Properties of gamma-aminobutyraldehyde dehydrogenase from Escherichia coli

gamma-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an Mr of 195,000 +/- 10,000 in its dimeric form with an Mr of 95,000 +/- 1,000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. Km values are 31.3 +/- 6.8 microM and 53.8 +/- 7.4 microM for delta-1-pyrroline and NAD+, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

Purification and characterization of bovine brain gamma-aminobutyraldehyde dehydrogenase.

gamma-Aminobutyraldehyde dehydrogenase was purified to homogeneity from bovine brain. The molecular weight of the native enzyme and subunit were 230,000 and 58,000, respectively. The Km value for gamma-aminobutyraldehyde and NAD+ were 154 microM and 53 microM, respectively. The optimum pH and temperature were 8.0 and 37 degrees C, respectively. N-terminal sequence of the enzyme is as follows: NH2-S-A-A-T-Q-A-V-P-T-P-N-Q-Q-COOH. The enzyme migrates on isoelectric focusing with pI = 6.5. Enhancement of the enzyme activity by polyamine, Mn2+, Mg2+ and inhibition by gamma-aminobutyric acid and Zn2+ will enhance the limited information on regulation of the gamma-ABALDH activity and GABA metabolism to some extent.     

Identification of Escherichia coli K12 YdcW protein as a gamma-aminobutyraldehyde dehydrogenase

Gamma-aminobutyraldehyde dehydrogenase (ABALDH) from wild-type E. coli K12 was purified to apparent homogeneity and identified as YdcW by MS-analysis. YdcW exists as a tetramer of 202+/-29 kDa in the native state, a molecular mass of one subunit was determined as 51+/-3 kDa. Km parameters of YdcW for gamma-aminobutyraldehyde, NAD+ and NADP+ were 41+/-7, 54+/-10 and 484+/-72 microM, respectively. YdcW is the unique ABALDH in E. coli K12. A coupling action of E. coli YgjG putrescine transaminase and YdcW dehydrogenase in vitro resulted in conversion of putrescine into gamma-aminobutyric acid.     

Aldehyde dehydrogenase from rat intestinal mucosa: purification and characterization of an isozyme with high affinity for gamma-aminobutyraldehyde.

In rat adrenal gland and gastric mucosa putrescine is efficiently oxidized to GABA via gamma-aminobutyraldehyde (ABAL) by action of diamine oxidase and aldehyde dehydrogenase. Having turned our attention on the rat intestinal mucosa, where putrescine uptake and diamine oxidase are active, we have purified and characterized an aldehyde dehydrogenase optimally active on gamma-aminobutyraldehyde. A dimer with a subunit molecular weight of 52,000, the native enzyme binds ABAL and NAD+ with high affinity: at pH 7.4, Km values are equal to 18 and 14 microM, respectively. Affinity for betaine aldehyde is much lower (Km = 285 microM), but the efficiency is equally good, thanks to a high value of V. Unaffected by disulfiram and Mg2+, the enzyme is activated by high NAD+ concentrations (Vnn = 1.6 x Vn) and is competitively inhibited by NADH. According to the best fitting model, the dimeric enzyme only binds one NADH and the mixed complex enzyme-NAD(+)-NADH is inactive. The increase of activity promoted by NAD+ can therefore be ascribed to an allosteric effect, rather than to the activation of a second reaction center. Highly stable at pH 6.8 in the presence of dithiothreitol and high phosphate concentrations, ABALDH is inactivated by ion-exchange resins and by cationic buffers. Our results show that the enzyme can be effectively involved in the metabolism of biogenic amines and, with a K(m) for ABAL lower than 20 microM, in the synthesis of GABA.     

Purification and characterization of a rat brain aldehyde dehydrogenase able to metabolize gamma-aminobutyraldehyde to gamma-aminobutyric acid.

An enzyme which catalyses dehydrogenation of gamma-aminobutyraldehyde (ABAL) to gamma-aminobutyric acid (GABA) was purified to homogeneity from rat brain tissues by using DEAE-cellulose and affinity chromatography on 5'-AMP-Sepharose, phosphocellulose and Blue Agarose, followed by gel filtration. Such an enzyme was first purified from mammalian brain tissues, and was identified as an isoenzyme of aldehyde dehydrogenase. It has an Mr of 210,000 determined by polyacrylamide-gradient-gel electrophoresis, and appeared to be composed of subunits of Mr 50,000. The close similarity of substrate specificity toward acetaldehyde, propionaldehyde and glycolaldehyde between the enzyme and other aldehyde dehydrogenases previously reported was observed. But substrate specificity of the enzyme toward ABAL was higher than those of aldehyde dehydrogenases from human liver (E1 and E2), and was lower than those of ABAL dehydrogenases from human liver (E3), Escherichia coli and Pseudomonas species. The Mr and relative amino acid composition of the enzyme are also similar to those of E1 and E2. The existence of this enzyme in mammalian brain seems to be related to a glutamate decarboxylase-independent pathway (alternative pathway) for GABA synthesis from putrescine.     

Effect of dietary putrescine on whole body growth and polyamine metabolism

Putrescine (1,4-diaminobutane) is the simplest of the mammalian polyamines. These are small, positively charged molecules which are essential for cell growth and are thought to play a role in regulation of anabolic events such as synthesis of DNA, RNA, and protein. Recent reports have indicated the potential for dietary precursor amino acids of putrescine to alter tissue putrescine concentrations. The current study was conducted to determine the physiologic significance of these effects by feeding up to flooding doses of putrescine to determine any influence on whole body growth and polyamine metabolism. A total of 96 chicks were fed purified crystalline amino acid diets containing 0.0, 0.2, 0.4, 0.6, 0.8, or 1.0% purified putrescine (four birds per pen, four pens per diet) for 14 days. The feeding of 0.2% putrescine increased growth rate beyond that of controls while further supplements reduced growth and were toxic when 0.8 and 1.0% putrescine were fed. Hepatic and muscle concentrations of ornithine increased with dietary putrescine while the effect in kidney was much less. Putrescine concentrations in liver, kidney, and muscle rose when 0.4% putrescine or more was fed. This effect was particularly obvious in muscle in which there were also increases in the concentrations of spermidine and spermine. In a subsequent similar experiment, putrescine was fed at 0.0, 0.1, 0.2, 0.3, 0.4, or 0.5% to determine the effect on the activities of the key enzymes regulating polyamine synthesis. The feeding of putrescine at even 0.1% caused a rapid reduction in hepatic ornithine decarboxylase activity while S-adenosylmethionine decarboxylase and arginase activities were not influenced by diet. It was concluded that excess tissue putrescine can be toxic to whole organisms but small, orally administered doses of this metabolite can promote growth.     

Polyamine metabolism in Acanthamoeba: polyamine content and synthesis of ornithine, putrescine, and diaminopropane

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

GABA and its relationship to putrescine metabolism in the rat brain and pancreas

The possibility that GABA may have its origin in putrescine was investigated in the rat pancreas, relative to the brain. These studies show that radioactive putrescine is converted to GABA at a similar rate in both the pancreas and brain, but that putrescine accounts for only a small fraction of the GABA found in these organs. Inhibitors of diamine and monoamine oxidases do not significantly change the GABA level in the pancreas. In contrast to the brain, where putrescine is catabolized to GABA via monoamine oxidase, the primary catabolic pathway of putrescine to GABA in the pancreas is via diamine oxidase. In vivo studies show that AOAA inhibits GABA-T activity to the same degree in the pancreas as in the brain, elevating GABA levels more than 2-fold in 4 h. GABA is metabolized more rapidly in the brain than the pancreas. Turnover times of GABA in the pancreas and brain are 1.9 and 1.0 h, respectively. The slower turnover of GABA in the pancreas than in the brain may relate to a neuromodulatory role for GABA, similar to that for neuropeptides. Developmental studies in the postnatal pancreas suggest a role for GABA in the maturation of insulin secretion.     

Polyamine metabolism in ripening tomato fruit : I. Identification of metabolites of putrescine and spermidine

The metabolism of [1,4-¹⁴C]putrescine and [terminal methylene-³H]spermidine was studied in the fruit pericarp (breaker stage) discs of tomato (Lycopersicon esculentum Mill.) cv Rutgers, and the metabolites identified by high performance liquid chromatography and gas chromatography-mass spectrometry. The metabolism of both putrescine and spermidine was relatively slow; in 24 hours about 25% of each amine was metabolized. The ¹⁴C label from putrescine was incorporated into spermidine, γ-aminobutyric acid (GABA), glutamic acid, and a polar fraction eluting with sugars and organic acids. In the presence of gabaculine, a specific inhibitor of GABA:pyruvate transaminase, the label going into glutamic acid, sugars and organic acids decreased by 80% while that in GABA increased about twofold, indicating that the transamination reaction is probably a major fate of GABA produced from putrescine in vivo. [³H]Spermidine was catabolized into putrescine and β-alanine. The conversion of putrescine into GABA, and that of spermidine into putrescine, suggests the presence of polyamine oxidizing enzymes in tomato pericarp tissues. The possible pathways of putrescine and spermidine metabolism are discussed.     

Analysis of polyamine metabolism in soybean seedlings using 15N-labelled putrescine

The translocation and metabolism of polyamines during soybean germination were studied using 15N-labelled putrescine as a precursor. Both 15N-labelled and unlabelled polyamines were simultaneously detected using a novel application of ionspray ionization-mass spectrometry. 15N-putrescine was rapidly transported to the shoots and roots, where it was converted to spermidine and spermine. The main 15N-polyamine that accumulated in the root was 15N-spermine. It was found that there were differences in the way endogenous putrescine and exogenous 15N-putrescine were metabolized in soybean seedlings.     

Affinity chromatography of putrescine oxidase from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens.

Putrescine oxidase [EC 1.4.3.4], putrescine : oxygen oxidoreductase (deaminating) (flavin-containing), from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens were adsorbed on amine-Sepharose 4B in which one of the terminal amino groups of diamine or triamine was covalently bound to Sepharose 4B leaving the other terminal amino group(s) free. The affinities of these enzymes for the amine-Sepharose 4B increased on increasing the chain length of the methylene groups in the immobilized amines and fell upon addition of the substrate. The affinity of putrescine oxidase modified with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC) was reduced in comparison with that of the native enzyme so far as 1,12-diaminododecane-Sepharose 4B was concerned. From these results, it can be concluded that the interactions between the enzyme and the amine-Sepharose result from specific affinities mediated through the active sites of the enzymes. It is suggested that spermidine dehydrogenase as well as putrescine oxidase has as anionic point and a hydrophobic region in the active site. On the basis of these results, the applicability of the enzyme affinities to purification procedures was examined. When partially purified enzymes were subjected to affinity chromatography, the following results were obtained. Putrescine oxidase gave a purification factor of 40-fold with about 100% recovery on a 1,12-diaminododecane-Sepharose column. In the case of spermidine dehydrogenase, the purification factor and recovery on a 1,8-diaminooctane-Sepharose column were about 1,200-fold and 86%, respectively. By introducing affinity chromatography as a purification step, each enzyme could be purified more simply and with higher recovery.     

Uptake,efflux and metabolism of the polyamine putrescine in rabbit lung slices

The herbicide paraquat is a selective pulmonary toxin in many mammals, including man, and its pulmonary toxicity has been attributed to selective uptake by a polyamine transport system in lung. In the present study, we investigated the characteristics of this transport process in rabbit lung slices. [¹⁴C]Putrescine was accumulated by both saturable and non-saturable processes and the accumulated putrescine was non-effluxable over 60 min. The saturable component was inhibited by spermine and paraquat. Moreover, uptake studies in Na⁺-deficient medium indicated that the lack of Na⁺ may selectively enhance uptake via the non-saturable process. The two components also differed in the metabolic fate of accumulated substrate. At 0.6 μM putrescine, where the saturable process predominated, 98% of the ¹⁴C in the perchloric acid-soluble fraction of tissue homogenates was present as putrescine, whilst 3% of the accumulated substrate was found in the acid-insoluble fraction. With 500 μM putrescine, where the non-saturable process predominated, 82% of the ¹⁴C in the acid-soluble fraction was present as putrescine and 15% of accumulated putrescine was found in the acid-insoluble fraction. The acid-insoluble ¹⁴C was localised mainly in the 700 g and 4500 g pellets obtained after homogenising the tissue. We conclude that there are two components to putrescine uptake in rabbit lung slices, both of an apparently irreversible nature. We suggest that the components represent compartmentalisation of putrescine in selective pulmonary cell-types or separate subcellular organelles. The observed metabolism and covalent binding of putrescine appeared to be associated with the non-saturable component only.     

Effect of heparin on the metabolism of putrescine in vivo.

Rats and guinea pigs which are given heparin metabolize intraperitoneally injected [14C]-putrescine to 14CO2 at reduced rates. The results have been considered in relation to the heparin-induced liberation of diamine oxidase from tissues into the blood stream.     

Role of pyridoxine in the metabolism of putrescine in the rat.

1. The ability of rats to metabolize radioactive putrescine to 14CO2 in vivo has been studied. 2. Animals made deficient in pyridoxine exhibit a significantly lower rate of catabolism of the diamine. 3. There dose not appear to be an important interaction between the effects of the deficiency and those stemming from treatment of the animals with the diamine oxidase inhibitor aminoguanidine. 4. These results favour the concept of a role of pyridoxal cofactor in the metabolism of diamines, presumably at the diamine oxidase stage.     

Transport kinetics and metabolism of exogenously applied putrescine in roots of intact maize seedlings

Putrescine metabolism, uptake, and compartmentation were studied in roots of hydroponically grown intact maize (Zea mays L.) seedlings. In vivo analysis of exogenously applied putrescine indicated that the diamine is primarily metabolized by a cell wall-localized diamine oxidase. Time-dependent kinetics for putrescine uptake could be resolved into a rapid phase of uptake and binding within the root apoplasm, followed by transport across the plasma membrane that was linear for 30 to 40 minutes. Concentration-dependent kinetics for putrescine uptake (between 0.05 and 1.0 millimolar putrescine) appeared to be nonsaturating but could be resolved into a saturable (V_max 0.397 micromoles per gram fresh weight per hour; K_m 120 micromolar) and a linear component. The linear component was determined to be cell wall-bound putrescine that was not removed during the desorption period following uptake of [³H]putrescine. These results suggest that a portion of the exogenously applied putrescine can be metabolized in maize root cell walls by diamine oxidase activity, but the bulk of the putrescine is transported across the plasmalemma by a carrier-mediated process, similar to that proposed for animal systems.     

Aldehyde dehydrogenase (EC 1.2.1.3): comparison of subcellular localization of the third isozyme that dehydrogenates gamma-aminobutyraldehyde in rat, guinea pig and human liver.

1. Subcellular fractionation of rat, guinea pig and human livers showed that aldehyde dehydrogenase metabolizing gamma-aminobutyraldehyde was exclusively localized in the cytoplasmic fraction in all three mammalian species. 2. Total gamma-aminobutyraldehyde activity of aldehyde dehydrogenase was found to be ca 0.41, 0.3 and 0.24 mumol NADH min-1 g-1 tissue, respectively in rat, guinea pig and human liver, with more than 95% of activity in the cytoplasm. 3. Partially purified cytoplasmic isozyme from rat liver showed similar chromatographic behavior and kinetic properties to the E3 isozyme isolated from human liver. 4. The rat isozyme was insensitive to disulfiram (40 microM) and to magnesium (160 microM) and had Km values of 5 microM (pH 7.4) for gamma-aminobutyraldehyde, 7.5 microM (pH 9.0) for propionaldehyde and 4 microM (pH 7.4) for NAD.