Immunological Studies of Betaine Aldehyde Dehydrogenase in Barley

The changes in the level of the protein for betaine aldehydedehydrogenase, which catalyzes the last step in the synthesisof glycinebetaine, were analyzed with antiserum raised againstSDS-denatured betaine aldehyde dehydrogenase from spinach. Inbarley leaves, the levels of betaine aldehyde dehydrogenaseprotein were found to be enhanced by the addition of 200 mMNaCl to the growth medium. These changes in the level of theenzyme protein corresponded to those in the activity of theenzyme, as described in our previous study (Arakawa et al. 1990).The extent of this enhancement was reduced when barley plantswere relieved from salt stress. An increase in the level ofthe protein was also induced by water stress, such as the withholdingof water or the addition of polyethylene glycol 6000. Betainealdehyde dehydrogenase protein was detected in etiolated leavesand roots, as well as in green leaves. In etiolated leaves,the level of betaine aldehyde dehydrogenase protein was notaffected by salt stress. ¹ This work was supported by a grant from the Bio-Media Projectof the Japanese Ministry of Agriculture, Forestry and Fisheries(BMP92-III-l-1).     

Oxidation of Betaine Aldehyde by Betaine Dehydrogenase from Spinach Leaves

Betaine content in leaves of fifteen plant species was determined. The results showed higher betaine levels in those salt-, drought-, and chilling-resistant species. Betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8 ) was isolated and partially purified from spinach leaves. Some properties of this enzyme were studied. BADH was precipitated by 60% saturation of (NH4)2SO4. Its activity was not detected in 70% saturation of (NH4)2SO4. BADH has two isoenzymes. The activity of BADH was quite stable below –80℃. It was inhibited by 0.125–1.0 mol/L NaG1 or KC1 but not by Mn2+ and Mo6+, and slightly increased by Mg2+.     

Betaine Aldehyde Dehydrogenase in the Gramineae: Levels in Leaves Both Betaine-Accumulating and Nonaccumulating Cereal Plants

The accumulation of betaine and the distribution of betainealdehyde dehydrogenase, which catalyzes the last step in thesynthesis of betaine, were analyzed in leaves of control andsalt-stressed cereal plants of the Gramineae. BADH protein waspresent in both betaine-accumulating and nonaccumulating leaves. ¹This research was supported by a Grant-in-Aid for ScientificResearch on Priority Area from the Ministry of Education, Scienceand Culture, Japan.     

Betaine aldehyde dehydrogenase from spinach leaves: purification, in vitro translation of the mRNA, and regulation by salinity

Spinach (Spinacia oleracea L.) leaves contain a nuclear-encoded chloroplastic betaine aldehyde dehydrogenase (EC 1.2.1.8) which is induced several-fold by salinization. Betaine aldehyde dehydrogenase was purified 2400-fold to homogeneity with an overall yield of 14%. The procedure included fractional precipitation with ammonium sulfate, followed by ion-exchange, hydrophobic interaction, and hydroxyapatite chromatography in open columns, and ion-exchange and hydrophobic interaction chromatography in a fast-protein liquid chromatography system. The betaine aldehyde dehydrogenase had a pI of 5.65, and a broad pH optimum between 7.5 and 9.5. The Km values for NAD+ and NADP+ were 20 and 320 microM, respectively; the Vmax of the reaction with NADP+ was 75% of that with NAD+. The native enzyme is a dimer with subunits of Mr 63,000. Highly specific antiserum was raised against the native enzyme, and was used in conjunction with cell-free translation of leaf poly(A)+ RNA to show (a) that betaine aldehyde dehydrogenase is synthesized as a precursor of Mr 1200 higher than the mature polypeptide, and (b) that both chronic salt stress and salt shock provoke a several-fold increase in the level of translatable message for the enzyme.     

Betaine Synthesis from Radioactive Precursors in Attached, Water-stressed Barley Leaves

In wilted barley leaves, betaine accumulates at about 200 nanomoles per 10 centimeters leaf per day. Results with ¹⁴C-labeled precursors were qualitatively and quantitatively consistent with de novo synthesis of this betaine from serine via ethanolamine, choline, and betaine aldehyde and indicated that water stress may increase the activities of all steps in this pathway except the last.     

植物甜菜碱合成酶的分子生物学和基因工程

甜菜碱是一种非毒性的渗透调节剂。多种高等植物在盐碱或缺水的环境下在细胞中积累甜菜碱 ,以维持细胞的正常膨压。甜菜碱的积累使得许多代谢中的重要酶类在渗透胁迫下能保持活性。在植物中甜菜碱由胆碱经两步氧化得到 ,催化第一步反应的酶是胆碱单加氧酶 (CMO) ,催化第二步反应的酶是甜菜碱醛脱氢酶 (BADH)。本文综述了这两种酶的分子生物学及基因工程研究的最新进展 ,讨论了其基因工程研究的意义。     

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme.

Betaine aldehyde dehydrogenase has been purified to homogeneity from rat liver mitochondria. The properties of betaine aldehyde dehydrogenase were similar to those of human cytoplasmic E3 isozyme in substrate specificity and kinetic constants for substrates. The primary structure of four tryptic peptides was also similar; only two substitutions, at most, per peptide were observed. Thus, betaine aldehyde dehydrogenase is not a specific enzyme, as formerly believed; activity with betaine aldehyde is a property of aldehyde dehydrogenase (EC 1.2.1.3), which has broad substrate specificity. Up to the present time the enzyme was thought to be cytoplasmic in mammals. This report establishes, for the first time, mitochondrial subcellular localization for aldehyde dehydrogenase, which dehydrogenates betaine aldehyde, and its colocalization with choline dehydrogenase. Betaine aldehyde dehydrogenation is an important function in the metabolism of choline to betaine, a major osmolyte. Betaine is also important in mammalian organisms as a major methyl group donor and nitrogen source. This is the first purification and characterization of mitochondrial betaine aldehyde dehydrogenase from any mammalian species.     

Betaine aldehyde dehydrogenase in sorghum.

The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa.     

Betaine aldehyde oxidation by spinach chloroplasts

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H₂O₂-mediated reaction cannot be ruled out.