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:1.1.1.1 (alcohol dehydrogenase)
9,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.
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PMID:Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching? 131 64

The existence of circular polarization in room-temperature protein phosphorescence is demonstrated, and time-resolved circularly polarized phosphorescence (TR-CPP) is used to characterize unique tryptophan environments in multitryptophan proteins. Circularly polarized luminescence studies provide information regarding the excited state chirality of a lumiphore which can be used to extract sensitive structural information. It is shown by time resolving the circular polarization that it is possible to correlate the excited state chirality with unique decay components in a multiexponential phosphorescence decay profile. The present study presents a concurrent analysis of room-temperature time-resolved phosphorescence and TR-CPP of bacterial glucose-6-phosphate dehydrogenase as well as those of horse liver alcohol dehydrogenase. Only one of the two tryptophan residues per subunit of dimeric alcohol dehydrogenase is believed to phosphorescence, while the dimeric glucose-6-phosphate dehydrogenase has eight tryptophan residues per subunit and shows a corresponding complexity in its phosphorescence decay profile. The anisotropy factor [g(em) = delta I/(Itotal/2); delta I = Ileft circular-Iright circular] for alcohol dehydrogenase is time independent, suggesting a unique excited state chirality. The phosphorescence decay of glucose-6-phosphate dehydrogenase can be well fitted with four exponential terms of 4, 23, 76, and 142 msec, and the TR-CPP of this enzyme shows a strong time dependence that can be resolved into four individual time-independent anisotropy factors of -4.0, -2.1, +6.5, and +6.9 (x10(-3)), each respectively associated with one of the four lifetime components. These results demonstrate how the use of TR-CPP can facilitate the study of proteins with multiple lumiphores.
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PMID:Time-resolved circularly polarized protein phosphorescence. 143 4

The G-box element is a moderately conserved component of the promoter of many inducible genes, including the alcohol dehydrogenase genes of Arabidopsis and maize. We used monoclonal antibodies generated against partially purified G-box binding factor (GBF) activity to characterize maize proteins that are part of the DNA binding complex. Antibodies interacted with partially purified maize GBF complexes to produce a slower migrating complex in the gel retardation assay. Immunoprecipitation experiments suggested that the protein recognized by the antibody is not a DNA binding protein in and of itself, but rather is associated with the DNA binding complex. These monoclonal antibodies were used to isolate cDNA clones encoding a protein that we have designated GF14. Maize GF14 contains a region resembling a leucine zipper and acidic carboxy and amino termini, of which the latter can form an amphipathic alpha-helix similar to known transcriptional activators such as VP16 and GAL4. Protein gel blot analysis of cell culture extract showed that a single, major protein of approximately 30 kD is recognized by anti-GF14; the protein is also present predominantly in the kernel and root. The deduced amino acid sequence of maize GF14 is more than 80% identical to Arabidopsis GF14 and Oenothera PHP-O, and is more than 60% identical to a class of mammalian brain proteins described as both protein kinase C inhibitors and activators of tyrosine and tryptophan hydroxylases. GF14 is found in a variety of monocotyledons and dicotyledons, gymnosperms, and yeast. This suggests a deep evolutionary conservation of a potential regulatory protein associated with a core sequence found in the promoter region of many genes.
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PMID:A maize protein associated with the G-box binding complex has homology to brain regulatory proteins. 144 70

The G box is a well-characterized cis-acting DNA regulatory element found in the promoters of several seemingly unrelated plant genes, including the alcohol dehydrogenase (Adh) gene of Arabidopsis thaliana. Using a monoclonal antibody screening approach coupled with electrophoretic mobility shift assays, we have isolated a cDNA clone encoding a protein that is part of the in vitro protein/G box complex. The derived amino acid sequence is homologous to a class of proteins in mammalian brains described as protein kinase C inhibitors and as activators of tyrosine and tryptophan hydroxylases, the rate-limiting enzymes in the pathways leading to the catecholamines and serotonin. The fact that a homologous member of this regulatory protein family is found in plants and is associated with binding to transcriptional regulatory elements suggests a much wider role for these proteins.
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PMID:Brain proteins in plants: an Arabidopsis homolog to neurotransmitter pathway activators is part of a DNA binding complex. 145 38

The horse EE and human beta 1 beta 1 alcohol dehydrogenase isoenzymes have almost identical protein backbone folding patterns and contain 2 tryptophans per subunit (Trp-15 and Trp-314). Tyr-286, which had been proposed to quench the fluorescence of Trp-314 by resonance energy transfer at alkaline pH in EE, is substituted by Cys in beta 1 beta 1. The proposed role of Tyr-286 in pH-dependent quenching of EE is confirmed by our observation that tryptophan fluorescence of beta 1 beta 1 is not substantially quenched at alkaline pH. Tyr-286 had also been implicated in the quenching of Trp-314 upon formation of the EE-NAD(+)-trifluoroethanol ternary complex. However, beta 1 beta 1 exhibits the same extent of tryptophan fluorescence quenching as EE upon complexation, which strongly suggests that Tyr-286 is not involved in ternary complex quenching.
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PMID:Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human beta 1 beta 1 and horse EE alcohol dehydrogenases. 155 56

The dependence of the fluorescence emission maximum of the tryptophan residues in several two-tryptophan-containing proteins (horse liver alcohol dehydrogenase, yeast 3-phosphoglycerate kinase, Staphylococcus aureus metalloprotease and bee venom phospholipase A2) on the excitation wavelengths has been studied. Using fluorescence-resolved spectroscopy, we have dissected the contributions of particular tryptophan residues located in different parts of the protein molecule. The results demonstrate that dipolar structural relaxation can occur in the environment of tryptophan residues buried within protein molecules. The observed spectral shifts upon red-edge excitation of these residues can depend on temperature or ligand binding, as demonstrated in case of metalloprotease and alcohol dehydrogenase. No spectral shifts upon red-edge excitation have been observed for tryptophan residues totally exposed to the rapidly relaxing aqueous solvent.
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PMID:Red-edge excitation fluorescence measurements of several two-tryptophan-containing proteins. 158 74

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan. 163 65

The phosphorescence yield and decay kinetics of tryptophan (Trp) in apoazurin from Pseudomonas aeruginosa, subtilisin Carlsberg, Staphylococcal nuclease and liver alcohol dehydrogenase were determined as a function of temperature from 150 K (glassy matrix) to 300 K (fluid solution). The constancy of the lifetime-normalized phosphorescence yield with apoazurin and with Trp-314 in alcohol dehydrogenase establishes that the intersystem crossing quantum yield is practically unaffected across the temperature range. Consequently, any decrease in phosphorescence intensity not accounted for by lifetime-shortening is a signal either of the selective quenching of specific Trp residues in the same macromolecule or that the protein sample is heterogeneous in its emission properties. From an analysis of the thermal profile it is concluded that subtilisin Carlsberg and S. nuclease, as opposed to apoazurin, are not phosphorescent at ambient temperature, their residual emission probably arising from protein impurities. Criteria for distinguishing conformer emission from a contribution by protein impurities are discussed.
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PMID:Temperature dependence of tryptophan phosphorescence in proteins. 219 61

Multiple freeze-thaw (FT) cycles, with complete melting between cycles, resulted in an exponential decline in liver alcohol dehydrogenase (LADH) enzyme activity. The reduction in activity of LADH as a result of FT damage was proportional to the decrease in the intensity of the tryptophan fluorescence of the enzyme. Treatment with urea resulted in a similar relationship between tryptophan fluorescence intensity and inactivation. Evidence from fluorescence and activity studies from the same sample, as well as gel electrophoresis, indicates that damage to LADH from a FT cycle, resulting in inactivation, is likely an unfolding of the enzyme rather than separation of subunits or aggregation of enzymes at the enzyme concentrations and cooling rates used. A nonexponential decline in enzyme activity, as a function of the number of FT cycles, can be achieved if complete melting between cycles is not allowed or if the samples are stored at +4 degrees C for 24 hr following the last FT cycle, prior to assay. In the latter case, a partial recovery in enzyme activity is seen. "Seeding," while lowering the enzyme activity, is desirable to achieve consistent results without the artifacts that are introduced if not used. Amino acids were tested for their effectiveness as cryoprotectants. From the results of this study, the mean fractional area loss of amino acid residues upon incorporation in globular proteins (f) is inversely proportional to the FT protection by these free amino acids. Thus, amino acid residues which tend to be found at the surface of proteins (e.g., glutamate) improve the FT survival of LADH, when added as the free amino acid, while those amino acids which are found in the interior of proteins (e.g., valine, leucine) sensitize LADH to FT damage. The pattern of protection ("fingerprint") of LADH by various amino acids is different from that of living cells. Furthermore, unlike the case with cells, glutamine and DMSO do not act independently when protecting LADH.
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PMID:Mechanism of freeze-thaw damage to liver alcohol dehydrogenase and protection by cryoprotectants and amino acids. 224 55

Class III alcohol dehydrogenase, whose activity toward ethanol is negligible, has defined, specific properties and is not just a "variant" of the class I protein, the traditional liver enzyme. The primary structure of the horse class III protein has now been determined, and this allows the comparison of alcohol dehydrogenases from human, horse, and rat for both classes III and I, providing identical triads for both these enzyme types. Many consistent differences between the classes separate the two forms as distinct enzymes with characteristic properties. The mammalian class III enzymes are much less variable in structure than the corresponding typical liver enzymes of class I: there are 35 versus 84 positional differences in these identical three-species sets. The class III and class I subunits contain four versus two tryptophan residues, respectively. This makes the differences in absorbance at 280 nm a characteristic property. There are also 4-6 fewer positive charges in the class III enzymes accounting for their electrophoretic differences. The substrate binding site of class III differs from that of class I by replacements at positions that form the hydrophobic barrel typical for this site. In class III, two to four of these positions contain residues with polar or even charged side chains (positions 57 and 93 in all species, plus positions 116 in the horse and 140 in the human and the horse), while corresponding intraclass variation is small. All these structural features correlate with functional characteristics and suggest that the enzyme classes serve different roles. In addition, the replacements between these triad sets illustrate further general properties of the two mammalian alcohol dehydrogenase classes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characteristics of mammalian class III alcohol dehydrogenases, an enzyme less variable than the traditional liver enzyme of class I. 269 Sep 42


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