PubMed · 13619425
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
G MACHATA. 1958. [Alcohol dehydrogenase method].. https://pubmed.ncbi.nlm.nih.gov/13619425/
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Of 17 genes annotated in the Arabidopsis genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an in silico analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine putative CADs, two (AtCAD5 and AtCAD4) had the highest activity and homology (approximately 83% similarity) relative to bona fide CADs from other species. AtCAD5 used all five substrates effectively, whereas AtCAD4 (of lower overall catalytic capacity) poorly used sinapyl aldehyde; the corresponding 270-fold decrease in k(enz) resulted from higher K(m) and lower k(cat) values, respectively. No CAD homologue displayed a specific requirement for sinapyl aldehyde, which was in direct contrast with unfounded claims for a so-called sinapyl alcohol dehydrogenase in angiosperms. AtCAD2, 3, as well as AtCAD7 and 8 (highest homology to sinapyl alcohol dehydrogenase) were catalytically less active overall by at least an order of magnitude, due to increased K(m) and lower k(cat) values. Accordingly, alternative and/or bifunctional metabolic roles of these proteins in plant defense cannot be ruled out. Comprehensive analyses of lignified tissues of various Arabidopsis knockout mutants (for AtCAD5, 6, and 9) at different stages of growth/development indicated the presence of functionally redundant CAD metabolic networks. Moreover, disruption of AtCAD5 expression had only a small effect on either overall lignin amounts deposited, or on syringyl-guaiacyl compositions, despite being the most catalytically active form in vitro.
Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.
The assessment of the variability of human responses to foreign chemicals is an important step in characterizing the public health risks posed by nontherapeutic hazardous chemicals and the risk of encountering adverse reactions with drugs. Of the many sources of interindividual variability in chemical response identified to date, hereditary factors are some of the least understood. Physiologically based pharmacokinetic modeling linked with Monte Carlo sampling has been shown to be a useful tool for the quantification of interindividual variability in chemical disposition and/or response when applied to biological processes that displayed single genetic polymorphisms. The present study has extended this approach by modeling the complex hereditary control of alcohol dehydrogenase, which includes polygenic control and polymorphisms at two allelic sites, and by assessing the functional significance of this hereditary control on ethanol disposition. The physiologically based pharmacokinetic model for ethanol indicated that peak blood ethanol levels and time-to-peak blood ethanol levels were marginally affected by alcohol dehydrogenase genotypes, with simulated subjects possessing the B2 subunit having slightly lower peak blood ethanol levels and shorter times-to-peak blood levels compared to subjects without the B2 subunit. In contrast, the area under the curve (AUC) of the ethanol blood decay curve was very sensitive to alcohol dehydrogenase genotype, with AUCs from any genotype including the ADH1B2 allele considerably smaller than AUCs from any genotype without the ADH1B2 allele. Furthermore, the AUCs in the ADH1C1/C1 genotype were moderately lower than the AUCs from the corresponding ADH1C2/C2 genotype. Moreover, these simulations demonstrated that interindividual variability of ethanol disposition is affected by alcohol dehydrogenase and that the degree of this variability was a function of the ethanol dose.