Enzyme variation between syngens in Paramecium aurelia

Five enzymes (succinic dehydrogenase isocitrate dehydrogenase, glutamate dehydrogenase, -hydroxybutyrate dehydrogenase, and fumarase) in the ciliate Paramecium aurelia have been examined by starch gel electrophoresis. Relatively few variants were found among stocks belonging to a given syngen and only in two out of the five enzymes studied. Comparison of stocks belonging to different syngens, however, revealed many enzyme differences, which did not resemble intrasyngen variants. By studying the electrophoretic patterns of the enzymes of the 14 described syngens of Paramecium aurelia, it was found that only two syngens (Nos. 1 and 5) were indistinguishable. It is concluded that the use of starch gel electrophoresis of suitable enzymes provides a method of assigning a stock of paramecia to its syngen. Such a method may, in some cases, be less laborious than the standard one of making test matings and would, of course, be available for organisms which show no mating reaction.This work was carried out during the tenure of an M.R.C. Postgraduate Research Scholarship, and forms part of the material for a Ph.D. thesis at the University of Edinburgh.     

Peroxisomal Localization of Enzymes Related to Fatty Acid β-Oxidation in an n-Alkane-grown Yeast,Candida tropicalis

In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β—oxidation—acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase—were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.

The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.     

Glucose-dependent enzyme activities in different yeast species

Summary The activities of several enzymes involved in the oxidation of ethanol and in the formation of oxaloacetate from acetate were compared in the yeasts Saccharomyces cerevisiae, Hansenula anomala and Rhodotorula glutinis grown on glucose and acetate, respectively. The most striking differences in the regulation of enzyme activities were found for alcohol dehydrogenase, acetaldehyde dehydrogenase, and malate dehydrogenase. The activities of the other enzymes tested behaved rather similar; in all three yeast species the enzymes of the glyoxylic acid by-pass showed the most extensive increase of activity in cells grown on acetate.     

Chlorate Reducing Activity of Spinach Nitrate Reductase

The activities of 2-oxoaldehyde-metabolizing enzymes (glyoxalase I, glyoxalase II, methyl- glyoxal reductase, methylglyoxal dehydrogenase and lactaldehyde dehydrogenase) were found to be widely distributed among microorganisms. One of the enzymes, methylglyoxal reductase, which catalyzes the reductive conversion of methylglyoxal into lactaldehyde, was purified from Escherichia coli cells. The enzyme was judged to be homogeneous on polyacrylamide gel electrophoresis and was a monomer with a molecular weight of 43000. The enzyme was most active at pH 6.5 and 45°C. The enzyme utilized both NADPH and NADH for the reduction of 2- oxoaldehydes (glyoxal, methylglyoxal, phenylglyoxal and 4,5-dioxovalerate) and some aldehydes (glycolaldehyde, D,l-glyceraldehyde, propionaldehyde and acetaldehyde). The Km values of the enzyme for methylglyoxal, NADPH and NADH were 4.0 mm, 1.7 fiM and 2.8 /¿m, respectively. The product of methylglyoxal reduction was identified as lactaldehyde. The enzyme from E. coli cells was different from the yeast and goat liver enzymes in both molecular structure and substrate specificity.     

Purification of Alcohol Dehydrogenase fromEntamoeba histolyticaandSaccharomyces cerevisiaeUsing Zinc-Affinity Chromatography

We have developed a single-step method for the purification of NADP⁺-dependent alcohol dehydrogenase fromEntamoeba histolyticaand NAD⁺-dependent alcohol dehydrogenase fromSaccharomyces cerevisiae.It is based on the affinity for zinc of both enzymes. The amebic enzyme was purified almost 800 times with a recovery of 54% and the yeast enzyme was purified 30 times with a recovery of 100%. The kinetic constants of the purified enzymes were similar to those reported for other purification methods. With mammalian alcohol dehydrogenase, we obtained a 40-kDa band suggestive of purified alcohol dehydrogenase, but we failed to retain enzymatic activity in this preparation. Our results suggest that the described method is more applicable to the purification of tetrameric alcohol dehydrogenases.     

Genetic tags in the New Zealand hoki Macruronus novaezelandiae1

Twelve enzymes and liver proteins were examined by starch and cellulose acetate electrophoresis in two samples of hoki Macruronus novaezelandiae from New Zealand. Two polymorphic enzymes, a-glycerophosphate dehydrogenase and sorbitol dehydrogenase, were found giving a mean heterozygosity of 0.027 over 12 enzyme loci or 0.016 over 15 protein loci. No differences in gene frequency were detected in nine regional samples collected from the New Zealand 200-mile exclusive economic zone. Together with existing biological information on spawning grounds these data suggest that hoki in the New Zealand fishery form one stock.     

The effect of yeast dehydration on the activity of a number of enzymes of cell energetic metabolism

Summary It has been shown that dehydration markedly affects the activity of a number of enzymes connected with energy metabolism in the yeastSaccharomyces cerevisiae. Independently of the drying method used, there was found to be an inverse relationship between the activity of mitochondrial enzymes — NADH-dehydrogenase (EC 1.6.2.1), succinate dehydrogenase (EC 1.3.99.1) and cytochrome C oxidase (EC 1.9.3.1) - and the viability of yeast cells at the stationary growth phase. Dehydration led to an increase in activity only in exogenous NADH-dehydrogenase compared with activity in the initial compressed yeast. On the basis of alcohol dehydrogenase (EC 1.1.1.1) and catalase (EC 1.11.1.6) as examples, an ambivalent effect of the dehydration process on the activity of cytoplasmic enzymes has been demonstrated. The results obtained lead to the conclusion that the activity of individual electron-transport enzymes in yeastSaccharomyces cerevisiae is a sufficiently sensitive to be used as an indicator of the physiological state and to monitor a microbial biomass dehydration procedure.     

Acquisition of a potential marker for insect transformation: isolation of a novel alcohol dehydrogenase gene from Bactrocera oleae by functional complementation in yeast

The alcohol dehydrogenase genes make up one of the best studied gene families in Drosophila, both in terms of expression and evolution. Moreover, alcohol dehydrogenase genes constitute potential versatile markers in insect transformation experiments. However, due to their rapid evolution, these genes cannot be cloned from other insect genera by DNA hybridization or PCR-based strategies. We have therefore explored an alternative strategy: cloning by functional complementation of appropriate yeast mutants. Here we report that two alcohol dehydrogenase genes from the medfly Ceratitis capitata can functionally replace the yeast enzymes, even though the medfly and yeast genes have evolved independently, acquiring their enzymatic function convergently. Using this method, we have cloned an alcohol dehydrogenase gene from the olive pest Bactrocera oleae. We conclude that functional complementation in yeast can be used to clone alcohol dehydrogenase genes that are unrelated in sequence to those of yeast, thus providing a powerful tool for isolation of dominant insect transformation marker genes. Received: 29 June 1999 / Accepted: 27 October 1999     

A cyclophilin A CPR1 overexpression enhances stress acquisition in Saccharomyces cerevisiae

Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. We found the expression of cyclophilin A, Cpr1, changes in response to exposure to yeast Saccharomyces cerevisiae to abiotic stress conditions. The effect of Cpr1 overexpression in stress responses was therefore examined. The CPR1 gene was cloned to the yeast expression vector pVTU260 under regulation of an endogenous alcohol dehydrogenase (ADH) promoter. The overexpression of Cpr1 drastically increased cell viability of yeast in the presence of stress inducers, such as cadmium, cobalt, copper, hydrogen peroxide, tert-butyl hydroperoxide (t-BOOH), and sodium dodecyl sulfate (SDS). The Cpr1 expression also enhanced the cell rescue program resulting in a variety of antioxidanr enzymes including thioredoxin system (particularly, thioredoxin peroxidase), metabolic enzymes (glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase), and molecular chaperones (Hsp104, Hsp90, Hsp60 and Hsp42). Thus, our study illustrates the importance of Cpr1 as a molecular chaperone that improves cellular stress responses through collaborative relationships with other proteins when yeast cells are exposed to adverse conditions, and it also premises the improvement of yeast strains.     

Inhibition of glycolysis by furfural in Saccharomyces cerevisiae

Summary Furfural, a Maillard reaction product, was found to inhibit growth and alcohol production by Saccharomyces cerevisiae. Furfural concentrations above 1 mg ml^–1 significantly decreased CO₂ evolution by resuspended yeast cells. Important glycolytic enzymes such as hexokinase, phosphofructokinase, triosephosphate dehydrogenase, aldolase and alcohol dehydrogenase were assayed in presence of furfural. Dehydrogenases appeared to be the most sensitive enzymes and are probably responsible for the observed inhibition of alcohol production and growth.     

Alkali Protease and Alcohol Dehydrogenase Immobilized to Weakly Basic Anion Exchange Resins Using 2-Car-boxymethylamino-4,6-dichloro-s-triazine

Alkali protease partially purified from a strain of Bacillus subtilis and yeast alcohol dehydrogenase were immobilized to weakly basic anion exchange resins using a bifunctional reagent, 2-carboxymethyIamino-4,6-dichloro-s-triazine.

Properties of these immobilized enzymes were studied both in batchwise operation and in packed bed reactor systems.     

Unusual evolution of 11β- and 17β-hydroxysteroid and retinol dehydrogenases

11β-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17β-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals. Phylogenetic analysis of the sequences from two 11β-hydroxysteroid dehydrogenases and four mammalian 17β-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes. Type 1 11β- and 17β-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with β-hydroxybutyrate dehydrogenase, 11-cis retinol dehydrogenase and retinol dehydrogenase; Type 3 17β-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch. Pig 17β-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17β-hydroxysteroid dehydrogenase dehydrogenases; in which case, the evolution of 17β-hydroxysteroid dehydrogenase activity is an example of functional convergence. The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11β- and 17β-hydroxysteroid dehydrogenases.     

Activities of the enzymes of formaldehyde catabolism in recombinant strains of <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Activities of the enzymes of formaldehyde (FA) catabolism in recombinant strains of the methylotrophic yeast Hansenula polymorpha overproducing NAD⁺- and glutathione-dependent formaldehyde dehydrogenase (FADH) were studied under different cultivation conditions and at elevated FA content. Southern dot-blot analysis confirmed the presence of six to eight copies of the target FLD1 gene in stable recombinant clones of H. polymorpha. Under certain cultivation conditions, the transformants resistant to elevated FA concentrations were shown to produce FADH and other bioanalytically important enzymes: formate dehydrogenase, alcohol dehydrogenase, alcohol oxidase, and formaldehyde reductase. The optimal cultivation conditions for recombinants were determined, resulting in maximum synthesis of FADH: methanol as a carbon source, methylamine as a nitrogen source, FA as an inducer, temperature of 37°C, and cells in the early exponential phase of growth.     

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD⁺-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%–70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.     

Enzydmatic Characterization of Babesia bovis1

ABSTRACT. Agarose gel electrophoresis was used to identify metabolic enzymes in Babesia bovis and B. bigemina. Glutamate dehydrogenase, lactate dehydrogenase, glucose phosphate isomerase, and hexokinase were identified in B. bovis- and B. bigemina-infected erythrocytes and B. bovis merozoite preparations. A specific electrophoretic mobility was observed for each enzyme. Malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and adenylate kinase were only detected in normal erythrocyte preparations. Inter-species, but not intra-species, variation was noted when comparing electrophoretograms of both species. Kinin-activating activity was not detected in B. bovis-infected erythrocyte or merozoite preparations at pH 4.2 or 7.6.     

Electrophoretic studies on three enzymes from Sarcocystis species in sheep

Summary Thin-layer starch gel electrophoresis was used to distinguish isoenzymic differences between three species of Sarcocystis from sheep, Sarcocystis tenella, S. gigantea and S. medusiformis. Three enzymes, NADP dependent glutamate dehydrogenase, phosphoglucomutase and 6-phosphogluconate dehydrogenase showed distinctive enzyme banding patterns for the three species. ac]19800301     

Enzymatic and Molecular Characterization of Arabidopsis ppGpp Pyrophosphohydrolase,AtNUDX26

Three α-keto ester reductases (yeast keto ester reductase, YKER-II, -IV, -V) were purified from bakers’ yeast. YKER-II, -IV, and -V are dimeric, monomeric, and dimeric enzymes, respectively, and molecular masses are estimated to be 58, 31–39, and 83kDa, respectively, based on gel filtration and SDS- polyacrylamide electrophoresis. Kinetic parameters and stereoselectivities in reduction of α-keto esters have been measured. YKER-IV contributes mainly to reduction by bakers’ yeast at low substrate concentrations, and is useful for synthetic purposes.     

Morphology-associated expression of NADP-dependent glutamate dehydrogenases during yeast-mycelium transition of a dimorphic fungus Benjaminiella poitrasii

Benjaminiella poitrasii, a dimorphic zygomycetous fungus possesses three glutamate dehydrogenases, one requiring NAD while the other two use NADP as a coenzyme. In the activity staining after electrophoresis on native polyacrylamide gel NAD- dependent glutamate dehydrogenase revealed the presence of one enzyme that was expressed in both, yeast- and mycelium-form cells. While in case of NADP- dependent glutamate dehydrogenase two distinct activity bands that were differentially expressed in yeast- and mycelium-form cells were seen. Interestingly, during yeast-mycelium transition and reverse, quantitative changes in form-specific native NADP-dependent glutamate dehydrogenase activities were seen. The biochemical data on temperature and pH optima, thermostability, and kinetic properties confirmed the presence of two NADP-dependent proteins in B. poitrasii, parent strain. The monomorphic mutant (Y-5, yeast form) showed NADP- glutamate dehydrogenase similar to parent yeast-form enzyme. For the first time the significance of differential expression of these enzymes during morphological transition in B. poitrasii has been suggested.     

Optically Active Bis-β-keto Sulfonium Ylid

Particulate alcohol dehydrogenase of acetic acid bacteria that is mainly participated in vinegar fermentation was purified to homogeneous state from Gluconobacter suboxydans IFO 12528. Solubilization of enzyme from the bacterial membrane fraction by Triton X-100 and subsequent fractionation on DEAE-Sephadex A-50 and hydroxylapatite was successful in enzyme purification. A cytochrome c-like component was tightly bound to the dehydrogenase protein and existed as an enzyme-cytochrome complex. It was also confirmed that the alcohol dehydrogenase is not a cytochrome component itself. The molecular weight of the enzyme was determined to be 150,000, and gel electrophoresis showed the presence of three subunits having a molecular weight of 85,000, 49,000 and 14,400. The smallest subunit was corresponded to the cytochrome c-like component. Ethanol was oxidized in the presence of dyes in vitro but NAD or NADP were not required as hydrogen acceptor. Unlike NAD- linked alcohol dehydrogenase in yeast or liver and other primary alcohol dehydrogenases in methanol utilizing bacteria, the enzyme from the acetic acid bacteria showed its optimum pH at fairly acidic pH.     

Purification of full-length recombinant human and rat type 1 11β-hydroxysteroid dehydrogenases with retained oxidoreductase activities

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum. This enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase. Previous attempts to purify the native enzyme have yielded a protein without reductase activity. To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11β-HSD1 with retention of their natural oxidoreductase activities. This procedure involved recombinant expression of these histidine-tagged enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography. Both enzymes were 90–95% pure and exhibited dehydrogenase and reductase activities with K_M values in agreement with those reported in the literature.