Thermodynamic,kinetic, and operational stabilities of yeast alcohol dehydrogenase in sugar and compatible osmolyte solutions

Thermodynamic, kinetic, and operational stabilities of yeast alcohol dehydrogenase (YADH) were measured and compared in aqueous solutions containing various sugars (sucrose, glucose, and ribose) and compatible osmolytes (betaine and sarcosine). In the measurement of operational stability, native YADH was entrapped and physically immobilized in an ultrafiltration hollow fiber tube to retain the native characteristics of the enzyme. All the additives tested increased thermodynamic stability and kinetic stability of YADH. The order of the magnitude of stabilization effect among additives was different between thermodynamic and kinetic stabilities. Compared to the thermodynamic and kinetic stabilities, the effects of additives were much different in operational stability. Sucrose, glucose, and betaine stabilized YADH substantially while ribose and sarcosine destabilized the enzyme. These results show that the thermodynamic and kinetic stabilities do not necessarily guarantee the operational stability of YADH. The coexistence of stabilizing solute was proved effective to increase the productivity of the bioreactor with immobilized YADH.     

Regeneration of NAD(H) by alcohol dehydrogenase in gel-entrapped yeast cells

Anaerobically grown cells of Saccharomyces cerevisiae entrapped in polyacrylamide gel have been shown to provide a stable source of alcohol dehydrogenase [(ADH) alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1] for effective regeneration of NAD(H). This system was able to provide the coenzyme required for the operation of other dehydrogenases, such as lactate dehydrogenase [(LDH) l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27] and malate dehydrogenase [(MDH) l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37]. Yeast cells coimmobilized with a dehydrogenase are capable of the reversible regeneration of the reduced or oxidized coenzyme, depending on the additions made. A two-cell system can also be constituted using the same strain of yeast, adapted differently. Cells grown anaerobically and aerobically as sources of ADH and MDH, respectively, can operate efficiently on coimmobilization. The system can be used repeatedly without measurable loss of efficiency.     

Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307deg;C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.     

The expression and anaerobic induction of alcohol dehydrogenase in cotton

The alcohol dehydrogenase (ADH) system in cotton is characterized, with an emphasis on the cultivated allotetraploid speciesGossypium hirsutum cv. Siokra. A high level of ADH activity is present in seed of Siokra but quickly declines during germination. When exposed to anaerobic stress the level of ADH activity can be induced several fold in both roots and shoots of seedlings. Unlike maize andArabidopsis, ADH activity can be anaerobically induced in mature green leaves. Three major ADH isozymes were resolved in Siokra, and it is proposed that two genes,Adh1 andAdh2, are coding for these three isozymes. The genes are differentially expressed. ADH1 is predominant in seed and aerobically grown roots, while ADH2 is prominent in roots only after anaerobic stress. Biochemical analysis demonstrated that the ADH enzyme has a native molecular weight of approximately 81 kD and a subunit molecular weight of approximately 42 kD, thus establishing that ADH in cotton is able to form and is active as dimers. Comparisons of ADH activity levels and isozyme patterns between Siokra and other allotetraploid cottons showed that the ADH system is highly conserved among these varieties. In contrast, the diploid species of cotton all had unique isozyme patterns.This work was generously supported by an Australian Cotton Research Council Postgraduate Studentship.     

Subcellular localization of long-chain alcohol dehydrogenase and aldehyde dehydrogenase in n-alkane-grown Candida tropicalis

Long-chain alcohol dehydrogenase and longchain aldehyde dehydrogenase were induced in the cells of Candida tropicalis grown on n-alkanes. Subcellular localization of these dehydrogenases, together with that of acyl-CoA synthetase and glycerol-3-phosphate acyltransferase, was studied in terms of the metabolism of fatty acids derived from n-alkane substrates. Both longchain alcohol and aldehyde dehydrogenases distributed in the fractions of microsomes, mitochondria and peroxisomes obtained from the alkane-grown cells of C. tropicalis. Acyl-CoA synthetase was also located in these three fractions. Glycerol-3-phosphate acyltransferase was found in microsomes and mitochondria, in contrast to fatty acid -oxidation system localized exclusively in peroxisomes. Similar results of the enzyme localization were also obtained with C. lipolytica grown on n-alkanes. These results suggest strongly that microsomal and mitochondrial dehydrogenases provide long-chain fatty acids to be utilized for lipid synthesis, whereas those in peroxisomes supply fatty acids to be degraded via -oxidation to yield energy and cell constituents.     

Distribution of aldehyde dehydrogenase and alcohol dehydrogenase in summer-acclimatized crucian carp, Carassius carassius L.

The tissue distribution of aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) in summer-acclimatized crucian carp showed almost the same exceptional pattern as previously found in winter-acclimatized specimens. There was a nearly complete spatial separation of ALDH and ADH; in other vertebrates these enzymes occur together. This exceptional enzyme distribution is probably an adaptation to the extraordinary ability of Carassius to produce ethanol as the major metabolic end product during anoxia. Since the crucian carp is less likely to encounter anoxia during the summer, the present results suggest that the crucian carp is unable to switch over to a 'normal' ALDH and ADH distribution in the summer. However, it is also possible that there is an advantage for the summer-acclimatized crucian carp in keeping ALDH and ADH separate, because of occasional anoxic periods.     

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

A method for determining the in vivo stability of Drosophila alcohol dehydrogenase (E.C.1.1.1.1.)

A rapid and accurate method of measuring the relative in vivo stability of Drosophila alcohol dehydrogenase is presented. The potential of this technique for examining posttranslational control of in vivo enzyme concentrations is discussed.This work was supported by NSF grant #DEB 7815466 to J.M.Journal Paper No. J-9977 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2272.     

Differences in charge and kinetic properties of alcohol dehydrogenase 4 from C57BL/6 mice compared to other inbred strains are associated with a cysteine120 to arginine120 substitution

Alcohol dehydrogenase class IV (ADH4) participates in retinol metabolism and is expressed primarily in ocular, digestive, and reproductive tissues of the mouse. A naturally occurring genetic variant in C57BL/6J mice results in a faster migrating ADH4 enzyme during electrophoresis when compared to other non-C57BJ/6J strains. The C57BL/6 ADH4 gene coding sequence is found to have two nucleotide substitutions when compared to the gene from C3HeB/FeJ mice. The substitution in exon 5 encodes Arg120 instead of Cys120 in C57BL/6 ADH4 polypeptide; that would account for the protein electrophoretic phenotype. Arg120 is present in all published mammalian ADH4 sequences but is only in a limited number of mouse strains. The Arg120 residue is part of the outer loop of the substrate binding pocket and appears to have an effect on the affinity of the enzyme for several substrates.     

Genetic analysis of cinnamyl alcohol dehydrogenase in loblolly pine: single gene inheritance, molecular characterization and evolution

The gene encoding the monolignol biosynthetic enzyme cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) can be expressed in response to different developmental and environmental cues. Control of Cad gene expression could involve either differential regulation of more than one Cad gene or, alternatively combinatorial regulation of a single Cad gene. In loblolly pine (Pinus taeda L.), we found several electrophoretic variants (allozymes) of CAD and a high level of heterozygosity (h_e=0.46). Analysis of inheritance patterns of pine CAD allozymes gave segregation ratios that were consistent with Mendelian expectations for a single functional gene. The identity of the full-length Cad cDNA sequence was confirmed by alignment with peptide sequences obtained from purified active enzyme and by extensive similarity to Cad sequences from other species. Southern blot analysis of genomic DNA using the Cad cDNA as a hybridization probe gave simple patterns, consistent with our interpretation that pine Cad is a single-copy gene. Phylogenetic analysis and evolution rate estimates showed that Cad sequences are diverging less rapidly in the gymnosperms than in the angiosperms. The Cad mRNA was present in both lignifying tissues and a non lignifying tissue (the megagametophyte) of pine. The presence of a single gene suggests that different regulatory mechanisms for a single Cad gene, rather than differential regulation of several genes, can account for its expression in response to different cues.     

Root and vascular tissue-specific expression of glycine-rich protein AtGRP9 and its interaction with AtCAD5, a cinnamyl alcohol dehydrogenase,in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The vascular tissue of roots performs essential roles in the physical support and transport of water, nutrients, and signaling molecules in higher plants. The molecular mechanisms underlying the function of root vascular tissue are poorly understood. In this study, we analyzed the expression pattern of AtGRP9, a salt stress-responsive gene encoding a glycine-rich protein, and its interacting partner, in Arabidopsis thaliana. Analysis of GUS or GFP expression under the control of the AtGRP9 promoter showed that AtGRP9 was expressed in the vascular tissue of the root; subcellular localization analysis further demonstrated that AtGRP9 proteins were localized in the cell wall and in the cytoplasm. Yeast two-hybrid analysis revealed that AtGRP9 interacted with AtCAD5, a major cinnamyl alcohol dehydrogenase (CAD) involved in lignin biosynthesis, for which tissue-specific distribution was comparable with that of AtGRP9. These results suggest that AtGRP9 may be involved in lignin synthesis in response to salt stress as a result of its interaction with AtCAD5 in A. thaliana.