Ethanol-resistant mutants of Nicotiana plumbaginifolia are deficient in the expression of pollen and seed alcohol dehydrogenase activity

Summary Six independent mutant lines ofNicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed againstArabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designatedAdh1.Abbreviations ADH alcohol dehydrogenase - EMS ethyl methane-sulfonate - MTT dimethyl thiazol tetrazolium - NAD nicotinamide adenine dinucleotide - NBT nitro blue tetrazolium - p-cells protoplast-derived cells - PMS phenazine methosulfate - SDS sodium dodecyl sulfate     

Artifactual staining of proteins on polyacrylamide gels by nitrobluetetrazolium chloride and phenazine methosulfate

Different purified proteins were shown to give purple formazan bands corresponding to the protein stain following electrophoresis on polyacrylamide gels, in the presence of nitrobluetetrazolium (NBT) and phenazine methosulfate (PMS). Both PMS and NBT are needed for formazan production which has a favorable pH at 8.5. Sulfhydryl blockers in the incubation medium inhibited this color development to different extents. While proteins with free SH groups like bovine serum albumin, ovalbumin, and urease showed this pyridine nucleotide independent artifact, nonthiol proteins, viz., bovine pancreatic ribonuclease A, and riboflavin-binding protein from chicken egg white failed to do so. The nonenzymatic formazan formation observed with different proteins could also be shown in an in vitro assay system. It is clear that the “nothing dehydrogenase” phenomenon observed in several cases may be due to the thiol group-mediated artifactual staining of proteins.     

Glycerol metabolism in the methylotrophic yeast Hansenula polymorpha: phosphorylation as the initial step

In hansenula polymorpha glycerol is metabolized via glycerol kinase and NAD(P)-independent glycerol-3-phosphate (G3P) dehydrogenase, enzymes which hitherto were reported to be absent in this methylotrophic yeast. Activity of glycerol kinase was readily detectable when cell-free extracts were incubated at pH 7–8 with glycerol/ATP/Mg²⁺ and a discontinuous assay for G3P formation was used. This glycerol kinase activity could be separated from dihydroxyacetone (DHA) kinase activity by ion exchange chromatography. Glycerol kinase showed relatively low affinities for glycerol (apparent K _m=1.0 mM) and ATP (apparent K _m=0.5 mM) and was not active with other substrates tested. No inhibition by fructose-1,6-bisphosphate (FBP) was observed. Both NAD-dependent and NAD(P)-independent G3P dehydrogenases were present. The latter enzyme could be assayed with PMS/MTT and cosedimented with the mitochondrial fraction. Glucose partly repressed synthesis of glycerol kinase and NAD(P)-independent G3P dehydrogenase, but compared to several other non-repressing carbon sources no clear induction of these enzymes by glycerol was apparent. Amongst glycerolnegative mutants of H. polymorpha strain 17B (a DHA kinase-negative mutant), strains blocked in either glycerol kinase or membrane-bound G3P dehydrogenase were identified. Crosses between representatives of the latter mutants and wild type resulted in the isolation of, amongst others, segregants which had regained DHA kinase but were still blocked in the membrane-bound G3P dehydrogenase. These strains, employing the oxidative pathway, were only able to grow very slowly in glycerol mineral medium.Abbreviations DHA dihydroxyacetone - G3P glycerol-3-phosphate - EMS ethyl methanesulphonate - MTT 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide - PMS phenazine methosulphate - FBP fructose-1,6-bisphosphate     

Effect of experimental cryptorchism on testis L-iditol: NAD oxidoreductase (sorbitol dehydrogenase or ketose-reductase)

Summary The histochemioal distribution of sorbitol dehydrogenase in normal and cryptorchid rat testis has been studied. In the normal testis sorbitol dehydrogenase is localized in the spermatids, increases during their differentiation and is maximal in those spermatids attached to the Sertoli cells (stages V–VII). In the cryptorchid testis, sorbitol dehydrogenase activity of the spermatids, similarly to that of the Sertoli cells, is completely abolished. Therefore, we conclude that sorbitol dehydrogenase activity of the Sertoli cells depends on the spermatid differentiation.Abbreviations used SBDH sorbitol dehydrogenase - NAD nicotinamide adenine dinucleotide - NBT m-nitroneotetrazolium chloride - PMS phenazine methosulfate - Tris tris (hydroxymethyl) aminomethane     

Degradation pathway of 2-chloroethanol in <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain JJ under denitrifying conditions

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.     

Methanol dissimilation in Xanthobacter H4-14: activities, induction and comparison to Pseudomonas AM1 and Paracoccus denitrificans

Methanol dissimilatory enzymes detected in the methanol autotroph Xanthobacter H4-14 were a typical phenazine methosulphate-linked methanol dehydrogenase, a NAD+-linked formate dehydrogenase, and a dye-linked formaldehyde dehydrogenase that could be assayed only by activity stains of polyacrylamide gels. This same methanol dehydrogenase activity was found in ethanol-grown cells and was apparently utilized for ethanol oxidation. Formaldehyde dehydrogenase activities were investigated in Paracoccus denitrificans, Xanthobacter H4-14, and Pseudomonas AM1. P. denitrificans contained a previously reported NAD+-linked, GSH-dependent activity, but both Xanthobacter H4-14 and Pseudomonas AM1 contained numerous activities detected by activity stains of polyacrylamide gels. Induction studies showed that in Xanthobacter H4-14, a 10 kDal polypeptide, probably a dehydrogenase-associated cytochrome c, was co-induced with methanol dehydrogenase, but the formaldehyde and formate dehydrogenases were not co-regulated. Analogous induction experiments revealed similar patterns in P. denitrificans, but no evidence for co-regulation of dissimilatory activities in Pseudomonas AM1.     

Steady-state kinetic analysis for the reaction of ammonium and alkylammonium ions with methylamine dehydrogenase from bacterium W3A1

The steady-state kinetic mechanism for the reaction of n-alkylamines and phenazine ethosulfate (PES) or phenazine methosulfate (PMS) with methylamine dehydrogenase from bacterium W3A1 is found to be of the ping-pong type. This conclusion is based on the observations that 1/v versus 1/[methylamine] or 1/[butylamine] plots, at various constant concentrations of an oxidizing substrate, and 1/v versus 1/[PES] or 1/[PMS] plots, at various constant concentrations of a reducing substrate, are parallel. Additionally, the values of kcat/Km for four n-alkylamines are identical when PES is the oxidizing substrate, as were the kcat/Km values for four reoxidizing substrates when methylamine was the reducing substrate. Last, analysis of steady-state kinetic data obtained when methylamine and propylamine are presented to the enzyme simultaneously and PES and PMS are used simultaneously also supports the involvement of a ping-pong mechanism. The enzymic reaction with either methylamine or PES is dependent on the ionic strength, and the data indicate that each interacts with an anionic site on methylamine dehydrogenase. The presence of ammonium ion at low concentration activates the enzyme, but at high concentration this ion is a competitive inhibitor in the reaction involving methylamine and the enzyme. A complete steady-state mechanism describing these ammonia effects is presented and is discussed in light of the nature of the pyrroloquinoline quinone cofactor covalently bound to the enzyme.     

A tetrazolium technique for the histochemical localization of ATP: Creatine phosphotransferase

Summary A tetrazolium technique is presented that permits the study of ATP: Creatine phosphotransferase, or creatine kinase, in fixed skeletal muscle tissue sections, within the limits imposed by the properties of the chosen ditetrazole, nitro blue tatrazolium. There is a variation in creatine kinase activity between the muscle fibres. Those with high creatine kinase activity also have high succinate dehydrogenase activity.List of Abbreviations ADP Adenosine-5-diphosphate - ATP adenosine-5-triphosphate - CK creatine kinase - G-6-P glucose-6-phosphate - G-6-P-DH glucose-6-phosphate dehydrogenase - HK hexokinase - NADP nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PMS phenazine methosulphate - SDH succinate dehydrogenase     

Quantitative succinic dehydrogenases histochemistry

Summary Activity of succinic dehydrogenase (SD, EC 1.3.99.1) was investigated in the livers of 17 male albino rats by means of the tetrazolium salts NBT, INT, MTT, BT and TT, incubations carried out at a determined pH optimum of 7.9 and activities calculated with regard to activity of nothing dehydrogenase (ND) measured in control sections incubated without substrate. The highest activity could be achieved by means of NBT, the ratios of activities measured by NBT, INT, MTT, BT and TT being 100:86:82:29:18 and the order of activities corresponding to the order of redox potentials of the tetrazoles. A determination of ND was necessary, because of its influence on the real activity of SD measured in the slices by means of NBT, INT and MTT. Exact measurements could be made if activity was related to the dry weight of the slices or to the content of nucleic acid — bound phosphorus of reference slices; if activity was related to the content of protein of reference slices, the standard deviation would be the 1.4 fold. Meldola Blue (MB) was not a suitable soluble redox dye, the use of it leading to an activity only 23% of that determined when phenazine methosulphate (PMS) applied. A special measuring of the half-formazan of NBT, the molar extinction coefficient determined being 21,840 l/Mol/cm, produced higher accuracy of measurement, but had no influence on the amount of activity of SD. A comparison of the measured activity with reference values obtained by biochemical methods and histochemically quantified with PMS applied, showed a good conformity.     

Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains.

An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma-methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genesis of Drosophila ADH: the shaping of the enzymatic activity from a SDR ancestor

Drosophila alcohol dehydrogenase (ADH) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols and aldehydes. Structurally and biochemically distinct from all the reported ADHs (typically, the mammalian medium-chain dehydrogenase/reductase-ethanol-metabolizing enzyme), it stands as the only small-alcohol transforming system that has originated from a short-chain dehydrogenase/reductase (SDR) ancestor. The crystal structures of the apo, binary (E.NAD(+)) and three ternary (E.NAD(+).acetone, E.NAD(+).3-pentanone and E.NAD(+).cyclohexanone) forms of Drosophila lebanonensis ADH have allowed us to infer the structural and kinetic features accounting for the generation of the ADH activity within the SDR lineage.     

NaCl and wounding induced changes in NAD reductase in hypocotyls and root tips ofPhaseolus vulgaris L.

The effect of 300 mM NaCl and wounding on the nicotinamide adenine dinucleotide (NAD) kinase, revealed by the guanosine triphosphate-dependent NAD reductase activity, was studied in two differently resistant bean cultivars using densitometric analysis of electrophoretic gels. In the presence of NaCl the total activity of NAD reductase was increased, in hypocotyls and root tips of resistant cultivar. The contribution of each of NAD reductase isoforms to the total activity was not significantly different between cultivars. Conversely, after wounding the hypocotyl, an increase could be observed in both cultivars and differences were demonstrated in the contribution of the different isoforms.     

The use of phenazinemethosulphate as an electron carrier in the histochemical demonstration of dehydrogenases

Summary The use of phenazine methosulfate (PMS) has been investigated in the histochemical demonstration of dehydrogenase in some organs of the rat. The demonstration of nicotinamid-adenine-dinucleotide (NAD) linked dehydrogenases, which in the conventional methods without PMS is dependent upon the localisation of reduced NAD (NADH)-diaphorase, is greatly hindered by PMS. This inhibition is caused by the inactivation of the diaphorase by PMS. However in tissues or cells lacking the diaphorase, the nucleotide-linked dehydrogenases can be made visible by the addition of PMS to the conventional dehydrogenase reagents. PMS strongly activates the nucleotide-independent dehydrogenases such as succinate dehydrogenase.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

Isozymic patterns and functional states of cell lines ofDrosophila melanogaster cultured in vitro. II

Summary Our previous isoenzyme investigation ofDrosophila melanogaster cell lines in vitro has been completed with twelve further enzyme systems. The enzyme profiles seem to be in good agreement with a previous hypothesis concerning the precise origin of these cell lines (probably from imaginal discs or nervous tissues). Our results have been summarized with reference to the biochemical genetic map ofDrosophila melanogaster in order to consider a possible functional organization of the genome.Abbreviations NAD nicotine adenine nucleotide - NADP nicotine adenine nucleotide phosphate - NBT nitroblue tetrazolium - PMS phenazine methosulfate - EDTA ethylene diamine tetraacetic acid - GOT Glutamate-oxaloacetate transaminase - PGK Phosphoglycerate kinase - GPDH -glycerophosphate dehydrogenase - MDH Malate dehydrogenase - PGM Phosphoglucomutase - Aph Alkaline phosphatase - MDH-NADP Malic enzyme - Lap Leucine Amino-Peptidase - LDH Lactate dehydrogenase - -1-OHDH L-3-hydroxyacid dehydrogenase - ADH Alcohol dehydrogenase - Aldox Aldehyde oxydase - 6PGD 6 Phosphogluconate dehydrogenase - G6PD Glucose-6-Phosphate dehydrogenase - Hex3 Fructokinase - IDH Isocitrate dehydrogenase - Est 6 Esterase 6 - Est C Esterase C - ODH Octanol dehydrogenase - XDH Xanthine dehydrogenase - AcPh Acid Phosphatase 1     

Oxidation of C1-compounds in Pseudomonas C.

Extracts of Pseudomonas C grown on methanol as a sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts. The addition of D-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when D-ribulose 5-phosphate was present in the assay mixtures. The amount of radioactivity found in CO2, was 6;8-times higher when extracts of methanol-grown Pseudomonas C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate. These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Phenazine Methosulfate; its Use in Evaluating Activity of Dehydrogenase Systems of Avian Liver

Lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and suecinate dehydrogenase were demonstrated in livers of 15-day chick embryos. The addition of phenazine methosulfate (PMS) to the LDH and MDH incubation mixtures reduced diformazan deposition in the liver epithelium but not in connective tissue. A 30 sec formalin fixation, absence of PMS, or the addition of sodium azide or potassium cyanide to the PMS-containing incubation mixtures facilitated formazan deposition. These results are explained by assuming that, in the absence of PMS, dehydrogenase activity is demonstrated via endogenous diaphorase. When PMS is present, Nitro BT reduction occurs within the incubation mixture. A side effect of the azide or cyanide is an interference with, the action of PMS, thus allowing diformazan deposition via the endogenous diaphorase when this is present in the tissue.     

A simple plate-assay for the screening of l-malic acid producing microorganisms

A simple plate-assay has been developed to screen microorganisms for L-malic acid production. Acid producing organisms were identified, after microbial colony growth on media containing glucose or fumaric acid as sole carbons sources, by formation of a dark halo of formazan. The halo was observed when the plate was covered with a soft agar overlay containing NAD(+)-malate dehydrogenase, NAD+, phenazine methosulfate (PMS) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay developed is simple, specific for L-malic acid and therefore can be used to identify L-malic acid producing filamentous fungi using glucose as carbon source (e.g. Aspergillus strains). The assay is also applicable for screening bacteria with high fumarase activity, able to convert fumaric acid to L-malic acid.     

Study of a time lag in the assay of Escherichia coli membrane-bound dehydrogenases based on tetrazolium salt reduction

The Escherichia coli membrane-bound D-lactate dehydrogenase and succinate dehydrogenase were assayed on the basis of the phenazine methosulfate- (PMS-) mediated reduction of the tetrazolium salt, MTT. An initial slower phase (lag) in the time-course of the reaction was observed and analyzed. The results were as follows. (1) The time lag in the assay of the D-lactate dehydrogenase was eliminated by preincubating the membranes with PMS plus D-lactate, with PMS plus succinate, or with PMS plus NADH (conditions which implicated PMS reduction). (2) When the D-lactate dehydrogenase was assayed by another method based on the measurement of the pyruvate formed, neither was a time lag observed nor was the enzyme activity affected by membrane preincubation with PMS plus D-lactate. (3) Although the superoxide radical was involved in MTT reduction, this radical seemed not to participate in the generation of the time lag. (4) Membranes whose D-lactate dehydrogenase activity had previously been destroyed by heating at 80 degrees C for 1 min, were able to prolong the time lag in MTT reduction when added to the assay medium for the D-lactate dehydrogenase from untreated membranes, whereas membranes previously heated at 100 degrees C instead of 80 degrees C did not have this effect. It was concluded that the E. coli membranes interfered in the dehydrogenase assay based on the PMS-mediated reduction of MTT. The time lag was interpreted as a period during which the interfering substance reacted with reduced PMS inhibiting the reduction of MTT.     

Purification and characterization of the molybdoenzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini

The enzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini could be purified to homogeneity by means of anion exchange chromatography, hydrophobic interaction chromatography, gel filtration, and chromatography on hydroxylapatite. During enrichment procedures both enzymes showed a significant loss in specific activity. The molecular weight of nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase was determined to be about 300,000 and 120,000, respectively. They were highly substrate specific and transferred electrons only to artificial acceptors of high redox potential. The K _m for their specific substrates was about 1.0 mM for both enzymes, and their pH optimum was determined to be 7.5. For nicotinate dehydrogenase a content of 8.3 mol iron, 1.5 mol acid-labile sulfur, 2.0 mol flavin, and 1.5 mol molybdenum per mol of enzyme was determined. Both enzymes contained FAD and Fe/S center. After inhibition by KCN, thiocyanate was detected, and subsequently the initial nicotinate dehydrogenase activity was restored by the addition of Na₂S indicating the presence of cyanolyzable sulfur. 6-Hydroxynicotinate dehydrogenase seemed to contain the same type of constituents as determined for nicotinate dehydrogenase. A partial immunological identity of the enzymes could be shown by antibodies raised against nicotinate dehydrogenase.Abbreviations DCPIP 2,6-dichlorophenol-indophenol - EEO electroendosmosis - FTTC fluorescein isothiocyanate - HAP hydroxylapatite - 6-HDH 6-hydroxynicotinate dehydrogenase - NBT nitroblue tetrazolium chloride - NDH nicotinate dehydrogenase - MTT thiazolyl blue - PES phenazine ethosulfate - PMSF phenylmethyl sulfonyl fluoride - TEMED N,N,N',N'-tetramethyl-ethylenediamine