivatives (8-demethyl-FAD, 8-chloro-FAD, and 5-deaza-FAD) could replace FAD in holoenzyme formation. The failure of covalent attachment of 5-deaza-FAD to apo6-HDNO is in agreement with the assumption that the quinone methide form of the isolloxazine ring is an intermediate in the flavinylation reaction.
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PMID:Autoflavinylation of apo6-hydroxy-D-nicotine oxidase. 191 24

The sensitivity of lipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase E3) from Azotobacter vinelandii to inhibition by NADH requires measurement of the activity in the initial phase of the reaction. Stopped-flow turnover experiments show that kcat is 830 s-1 compared with 420 s-1 found in standard steady-state experiments. Mutations at the si-side of the flavin prosthetic group that cause severe inhibition by NADH were studied. Tyr16 was replaced by phenylalanine and serine, which causes the loss of two intersubunit H-bonds. [F16]E3 shows only 5.7% of wild-type activity in the standard assay procedure, but analyzed by stopped-flow the activity is 70% of the wild-type enzyme. The NADH-->Cl2Ind (dichloroindophenol) activity was normal or slightly increased. The inhibition by NADH is competitive with respect to NAD+, Ki = 50 microM. Spectral analysis show that electrons readily pass over from the disulfide to the FAD, indicating an increase in the redox potential of the flavin. It is concluded that subunit interaction plays an important role in the protection of the enzyme against over-reduction by decreasing the redox potential of the flavin. The interaction of wild-type or mutant enzymes with the core component of the pyruvate (E2p) or oxoglutarate (E2o) dehydrogenase multienzyme complex relieves the inhibition to a large extent. In the mutant enzymes, the mechanism of inhibition changes from competitive to the mixed-type inhibition observed for the wild-type enzyme. The stabilizing effect of E2 on [F16]E3 was used as an assay to analyze the stoichiometry of interaction of E3 with E2p as well as E2o. 1 mol E2p monomer was sufficient to saturate 1 mol E3 dimer with a Kd of about 1 nM. Similarly, 1 mol E2o saturated the E3 dimer with a Kd of 30 nM. From these experiments it is concluded that the E3-binding domain of E2 interacts with the subunit interface of E3 near the dyad axis, thus preventing sterically the interaction with a second molecule of the binding domain. This mode of interaction, which causes asymmetry in the complex, explains the stabilization against over-reduction by tightening the subunit interaction. Subgene cloning of the E2p component of the pyruvate dehydrogenase complex is described in order to obtain a complex between the lipoamide dehydrogenase component (E3) and the binding domain of E2p. A unique restriction site in the DNA encoding the flexible linker between the third lipoyl domain and the binding domain combined with timed digestion with exonuclease Bal31 was used to create a set of deletion mutants in the N-terminal region of the binding-catalytic didomain, fused to six N-terminal amino acids from beta-galactosidase. The expressed proteins, selected for E2p activity, were analyzed for binding of E3 and E1p. The shortest fusion protein containing a functional binding domain was expressed and purified. [F16]E3 was combined with this fusion protein in a stoichiometric ratio and the resulting complex was subjected to limited proteolysis to remove the catalytic domain. The resulting [F16]E3-binding domain preparation was purified to homogeneity.
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PMID:The interaction between lipoamide dehydrogenase and the peripheral-component-binding domain from the Azotobacter vinelandii pyruvate dehydrogenase complex. 857 46

The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in beta-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a beta-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a beta-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli beta-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (Adex1CAmGPDH) caused 2.1-fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDH cDNA (Adex1CAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120-130% by infection with Adex1CAmGPDH, but not with Adex1CAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with Adex1CAmGPDH together with Adex1CAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.
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PMID:Effect of mitochondrial and/or cytosolic glycerol 3-phosphate dehydrogenase overexpression on glucose-stimulated insulin secretion from MIN6 and HIT cells. 877 29

We described here the construction of two novel Saccharomyces cerevisiae strains in which the regulatory region of the SDH1 gene, coding for the succinate dehydrogenase flavoprotein subunit, was fused in frame to the reporter gene lacZ of E. coli, coding for beta-galactosidase. By this approach, SDH1 expression was studied in the yeast strain, flx1 delta-lacZ, lacking of a functional mitochondrial FAD translocator, Flx1p. The experiments described here are in line with the hypotesys that a correlation exists between defects in flavin cofactor homeostasis and mitochondrial apo-flavoprotein expression.
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PMID:Expression of succinate dehydrogenase flavoprotein subunit in saccharomyces cerevisiae studied by lacZ reporter strategy. Effect of FLX1 deletion. 1919 35