Dehydrogenase System Research Articles

Anaerobic oxidation of phenylacetate and 4-hydroxyphenylacetate to benzoyl-coenzyme A and CO2 in denitrifying Pseudomonas sp. Evidence for an alpha-oxidation mechanism.

Anaerobic degradation of (4-hydroxy)phenylacetate in denitrifying Pseudomonas sp. was investigated. Evidence is presented for alpha-oxidation of the coenzyme A (CoA)-activated carboxymethyl side chain, a reaction which has not been described. The C6-C2 compounds are degraded to benzoyl-CoA and furtheron to CO2 via the following intermediates: Phenylacetyl-CoA, phenylglyoxylate, benzoyl-CoA plus CO2; 4-hydroxyphenylacetyl-CoA, 4-hydroxyphenylglyoxylate, 4-hydroxybenzoyl-CoA plus CO2, benzoyl-CoA. Trace amounts of mandelate possibly derived from mandelyl-CoA were detected during phenylacetate degradation in vitro. The reactions are catalyzed by (i) phenylacetate-CoA ligase which converts phenylacetate to phenylacetyl-CoA and by a second enzyme for 4-hydroxyphenylacetate; (ii) a (4-hydroxy)-phenylacetyl-CoA dehydrogenase system which oxidizes phenylacetyl-CoA to (4-hydroxy)phenylglyoxylate plus CoA; and (iii) (4-hydroxy)phenylglyoxylate: acceptor oxidoreductase (CoA acylating) which catalyzes the oxidative decarboxylation of (4-hydroxy)phenylglyoxylate to (4-hydroxy)benzoyl-CoA and CO2. (iv) The degradation of 4-hydroxyphenylacetate in addition requires the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA, catalyzed by 4-hydroxybenzoyl-CoA reductase (dehydroxylating). The whole cell regulation of these enzyme activities supports the proposed pathway. An ionic mechanism for anaerobic alpha-oxidation of the CoA-activated carboxymethyl side chain is proposed.

The effects of pCO2 on yeast growth and metabolism under continuous fermentation

The effects of pCO2 were investigated by changing the aeration rate, the purging gas and the total pressure in a chemostat cultivatioa Under glucose supply limitation, an increase in pCO2 from 44 kPa to 195 kPa resulted in 25 % decrease in cell concentration, 8 % increase in ethanol concentration, and 50 % decrease in glycerol concentration. Under oxygen supply limitation, similar dependency of ethanol and glycerol on pCO2 was observed, however, no influence of pCO2 on the cell yield was observed. The change in ethanol yield by pCO2 appeared to be caused by the equilibrium shift of pyruvate dehydrogenase system.

Enzymatic synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid in a membrane reactor

Dehydrocholic acid (3,7,12-trioxo-5β-cholanic acid) (0.5% concentration) was completely and selectively reduced to 12-ketoursodeoxycholic acid (3α, 7β-dihydroxy-12-oxo- 5β-cholanic acid) in a membrane reactor by means of 3α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with the glucose-glucose dehydrogenase system.

Electrostimulation of the dehydrogenase system of yeast by alternating currents

The dehydrogenase system of Saccharomyces cerevisiae was electrostimulated utilizing Helmholtz coils (average 4 mT, 60 Hz) to induce a current density of microamps per square centimetre or a field strength of the order of millivolts per centimetre. As an indicator, the reduction rate of methylene blue (MB) was used, measured by its polarographic signal instantaneously in the range of minutes. Up to now the effect of field on the reduction yield of MB has been determined depending on the following parameters: the concentration of cells in suspension, the pre-exposure time before adding MB to the cells, the temperature of the solution, the influence of the physiological state of growing cells and the concentration of phosphate buffer. Non-linear responses were detected in some of these dependences. As a general rule one can conclude that the slower the reduction rate, the higher the stimulation effect. The combination of MB with the dehydrogenase system turns out to be a method for determination of the electromagnetic field effect. This technique may also be useful for other microorganisms.

Electrostimulation of the dehydrogenase system of yeast by alternating currents

The dehydrogenase system of Saccharomyces cerevisiae was electrostimulated utilizing Helmholtz coils (average 4 mT, 60 Hz) to induce a current density of microamps per square centimetre or a field strength of the order of millivolts per centimetre. As an indicator, the reduction rate of methylene blue (MB) was used, measured by its polarographic signal instantaneously in the range of minutes. Up to now the effect of field on the reduction yield of MB has been determined depending on the following parameters: the concentration of cells in suspension, the pre-exposure time before adding MB to the cells, the temperature of the solution, the influence of the physiological state of growing cells and the concentration of phosphate buffer. Non-linear responses were detected in some of these dependences. As a general rule one can conclude that the slower the reduction rate, the higher the stimulation effect.The combination of MB with the dehydrogenase system turns out to be a method for determination of the electromagnetic field effect. This technique may also be useful for other microorganisms.

Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system

A 3.7-kb DNA region encoding part of the Rhodospirillum rubrum CO oxidation (coo) system was identified by using oligonucleotide probes. Sequence analysis of the cloned region indicated four complete or partial open reading frames (ORFs) with acceptable codon usage. The complete ORFs, the 573-bp cooF and the 1,920-bp cooS, encode an Fe/S protein and the Ni-containing carbon monoxide dehydrogenase (CODH), respectively. The four 4-cysteine motifs encoded by cooF are typical of a class of proteins associated with other oxidoreductases, including formate dehydrogenase, nitrate reductase, dimethyl sulfoxide reductase, and hydrogenase activities. The R. rubrum CODH is 67% similar to the beta subunit of the Clostridium thermoaceticum CODH and 47% similar to the alpha subunit of the Methanothrix soehngenii CODH; an alignment of these three peptides shows relatively limited overall conservation. Kanamycin cassette insertions into cooF and cooS resulted in R. rubrum strains devoid of CO-dependent H2 production with little (cooF::kan) or no (cooS::kan) methyl viologen-linked CODH activity in vitro, but did not dramatically alter their photoheterotrophic growth on malate in the presence of CO. Upstream of cooF is a 567-bp partial ORF, designated cooH, that we ascribe to the CO-induced hydrogenase, based on sequence similarity with other hydrogenases and the elimination of CO-dependent H2 production upon introduction of a cassette into this region. From mutant characterizations, we posit that cooH and cooFS are not cotranscribed. The second partial ORF starts 67 bp downstream of cooS and would be capable of encoding 35 amino acids with an ATP-binding site motif.

16] Choline/ethanolamine kinase from rat brain

Choline kinase or ATP:choline phosphotransferase catalyzes the phosphorylation of choline with ATP yielding phosphorylcholine and ADP. This is the initial enzyme of the CDPcholine pathway, which serves as the principal pathway of phosphatidylcholine synthesis in animal tissues. Choline kinase has been highly purified from lung, kidney, liver and brain. The brain is one of the richest sources of choline kinase. The enzyme of the soluble fraction from brain has been purified to homogeneity and shown to mediate the phosphorylation of choline, N,N -dimethylethanolamine, N -monomethylethanolamine, and ethanolamine. Choline kinase activity is assayed by determining the rate of formation of either phosphorylcholine isotopically or ADP spectrophotometrically. In the first method, [ 14 C]choline is used as substrate and the phosphorylcholine formed is separated from unreacted choline by solvent extraction and then counted. In the second method, choline kinase is coupled with the pyruvate kinase–lactate dehydrogenase system, so that the ADP formed can be determined spectrophotometrically by measuring the decrease in NADH.

A possible defect in the inter-conversion between cortisone and cortisol in prepubertal patients with congenital adrenal hyperplasia receiving cortisone acetate therapy

Oral administration of cortisone acetate is widely used to treat prepubertal patients with congenital adrenal hyperplasia (CAH). However, efficient ‘first pass’ hepatic conversion of the biologically inactive cortisone (E) to cortisol (F) by the 11-reductase component of the 11β-hydroxysteroid dehydrogenase (11β-HSD) system is required for suppression of the hypothalamic—pituitary—adrenal (HPA) axis. 11-β-HSD activity can be assessed by measurement of urinary tetrahydroderivatives of E (tetrahydrocortisone, THE) and F (tetrahydrocortisol, THF), formed in separate hepatic compartments by reduction of the A ring. Inadequate HPA axis suppression is frequently encountered in peripubertal CAH patients receiving cortisone acetate therapy. In this paper, we describe THE and THF concentrations in 24 h urine samples collected every 3–6 months from 14 prepubertal patients with simple virilizing CAH. The patients had been receiving cortisone acetate and 9α-fluorohydrocortisone since diagnosis and were investigated for 2–4 years during which there was marked intra- and inter-individual variation in the level of suppression. Good and poor control of HPA axis suppression were defined on the basis of a profile of early morning serum 17-hydroxyprogesterone, androstenedione, plasma renin activity and 24 h urinary excretion of pregnanetriol, pregnanetriolone and 5β,17α-hydroxypregnanolone. Serum steroids were measured by RIA and urinary metabolites quantitated as methyloxime—trimethylsilylimidazole derivatives by gas chromatography and GC-mass spectrometry. There were no significant differences in the THE/THF ratio between male ( n = 9) and female ( n = 5) patients during either good or poor therapeutic control. The data were therefore analyzed without consideration of patient sex. Urinary THE/THF (mean ± SD) was significantly higher in patients during periods of poor control (6.56 ± 2.51, P < 0.001) compared with periods of good control (3.73 ± 0.96) in the same patients. THE/THF levels were also significantly ( P < 0.001) higher in CAH patients, irrespective of the level of control, than those for the normal subjects (1.79 ± 0.20). Furthermore, THE excretion was significantly higher during periods of poor control compared with good control at all doses of cortisone acetate administered (10–50 mg/day). There were no significant differences in THF excretion. THE levels also rose significantly ( P < 0.001) in response to increasing total dose during periods of poor control. The increase in THF excretion was slight and significant only at doses >40 mg/day compared with doses <15 mg/day. A significant linear correlation could be drawn between THE excretion and total daily dose during periods of both poor ( r = 0.67, P < 0.05) and good ( r = 0.68, P < 0.005) control. Excretion of 5α-THF, cortols and the cortolones, unlike that for normal subjects, was very low in cortisone acetate treated CAH patients. In contrast, the ratios of 5α 5β C19 steroid metabolites were no different from normal. The results from this study suggest that poor therapeutic control is unlikely to be due to: (i) failure of compliance with therapy; (ii) inefficient absorption from the intestine; or (iii) inefficient acetate group removal. The data are consistent with a hypothesis of rapid tetrahydroderivitization of E, present in excess of the capacity of 11β-HSD to form F; i.e. a preferential reduction of the A ring rather than the 11-keto group as a consequence of hepatic ‘first pass’ uptake of the exogenously administered E. The ability of prednisolone and dexamethasone, both of which are bioactive and not subject to significant A ring reduction, to suppress the HPA axis of patients poorly suppressed by cortisone acetate, lends support to this hypothesis.

Immobilization of Arthrobacter simplex in thermally reversible hydrogels: effect of gel hydrophobicity on steroid conversion.

Arthrobacter simplex cells have been immobilized in a series of thermally reversible hydrogels having different gel hydrophobicities. Steroid conversion from hydrocortisone to prednisolone via the delta 1-dehydrogenase system was greatly affected by the relative hydrophobicities of the gel matrices, which were prepared by copolymerizing varying ratios of N-isopropylacrylamide to acrylamide. The characteristics of the immobilized cells, such as optimal temperatures, Km values, and the effects of an added artificial electron acceptor, were largely influenced by the gel matrices and their different lower critical solution temperatures (LCST). The data indicate that the microenvironment of the dehydrogenation system is quite different within the different hydrophilic/hydrophobic gel matrices. The high partitioning of water-insoluble steroids into the hydrophobic regions and the reduced possibility of product inhibition within the more hydrophobic gel matrices may cause the observed higher steroid conversion in these gels. A possible model for immobilized A. simplex cells in such different gel matrices is proposed.

Two genes encoding putative mitochondrial alcohol dehydrogenases are present in the yeast Kluyveromyces lactis

Four structural genes encoding isozymes of the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis have been identified by hybridization to ADH2 DNA probes from Saccharomyces cerevisiae. In this paper we report on the isolation of KlADH4 and the complete sequencing of KlADH3 and KlADH4, two genes which show high homology to KlADH1, the ADH gene previously isolated in K. lactis, and to the ADH genes of S. cerevisiae. When compared with KlADH1, both KlADH3 and KlADH4 encode amino-terminal extensions which show the characteristics of the mitochondrial targeting sequences. These extensions are poorly conserved both at the nucleotide and the amino acid level. Surprisingly, the KlADH4 extension shows a higher identity at the amino acid level to the one encoded by ADH3 of S. cerevisiae than to the KlADH3 presequence. KlADH3 and KlADH4, in contrast to the ADH3 gene of S. cerevisiae, show a strong bias in the choice of codons.

Amphibian alcohol dehydrogenase, the major frog liver enzyme. Relationships to other forms and assessment of an early gene duplication separating vertebrate class I and class III alcohol dehydrogenases

Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of the different types of the mammalian enzyme. Two avian forms were recently reported, and we now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digests. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to the human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. Functionally, the amphibian enzyme exhibits properties typical for class I but has an unusually low Km for ethanol (0.09 mM) and Ki for pyrazole (0.15 microM) at pH 10.0. This correlates with a strictly hydrophobic substrate pocket and one amino acid difference toward the human class I enzyme at the inner part of the pocket. Coenzyme binding is highly similar, while subunit-interacting residues, as in other alcohol dehydrogenases, exhibit several differences.(ABSTRACT TRUNCATED AT 250 WORDS)

"Ermioni" a new Lemon Cultivar Resistant to Mal secco Disease (Phoma tracheiphila)

A lemon tree resistant to citrus tracheomycotic disease Mal secco (Phoma tracheiphila) found in a Greek orchard and its progenies were evaluated. Scions budded on sour orange and Volkameriana rootstocks or cuttings rooted in mist were placed under natural infections or inoculated artificially with Mal secco inoculum. No disease symptoms appeared on the original tree, while in bud-progenies only 16 out of approximately 2500 field grown trees were diseased by Mal secco. In 45 artificially inoculated trees only two were infected by the fungus. Analysis of leaf proteins using electrophoresis and further genetic control of gene/enzyme 6-Phosphogluconate dehydrogenase (6-PGD) system were carried out for the resistant selection: the Greek varieties Maglini, Adamopoulou, Karystini and the Italian Monachello and Interdonato. Protein analysis showed a close relationship of the resistant selection with var. Maglini, of which a scion is the resistant selection, while the isozyme genetic marker showed three different electrophoretic patterns, the resistant selection being in a separate pattern. Some phenotypical differences between the resistant selection and Maglini were also observed. The phenotypical differences, the resistance to Mal secco, the differences in proteins from all other varieties except Maglini and the different electrophoretic pattern of 6-PGD of this selections vs other varieties, suggest a new cultivar, for which the name “Ermioni” is proposed.

Mechanisms of generation of oxygen radicals and reductive mobilization of ferritin iron by lipoamide dehydrogenase.

The oxidase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO-OOH and DMPO-OH signals were observed. The DMPO-OOH signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO-OOH adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH3 signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO-OOH adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO-OOH signal, indicating that the superoxide radical interacted with ferritin iron.

Human class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase

The class III human liver alcohol dehydrogenase, identical to glutathione-dependent formaldehyde dehydrogenase, separates electrophoretically into a major anodic form (chi 1) of known structure, and at least one minor, also anodic but a slightly faster migrating form (chi 2). The primary structure of the minor form isolated by ion-exchange chromatography has now been determined. Results reveal an amino acid sequence identical to that of the major form, suggesting that the two derive from the same translation product, with the minor form modified chemically in a manner not detectable by sequence analysis. This pattern resembles that for the classical alcohol dehydrogenase (class I). Hence, the chi 1/chi 2 multiplicity does not add further primary forms to the complex alcohol dehydrogenase system but shows the presence of modified forms also in class III.

Avian alcohol dehydrogenase: the chicken liver enzyme

The major ethanol-active form of chicken liver alcohol dehydrogenase was characterized. The primary structure was determined by peptide analysis and, to a large part, was also deduced by cDNA analysis of a near full-length cDNA clone. The latter was detected by screening of a chicken liver cDNA library with antibodies raised against the purified dehydrogenase. The structure shows that the avian enzyme exhibits characteristics of the complex mammalian alcohol dehydrogenase system, tracing its origin and divergence, and allowing functional correlations. The chicken protein analyzed proves to be a class I alcohol dehydrogenase, with 74% residue identity to gamma chains of the human enzyme, a Km for ethanol of 0.5 mM and a Ki for 4-methyl pyrazole of 2.5 microM. Relationships to the other two classes are non-identical; residue exchanges towards the human classes increase in the order I less than III less than II, and human/chicken differences are less than inter-class differences. Consequently, the origins of the classes are more distant than the avian/mammalian separation. They reflect duplicatory events separated in time, and the lines that lead to present-day classes I and II deviate early. Integrated with the data for the quail enzyme, the structure of the chicken protein shows that within the avian enzymes the degree of variation is comparable to that within the mammalian class I enzymes, which are more variable than the class III forms. The coenzyme-binding and substrate-binding residues of this chicken alcohol dehydrogenase are largely identical to those in the mammalian class I counterparts. However, the subunit-interacting areas are more variable and suggest some relationships of the avian enzyme with both class I and III mammalian forms. One of the residues, Gly260 (mammalian class I numbering system), previously considered characteristic of all alcohol dehydrogenases, is replaced by Gln.

Avian alcohol dehydrogenase. Characterization of the quail enzyme, functional interpretations, and relationships to the different classes of mammalian alcohol dehydrogenase

The primary structure of the major quail liver alcohol dehydrogenase was determined. It is a long-chain, zinc-containing alcohol dehydrogenase of the type occurring also in mammals and hence allows judgement of the gene duplications giving rise to the classes of the human alcohol dehydrogenase system. The avian form is most closely related to the class I mammalian enzyme (72-75% residue identity), least related to class II (60% identity), and intermediately related to class III (64-65% identity). This pattern distinguishes the mammalian enzyme classes and separates classes I and II in particular. In addition to the generally larger similarities with class I, the avian enzyme exhibits certain residue patterns otherwise typical of the other classes, including an extra Trp residue, present in both class II and III but not in class I, with a corresponding increase in the UV absorbance. The avian enzyme further shows that a Gly residue at position 260 previously considered strictly conserved in alcohol dehydrogenases can be exchanged with Lys. However, zinc-binding residues, coenzyme-binding residues, and to a large extent substrate-binding residues are unchanged in the avian enzyme, suggesting its functional properties to be related to those of the class I mammalian alcohol dehydrogenases. In contrast, the areas of subunit interactions in the dimers differ substantially. These results show that (a) the vertebrate enzyme classes are of distant origin, (b) the submammalian enzyme exhibits partly mixed properties in relation to the classes, and (c) the three mammalian enzyme classes are not as equidistantly related as initially apparent but suggest origins from two sublevels.

Production of essential L‐branched‐chain amino acids in bioreactors containing artificial cells immobilized multienzyme systems and dextran–NAD+

We prepared artificial cells each containing leucine dehydrogenase (EC 1.4.1.9), urease (EC 3.5.1.5), soluble dextran-NAD(+), and one of the following coenzyme regenerating dehydrogenases: glucose dehydrogenase (EC 1.1.1.47); yeast alcohol dehydrogenase (EC 1.1.1.1); malate dehydrogenase (EC 1.1.1.37); or lactate dehydrogenase (EC 1.1.1.27). Artificial cells were packed in small columns. L-Leucine, L-valine, and L-isoleucine were continuously produced with simultaneous dextran-NADH regeneration. The maximum production ratios depended on the coenzyme regenerating systems used: 83-93% for D-glucose and glucose dehydrogenase system; 90% for ethanol and yeast alcohol dehydrogenase system; 45-55% for L-malate and malate dehydrogenase system; and 64-78% for L-lactate and lactate dehydrogenase system. Kinetic experiments were also carried out. The apparent K(m) values are as follows: 0.33 mM for alpha-ketoisocaproate (KIC); 0.51 mM for alpha-ketoisovalerate (KIV); 0.58 mM for DL-alpha-keto-beta-methyl-n-valerate (KMV); 3.52 mM for urea; 27.82 mM for D-glucose; 3.89 mM for ethanol; 3.02 mM for L-malate; and 16.67 mM for L-lactate. Kinetic analysis showed that KIC, KIV, and KMV were all competitive inhibitors in the reactions catalyzed by leucine dehydrogenase. Their inhibitor constants were the corresponding K(m) values.

Failure of glutamate dehydrogenase system to predict oxygenation state of human skeletal muscle

In a recent study, the total tissue contents of glutamate (Glu), ammonium (NH+4), and 2-oxoglutarate (2-OG) were used to estimate changes in the mitochondrial redox state ([NAD+]/[NADH]) of contracting skeletal muscle with intact circulation [Am. J. Physiol. 253 (Cell Physiol. 22): C263-C268, 1987]. These metabolites participate in the glutamate dehydrogenase (GDH) reaction, which, based on a number of assumptions, theoretically enables calculation of the mitochondrial redox state as follows (brackets indicate concentrations): [NAD+]/[NADH] = ([NH+4] [2-OG])/[( Glu]Kapp), where Kapp is the apparent equilibrium constant for GDH. The purpose of this study was to determine whether changes in the total tissue contents of Glu, NH+4, and 2-OG could be used to predict a reduction of the mitochondrial redox state in anoxic skeletal muscle. Anoxia was induced in the quadriceps femoris muscle by 10 min of circulatory occlusion (low metabolic rate) and isometric contraction to fatigue (high metabolic rate). The mean (+/- SE) value for the metabolite ratio ([NH+4][2-OG]/[Glu]) at rest was 6 +/- 3 mmol/kg dry wt (x 10(-4]. No significant change occurred after circulatory occlusion (4 +/- 2 x 10(-4); P greater than 0.05), whereas an almost 60-fold increase was observed after isometric contraction (P less than 0.05). Because the muscle was anoxic under both conditions, a significant decrease in the metabolite ratio should have occurred. These data demonstrate that changes in total tissue contents of Glu, NH+4, and 2-OG cannot be used to estimate changes in the redox and oxygenation state of mitochondria in intact human skeletal muscle.

The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis

We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomyces pombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named K1ADH1. The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence. We named this gene K1ADH3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K. lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.

Studies on saccharomyces cerevisiae cinder carbon-limiting growth transformed with plasmid pcyg4 that carries the gene for NADP-GDH

The gene (GDH1) coding for the NADP-linked glutamate dehydrogenase system (NADP-GDH) has been cloned from Saccharomyces cerevisiae strain. Cells being transformed by the NADP-GDH gene on a 2 micron bared vector (pCYG4) plasmid confering 11-fold higher level on expressed GDH activity over the wild-type cells. The behavior of these cells was investigated under chemostatic growth with a carbon rate-limiting nutrient. Specific growth rates of cells carrying plasmid pCYG4 were found to be slightly slower than wild type cells. Furthermore, the NADP-GDH activity increases proportionally with the dilution rate. In addition, oscillations in the NADP-GDH activity, especially at a dilution rate up to 0.15/h, are probably consequential on the appearance of a changing mixed population (cells with and without plasmids).