Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides

Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5-AAAGCT GCTCATGGTTCT-3 which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5-TGCAGCTTCCATCACCGT-3. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from – 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.Abbreviations ODC ornithine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO difluoromethylornithine     

Induction of de-novo synthesis of tryptophan decarboxylase in cell suspensions of Catharanthus roseus

Tryptophan decarboxylase (EC 4.2.1.27) is synthesized de-novo by Catharanthus roseus cells shortly after the cells have been transferred into culture medium in which monoterpenoid indole alkaloids are formed. The enzyme production, monitored by in-vivo labelling with [³⁵S]methionine and immunoprecipitation, precedes the apparent maximal enzyme activity by 10–12 h. From the time course of the descending enzyme activity after induction, a half-life of 21 h for tryptophan decarboxylase in C. roseus cell suspensions is calculated. A comparison of the polyadenylated-RNA preparations from C. roseus cells indicates that mRNA activity for tryptophan decarboxylase is only detected in cells grown in the production medium. The importance of tryptophan decarboxylase induction with respect to the accumulation of th corresponding alkaloids is discussed.Abbreviation TDC tryptophan decarboxylase     

Effect of phenylalanine derivatives on the main regulatory enzymes of hepatic cholesterogenesis

Phenylalanine, phenylpyruvate and phenylacetate produced a considerable inhibition of chick liver mevalonate 5-pyrophosphate decarboxylase while mevalonate kinase and mevalonate 5-phosphate kinase were not significantly affected. Phenolic derivatives of phenylalanine produced a similar inhibition of decarboxylase activity than that found in the presence of phenyl metabolites. The degree of inhibition was progressive with increasing concentrations of inhibitors (1.25–5.00 mM). Simultaneous supplementation of different metabolites in conditions similar to those in experimental phenylketonuria (0.25 mM each) produced a clear inhibition of liver decarboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. To our knowledge, this is the first report on the in vitro inhibition of both liver regulatory enzymes of cholesterogenesis in phenylketonuria-like conditions. Our results show a lower inhibition of decarboxylase than that of reductase but suggest an important regulatory role of decarboxylase in cholesterol synthesis.     

Effect of clofibrate on brain mevalonate-5-pyrophosphate decarboxylase

The effect of clofibrate on the activity of the three mevalonate-activating enzymes has been studied for the first time in brain by reactions carried out using [2-¹⁴C] mevalonic acid as substrate and 105,000g supernatants from 14-day-old chick brain. Mevalonate-5-pyrophosphate decarboxylase was clearly inhibited, while mevalonate kinase and mevalonate-5-phosphate kinase were not significantly affected. The effect of clofibrate on decarboxylase activity was progressive with increasing concentrations (1.25–5.00 mM) of the inhibitor. A transient inhibition and a subsequent activation as a function of clofibrate concentration seemed to occur for mevalonate kinase. Direct measurements of decarboxylase activity utilizing [2-¹⁴C] pyrophosphomevalonate as the specific substrate of this enzyme corroborated these results. Kinetic studies showed that clofibrate competes with the substrate ATP.     

Chromatin-associated ornithine decarboxylase at early stages of growth and differentiation of etiolated mono- and dicotyledonous plants

Ornithine decarboxylase (ODC) of barley, corn, bean and pea plants was associated with chromatin at early stages of growth. In corn, bean and pea plants the chromatin of roots possessed ODC with the highest specific activity of 30–65 units per mg protein. After 200 h of growth ODC activity of chromatin declined, while ODC activity of cytosol increased linearly to 4–19 units per mg protein. ODC activity of either chromatin or cytosol of shoots in the above mentioned plants was usually low, 1–2 units per mg protein and only at the beginning of shoot growth (96 h) did pea chromatin have high ODC activity (14 units per mg protein). In these plants ODC was tightly bound to chromatin and could not be extracted with different concentrations of NaCl (0.1–1.0 M) or non-ionic detergent (Triton X100, Tween 20) but could be extracted by freezing and thawing with satisfactory recovery.     

Affinity labeling of oxaloacetate decarboxylase by novel dichlorotriazine linked alpha-ketoacids

The 4-aminophenyloxanilic acid and -mercaptopyruvic acid linked to the reactive diclorotriazine ring, were studied as active site-direct affinity labels towards oxaloacetate decarboxylase (EC 4.1.1.3, OXAD). Oxaloacetate decarboxylase when incubated with 4-aminophenyloxanilic-diclorotriazine (APOD) or -mercaptopyruvic-diclorotriazine (MPD) at pH 7.0 and 25°C shows a time-dependent and concentration-dependent loss of enzyme activity. The inhibition was irreversible and activity cannot be recovered either by extensive dialysis or gel-filtration chromatography. The enzyme inactivation following the Kitz & Wilson kinetics for time-dependent irreversible inhibition. The observed rate of enzyme inactivation (k _obs) exhibits a non-linear dependence on APOD or MPD concentration with maximum rate of inactivation (k ₃) of 0.013 min^–1 and 0.0046 min^–1 and K _D equal to 20.3 and 156 M respectively. The inactivation of oxaloacetate decarboxylase by APOD and MPD is competitively inhibited by OXAD substrate and inhibitors, such as oxaloacetate, ADP and oxalic acid whereas Mn⁺² enhances the rate of inactivation. The rate of inactivation of OXAD by APOD shows a pH dependence with an inflection point at 6.8, indicating a possible histidine derivatization by the label. These results show that APOD and MPD demonstrate the characteristics of an active-site probe towards the oxaloacetate binding site of oxaloacetate decarboxylase.     

Alleviation of low temperature sweetening in potato by expressing Arabidopsis pyruvate decarboxylase gene and stress-inducible rd29A: A preliminary study

The acceptability of potatoes for processing chips and French fries is largely dependent on the color of the finished product. Most potato cultivars and varieties stored at temperatures below 9–10 °C are subjected to low temperature sweetening (LTS) which result in the production of bitter-tasting, dark colored chips and French fries which are unacceptable to consumers. However, storing tubers at low temperatures (i.e., <10 °C) has many advantages such as lowered weight loss during storage, natural control of sprouting, and reduction/elimination of chemical sprout inhibitors. Our earlier research results on LTS suggested a role for pyruvate decarboxylase (PDC) in LTS-tolerance. In the present study, the role of PDC was examined whereby the potato variety Snowden was transformed with Arabidopsis cold-inducible pyruvate decarboxylase gene 1 (AtPDC1) under the control of promoter rd29A. Two transgenic plants were selected and storage studies were conducted on tubers harvested from one of the transgenic lines grown under green house conditions. Transgenic tubers showed higher Agtron chip color score indicating lighter chip and lower reducing sugar and sucrose concentrations compared to the untransformed tubers during the storage periods studied at 12 °C and 5 °C. These results suggest that overexpression of pyruvate decarboxylase gene resulted in low temperature sweetening tolerance in the transgenic Snowden.     

Purification of DOPA decarboxylase from bovine striatum

Pyridoxal phosphate-dependent DOPA decarboxylase has been purified from bovine striatum to a specific activity of 1.6 U/mg protein. After ammonium sulfate precipitation (30–60%) it was purified by DEAE-Sephacel, Sephacryl S-200, and TSK Phenyl 5 PW chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. The bovine striatal DOPA decarboxylase is a dimer (subunit M_r = 56000 by SDS-PAGE) with a native M_r of 106000 as judged by chromatography on Sephacryl S-200 and by sedimentation analysis. Similar to the DOPA decarboxylase purified from non-CNS tissues, the bovine striatal enzyme requires free sulfhydryl groups for activity, is strongly inhibited by heavy metal ions, and can decarboxylate 5-hydroxytryptophan as well. It should be noted, however, that the final enzyme preparation is enriched in DOPA decarboxylase activity. The distribution of the DOPA decarboxylase and 5-HTP decarboxylase activities also varies among several bovine brain regions. In addition, heat treatment of the enzyme preparation inactivated the two decarboxylation activities at different rates.Abbreviations AADC Aromatic L-amino Acid Decarboxylase - CNS Central Nervous System - DOPA 3,4-dihydroxyphenylalanine - DTT Dithiothreitol, 5-HTP - 5-hydroxytryptophan - Mr relative molecular weight - PLP pyridoxal 5-phosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Part of this paper was presented at the 1987 Annual Pharmacology and Toxicology Conferences held at University of North Dakota School of Medicine, North Dakota, USA Res Commun Psychol Psychiat Behav 12: 227–228, 1987 (Abstr).     

Cloning, expression, purification and characterization of the cholera toxin B subunit and triple glutamic acid decarboxylase epitopes fusion protein in Escherichia coli

Induction of specific immunological unresponsiveness by oral autoantigens such as glutamic acid decarboxylase 65 (GAD65) is termed oral tolerance and may be a potential therapy for autoimmune diabetes. However, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. Mucosal adjuvants such as cholera toxin B subunit (CTB) may lower the level of autoantigens required. Here we describe cloning, expression, purification and identification study of the CTB and triple GAD_531–545 epitopes fusion gene. The fusion gene was ligated via a flexible hinge tetrapeptide and expressed as a soluble protein in Escherichia coli BL21 (DE3) driven by the T7 promoter. We purified the recombination protein from the cell lysate and obtained approximately 2.5 mg of CTB–GAD_(531–545)3 per liter of culture with greater than 90% purity by a Ni–NTA resin column. The bacteria produced this protein as the pentameric form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and GAD65. Further studies revealed that oral administration of bacterial CTB–GAD_(531–545)3 fusion protein showed the prominent reduction in pancreatic islet inflammation in non-obese diabetic mice. The results presented here demonstrate that the bacteria bioreactor is an ideal production system for an oral protein vaccine designed to develop immunological tolerance against autoimmune diabetes.     

The effect of a mealybug infestation on the activity of amino acid decarboxylases in orchid leaves

Changes in the activity of lysine decarboxylase (LDC), tyrosine decarboxylase (TyDC), and ornithine decarboxylase (ODC) within orchid (Phalaenopsis × hybridum ‘Innocence’) leaves, infested by two mealybug species: Pseudococcus longispinus (Targ. Tozz.) and Pseudococcus maritimus (Ehrh.) were quantified. The pattern of changes was dependent on the insect species and duration of infestation. P. longispinus feeding increased LDC and TyDC activity after one week during the total period of observations. This species inhibited ODC activity after one week but increased later. P. maritimus decreased LDC activity in orchid leaves at all studied terms. TyDC action also went up during the first week of the infestation and was reduced after two weeks, while ODC was decreased after one day and induced later. The mechanism for the participation of analysed amino acid decarboxylases in local and/or systemic steps of orchid responses to mealybug infestation is discussed.     

Inhibition of polyamine biosynthesis in Acanthamoeba castellanii and 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by chitosanoligosaccharide

Growth of Acanthamoeba castellaniiwas inhibited by chitosanoligosaccharide (up to 20 mg ml^–1) from the shells of crabs but was reversed by the polyamines, putrescine or spermidine, at 0.8 mM. Chitosanoligosaccharide strongly inhibited the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate, a key enzyme of polyamine biosynthesis, which is enhanced in tumour promotion.     

Autoradiographic localization of ornithine decarboxylase in mouse kidney by use of radiolabeled α-difluoromethylornithine

Summary Ornithine decarboxylase, a key enzyme in polyamine biosynthesis and cell growth, has been localized in mouse kidney by autoradiography after administration of radiolabeled -difluoromethylornithine. This drug is an enzyme-activated irreversible inhibitor of ornithine decarboxylase and forms a covalent bond with the enzyme. It was found that ornithine decarboxylase is present in all cell types studied but that the highest content occurs in the proximal convoluted tubules followed by the distal convoluted tubules and the collecting tubules. The majority of the enzyme is located in the cytoplasm but about 10–15% is present in the nuclei (often associated with nucleolus-like components) of the cells of the proximal and distal convoluted tubules. The labeled ornithine decarboxylase was lost rapidly from both nucleus and cytoplasm of all the cell types examined, and labeling by radioactive -difluoromethylornithine was greatly reduced if the mice were pretreated for 5 h with cycloheximide to block protein synthesis. These results indicate that ornithine decarboxylase turns over rapidly in all of the cells.     

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. α-Difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.     

Immunological comparison of microbial TPP-dependent non-oxidative α-keto acid decarboxylases

Abstract Antigenic, and hence possible evolutionary, relationships amongst various TPP-dependent non-oxidative α-keto acid decarboxylases were determined by the Ouchterlony double diffusion method and by measuring the degree of antibody-induced enzyme inhibition. The results show that: (a) phenylglyoxylate decarboxylases of various wild-type strains of Acinetobacter calcoaceticus are antigenically indistinguishable; (b) there seems to be no antigenic cross-reactivity between the phenylglyoxylate decarboxylase of A. calcoaceticus and of Pseudomonas aeruginosa or Pseudomonas putida ; and (c) no antigenic homology can be detected amongst phenylglyoxylate decarboxylase and phenylpyruvate decarboxylase of A. calcoaceticus and pyruvate decarboxylase of brewers' yeast.     

The Histidine Decarboxylase (HDC) Gene of Tetrahymena Pyriformis is Similar to the Mammalian One. A Study of HDC Expression

RNA was isolated from Tetrahymena pyriformis GL and using human histidine decarboxylase (HDC) gene primers, the RT-PCR product was sequenced. A fraction containing 207 base pairs was compared to the published sequences of prokaryotic and mammalian (rat, mouse and human) HDC cDNA (exons). The HDC-cDNA fraction of Tetrahymena was similar to the mammalian cDNA-s and it was completely different from the prokaryotic HDC-gene. The results indicate the presence of a mammalian-like HDC-gene already in a unicellular eukaryote organism and demonstrates also that the divergence of the prokaryotic–eukaryotic common gene took place already at this low evolutionary level.     

Development of a robust system for high-throughput colorimetric assay of diverse amino acid decarboxylases

Amino acid decarboxylases catalyze decarboxylation of amino acids into amines that possess wide industrial applications. As key enzymes in biobased production of industrially important amines such as cadaverine, putrescine and β-alanine, lysine decarboxylase, ornithine decarboxylase and aspartic acid decarboxylase have attracted increasing attention. To develop enzyme variants with superior catalytic properties, there is a great need for high-throughput assay of these decarboxylases. Here we report the development of assays based on the color change of pH indicator – chlorophenol red (CPR) or bromothymol blue (BTB) – in decarboxylation reactions, in which one proton was consumed per carboxylic group decarboxylated resulting in an increase in pH. First, two buffer-indicator pairs, 4-morpholineethanesulfonic acid (MES)-CPR and 3-morpholinopropanesulfonic acid (MOPS)-BTB, were chosen on the basis of their similar pK_a values at approximately pH 6.0 and 7.0, both of which are physiologically relevant. Next, the effects of buffer strength and indicator concentration on absorbance changes were examined in assay mixtures with NaOH titration, which mimicked proton consumption in decarboxylation reactions. Finally, high-throughput quantification of lysine decarboxylase, ornithine decarboxylase and aspartic acid decarboxylase was achieved using a microplate format. These results suggest that our indicator assay system may have potential applications for screening diverse decarboxylases.     

A continual spectrophotometric assay for amino acid decarboxylases

A spectrophotometric method for assaying the activity of three amino acid decarboxylases is reported. This method makes use of the coupled reaction of the decarboxylase with phosphoenolpyruvate carboxylase and malate dehydrogenase. The assay is simple and rapid and allows continuous monitoring of the reaction progress. The kinetic parameters obtained using this method for diaminopimelate decarboxylase, lysine decarboxylase, and arginine decarboxylase are comparable to values obtained by radiochemical methods.     

Effect of diabetes on the induction of ornithine decarboxylase by refeeding.

Refeeding of starved rats that had previously been schedule-fed increased ornithine decarboxylase activity 140-fold in liver and six-fold in skeletal muscle within three hours. In diabetic rats, refeeding caused a smaller increase in enzyme activity in liver and none at all in muscle. When insulin was administered together with food to the diabetic rats, ornithine decarboxylase in muscle increased to levels greater than those observed in refed controls. The activity of the enzyme in liver also increased; however, the increase was still less than that observed in refed control rats. The data indicate that the induction of ornithine decarboxylase in liver and muscle following food ingestion is altered in diabetes. In addition, they suggest that insulin, or a factor dependent on insulin, modulates the activity of ornithine decarboxylase in skeletal muscle.     

Anaerobic degradation of malonatevia malonyl-CoA bySporomusa malonica,Klebsiella oxytoca,andRhodobacter capsulatus

Anaerobic decarboxylation of malonate to acetate was studied withSporomusa malonica, Klebsiella oxytoca, andRhodobacter capsulatus. WhereasS. malonica could grow with malonate as sole substrate (Y=2.0 g·mol^–1), malonate decarboxylation byK. oxytoca was coupled with anaerobic growth only in the presence of a cosubstrate, e.g. sucrose or yeast extract (Y _s =1.1–1.8 g·mol malonate^–1).R. capsulatus used malonate anaerobically only in the light, and growth yields with acetate and malonate were identical. Malonate decarboxylation in cell-free extracts of all three bacteria was stimulated by catalytic amounts of malonyl-CoA, acetyl-CoA, or Coenzyme A plus ATP, indicating that actually malonyl-CoA was the substrate of decarboxylation. Less than 5% of malonyl-CoA decarboxylase activity was found associated with the cytoplasmic membrane. Avidin (except forK. oxytoca) and hydroxylamine inhibited the enzyme completely, EDTA inhibited partially. InS. malonica andK. oxytoca, malonyl-CoA decarboxylase was active only after growth with malonate; malonyl-CoA: acetate CoA transferase was found as well. These results indicate that malonate fermentation by these bacteria proceedsvia malonyl-CoA mediated by a CoA transferase and that subsequent decarboxylation to acetyl-CoA is catalyzed, at least withS. malonica andR. capsulatus, by a biotin enzyme.Abbreviations CoASH Coenzyme A - EDTA ethylenediamine tetraacetate     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.