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At least 19 recordsLinked to original sources
PubMed1980

A novel purification and some properties of rat liver mitochondrial choline dehydrogenase.

Choline dehydrogenase (choline:(acceptor) oxidoreductase, EC 1.1.99.1) was purified from rat liver mitochondria. An approx. 240-fold purification was achieved by chemically modified enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) through columns of DEAE-Sepharose CL-6B and the choline-Sepharose 4B with C3-spacer, and after the subsequent release of the thionitrobenzoate with dithiothreitol, through a second column of DEAE-Sepharose CL-6B in the presence of 0.1% Triton X-100. The purified preparation gave a specific activity of 6.3 mumol of O2 consumed/min per mg protein at 30 degrees C with phenazine methosulfate as the primary electron acceptor. After polyacrylamide gel electrophoresis in the presence of 0.5% sodium dodecyl sulfate, the enzyme showed activity in the gel. The preparation thus purified oxidized only choline and betaine aldehyde. The Km value for choline was 7.0 mM at an infinite concentration of phenazine methosulfate at pH 7.6 and 30 degrees C. 2-Dimethyl aminoethanol (Ki,app = 1.0 mM) and monoethanolamine did not work as substrates, inhibiting the enzyme competitively. The absolute requirement of any electron acceptor other than the molecular oxygen was confirmed. The Km value for phenazine methosulfate was about 1.1 mM at infinite concentration of choline. These findings suggested that coenzyme Q served as the primary electron acceptor in vivo.

Alcohol Oxidoreductases↗
PubMed1990

Spectra properties of rat liver mitochondrial choline dehydrogenase.

Choline dehydrogenase contains the prosthetic group FAD, non-haem iron and acid labile sulfur. However, the absorption spectra of the purified enzyme do not change after adding substrate. The reduced absorption spectra of choline dehydrogenase can only be determined after the addition of dithionite. Those choline dehydrogenases situated in the mitochondrial inner membrane can be reduced by substrate and exist in the reduced state. When cholate was used to solubilize the substrate-reduced choline dehydrogenase, the reduced spectra will gradually disappear. However, if solubilization is carried out under anaerobic conditions, the reduced spectra can be retained, suggesting that the solubilized choline dehydrogenase can use oxygen as an acceptor.

Alcohol Oxidoreductases↗
PubMed1985

The effects of inhibiting choline dehydrogenase on choline metabolism in mice.

3,3-Dimethylbutanol (Dimbunol), a competitive inhibitor of choline dehydrogenase (CDH), and ethylcholine mustard aziridinium (ECMA), an effective irreversible inhibitor of both CDH and choline transport, were investigated for their effects upon the uptake and metabolism of [3H]choline in mice. Thirty minutes after Dimbunol administration (i.p. 0.5 mmoles/kg) a reduction in the rate of choline oxidation was accompanied by an inhibition of choline phosphorylation in the kidney. Choline had accumulated to 5-fold the control level. After ECMA (i.v. 4 mumoles/kg), kidney choline was elevated 18-fold and both oxidation and phosphorylation rates were severely inhibited. In the liver Dimbunol inhibited oxidation and phosphorylation of choline and generated a 2-fold rise in tissue choline. Ethylcholine mustard aziridinium inhibited both oxidation and phosphorylation in the liver to the same extent as in the kidney but produced only a 3-fold elevation of choline. Dimbunol failed to elevate serum choline 30 min after administration and brain choline and acetylcholine levels were also unchanged. Serum choline was doubled by ECMA. These studies suggest that both transport across the renal tubules and oxidation may be important in choline regulation, that high levels of choline may accumulate in the liver and kidney which are not available for acetylcholine synthesis but that longer term studies on the effects of Dimbunol might reveal useful ways of facilitating sustained elevation of serum choline in precursor therapy.

Alcohol Oxidoreductases↗
PubMed2003

Functional expression and processing of rat choline dehydrogenase precursor.

Choline dehydrogenase (CHDH, EC 1.1.99.1) was purified from rat liver mitochondria, and the amino terminal sequence was determined and used to clone a full-length cDNA encoding a protein precursor (CHDHp) of 599 amino acids (64kDa). Sequence analysis identified a possible processing site that meets the requirements of IMP in comparison to the previously determined N-terminal sequence of mature rat CHDH. This suggested that the precursor might be processed in the intermembrane space. Confocal imaging showed that expression of the CHDHp-GFP fusion gene in NIH-3T3 cells led to fusion proteins being targeted to mitochondria. In addition, expression of a recombinant version of the CHDHp gene in Saccharomyces cerevisiae led to enrichment of the target protein in the mitochondrial inner membrane. The expressed protein conferred choline dehydrogenase activity, suggesting that both functional domains (FAD and the iron sulfur cluster) were properly assembled and that the mature CHDH was appropriately located in the inner mitochondria membrane.

3T3 Cells↗
PubMed1984

Effect of ethyl choline mustard on choline dehydrogenase and other enzymes of choline metabolism.

The effect of ethyl choline mustard (ECMA), and effective irreversible inhibitor of choline transport, was investigated on the enzymes of choline metabolism. ECMA at concentrations of 50 microM hardly affected choline acetyltransferase and caused only a 20% inhibition of choline kinase at a concentration of 1 mM. However, the mustard was an extremely effective inhibitor of choline dehydrogenase, producing 50% inhibition at concentrations of 6 microM. The inhibition was prevented by incubation in the presence of choline or by prior reaction of the mustard with thiosulphate. Separation of the components of the ECMA solution on TLC suggested that only the compound with an aziridine ring was an effective inhibitor of choline dehydrogenase. The inhibition was resistant to the washing out of excess unreacted mustard. The rate constant of inhibition was 395 M-1 X S-1. By the use of [3H]ECMA a single polypeptide in the enzyme preparation having a MW of 67,000 was labelled. The labelling was thiosulphate-sensitive and prevented by incubation with choline. It is concluded that ECMA is an irreversible inhibitor of choline dehydrogenase. It is at least as effective an inhibitor of choline dehydrogenase as of the choline transport system, although it does not appreciably inhibit choline acetyltransferase or choline kinase in the micromolar range.

Alcohol Oxidoreductases↗
PubMed1996

The Protection of Choline Dehydrogenase by Its Substrate.

Choline dehydrogenase, an enzyme bound to the mitochondrial inner membrane, plays an important role in the mitochondrial respiratory chain. The purified enzyme is different from the membrane bound enzyme in some properties. The inactivation effects of temperature and SDS on choline dehydrogenase were studied. It was found that the substrate choline had significant protective effect. It was suggested that its substrate could induce the conformational changes of choline dehydrogenase.

Journal Article↗
PubMed1997

Studies on the Denaturation and Conformation of Choline Dehydrogenase.

The substrate choline was able to improve the pH stability of choline dehydrogenase (CDH). It was found that during the thermal denaturation there were an increase of beta-structure and a decrease of the alpha-helix content. Changes in beta-structure were attributed to beta-turn and 3(10)-helix. The substrate had a protective effect on CDH thermal denaturation. The proportion of 3(10)-helix content of the SDS-denatured protein and that in the presence of substrate was 33% and 31.2% respectively; while, the corresponding proportion of beta-sheet was 29% and 10.6%, respectively. Moreover, when the absorption proportion of alpha-helix, random coil and the side-chain of tyrosine residues were concerned, it was found that the spectral property of the CDH treated by denaturants in the presence of substrate were rather similar to that of the non-denatured enzyme.

Journal Article↗
PubMed2003

Cloning, expression, and purification of choline dehydrogenase from the moderate halophile Halomonas elongata.

Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate. Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures. To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro. In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase. The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose. The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 micro mol of O(2) min(-1) mg(-1) mM(-1) at pH 7 and 25 degrees C, respectively. With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent V(max) values for choline and betaine-aldehyde were 10.9 and 5.7 micro mol of O(2) min(-1) mg(-1), respectively. These V(max) values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor. Altogether, the kinetic data are consistent with the conclusion that H. elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available.

Alcohol Oxidoreductases↗
PubMed1994

Use of hydrophobic chromatography for purification of the membrane-located choline dehydrogenase from a Pseudomonas strain.

Choline dehydrogenase has been purified using hydrophobic chromatography 250-fold from a Pseudomonas strain. Although the enzyme is associated with the cell membrane and could be extracted from membrane preparations, it was best purified from a complete cell extract made with a non-ionic detergent. Only phenazine methosulfate was able to act as electron acceptor; there was no evidence of bound flavin, but there was evidence of pyrroloquinoline quinone cofactor. The purified enzyme had a specific activity of up to 67 units/mg, which is at least ten times higher than the values reported for mitochondrial choline dehydrogenases, and up to 100 times higher than previous reports for the Pseudomonas enzyme. The estimated subunit size of 66 kDa, which corresponds with the native size, is close to that deduced from the gene sequence of the Escherichia coli betA gene, and preliminary N-terminal sequencing shows homology with this deduced sequence. The next enzyme in the degradation pathway of choline, betaine aldehyde dehydrogenase, was also purified from the same extract.

Alcohol Oxidoreductases↗
PubMed1989

Renal inner medullary choline dehydrogenase activity: characterization and modulation.

Betaine belongs to the trimethylamine class of osmolytes (osmotically active substances believed to play an important role in cell volume homeostasis) and has recently been identified in the inner medulla of the mammalian kidney. Trimethylamines accumulate in the renal inner medulla during hypertonic stress, and betaine content in the inner medulla has been shown recently to increase during hypernatremia, yet the mechanisms governing the modulation of trimethylamine content and, in particular, of betaine content are not well understood. In this study, we demonstrate the presence of choline dehydrogenase activity in the renal inner medullas of three separate rat strains. Choline dehydrogenase is the enzyme that catalyzes the first of two successive oxidation steps in the biosynthetic conversion of choline to betaine. The presence of choline dehydrogenase activity in the inner medulla suggests that betaine accumulation in the inner medulla may result, at least in part, through in situ synthesis. The Km and Vmax of the reaction in the inner medullas of Long-Evans rats are 4.7 +/- 0.5 mM and 36.9 +/- 5.0 nmol.mg protein-1.min-1, respectively. These values are similar to the characteristics of choline dehydrogenase in mammalian liver. During hypernatremia, when betaine content of the inner medulla has been shown to increase 1.5-fold, choline dehydrogenase activity remains unchanged (or slightly increased), whereas enzyme activity in the cortex increases approximately 50%. Possible mechanisms of inner medullary betaine accumulation are discussed.

Alcohol Oxidoreductases↗
PubMed2006

Liver choline dehydrogenase and kidney betaine-homocysteine methyltransferase expression are not affected by methionine or choline intake in growing rats.

Choline dehydrogenase (CHDH) and betaine-homocysteine methyltransferase (BHMT) are 2 enzymes involved in choline oxidation. BHMT is expressed at high levels in rat liver and its expression is regulated by dietary Met and choline. BHMT is also found in rat kidney, albeit in substantially lower amounts, but it is not known whether kidney BHMT expression is regulated by dietary Met or choline. Similarly, CHDH activity is highest in the liver and kidney, but the regulation of its expression by diet has not been thoroughly investigated. Sprague Dawley rats ( approximately 50 g) were fed, for 9 d in 2 x 3 factorial design (n = 8), an l-amino acid-defined diet varying in l-Met (0.125, 0.3, or 0.8%) and choline (0 or 25 mmol/kg diet). Liver and kidney BHMT and CHDH were assessed using enzymatic, Western blot, and real-time PCR analyses. Liver samples were also fixed for histological analysis. Liver BHMT activity was 1.3-fold higher in rats fed the Met deficient diet containing choline, which was reflected in corresponding increases in mRNA content and immunodetectable protein. Independent of dietary choline, supplemental Met increased hepatic BHMT activity approximately 30%. Kidney BHMT and liver CHDH expression were refractory to these diets. Some degree of fatty liver developed in all rats fed a choline-devoid diet, indicating that supplemental Met cannot completely compensate for the lack of dietary choline in growing rats.

Animals↗
PubMed1975

The reaction of choline dehydrogenase with some electron acceptors.

1. The choline dehydrogenase (EC 1.1.99.1) WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme.

2,6-Dichloroindophenol↗
PubMed1996

Determination of choline dehydrogenase activity along the rat nephron.

A radioenzymatic microassay was developed to quantitate choline dehydrogenase activity in single microdissected nephron segments. This enzyme is the rate limiting step in the biosynthesis of betaine, which serves as an intracellular osmoregulatory organic solute in mammalian kidney. The enzyme localized in renal mitochondrial inner membrane forms betaine aldehyde, which in the assay is converted to betaine by oxidative treatment. A histochemical procedure based on the formazan detection of tetranitroblue tetrazolium chloride was applied in parallel. The results show that activities in proximal convoluted and straight tubules are more than 5 times higher (21 to 25 pmol h-1 mm tubule-1) compared to distal nephron segments with no significant differences along the proximal tubule. Along the osmotic gradient from the outer medullary towards the papillary structures enzyme activities increased in ascending limbs of Henle's loop and collecting tubules. Collecting ducts showed two times higher activities than ascending loop segments when corrected for tubular cell volumes. The quantitative data were confirmed by the histochemical procedure. The results allow for the conclusion that betaine synthesis is sufficient to build up renal betaine, but cannot explain the distribution pattern of betaine along the corticopapillary axis. Additional mechanisms like intrarenal and tubular transport have to be postulated.

Alcohol Oxidoreductases↗
PubMed2000

Choline dehydrogenase kinetics contribute to glycine betaine regulation differences in chesapeake bay and atlantic oysters.

Choline dehydrogenase (CD), the first enzyme of the glycine betaine synthetic pathway, was measured in a mitochondrial lysate from gill tissue from Atlantic and Chesapeake Bay oysters acclimated to both 350 and 750 mosm. CD from both populations functions at its maximum rate at 30 degrees C and pH 8.75. Although CD from both populations has a similar affinity for its substrate, choline (K(m) = 15.7 mM), CD V(max) from Atlantic oysters is twice that from Bay oysters. In addition, the CD K(m )doubles and the V(max) increases four-fold in both oyster populations acclimated to 750 mosm. CD activity is competitively inhibited by both betaine aldehyde and glycine betaine. The differences in CD kinetics between the two oyster populations help to account for the lower glycine betaine synthesis rates and concentrations in Chesapeake Bay oysters. CD cannot function rapidly enough to saturate the enzyme, betaine aldehyde dehydrogenase (BADH), immediately downstream, and, therefore, CD kinetics limit the rate of glycine betaine synthesis in oysters. J. Exp. Zool. 286:250-261, 2000.

Alcohol Oxidoreductases↗