Identification and characterization of alkenyl hydrolase (lysoplasmalogenase) in microsomes and identification of a plasmalogen-active phospholipase A2 in cytosol of small intestinal epithelium

A lysoplasmalogenase (EC 3.3.2.2; EC 3.3.2.5) that liberates free aldehyde from 1-alk-1'-enyl-sn-glycero-3-phospho-ethanolamine or -choline (lysoplasmalogen) was identified and characterized in rat gastrointestinal tract epithelial cells. Glycerophosphoethanolamine was produced in the reaction in equimolar amounts with the free aldehyde. The microsomal membrane associated enzyme was present throughout the length of the small intestines, with the highest activity in the jejunum and proximal ileum. The rate of alkenyl ether bond hydrolysis was dependent on the concentrations of microsomal protein and substrate, and was linear with respect to time. The enzyme hydrolyzed both ethanolamine- and choline-lysoplasmalogens with similar affinities; the Km values were 40 and 66 microM, respectively. The enzyme had no activity with 1-alk-1'-enyl-2-acyl-sn-glycero-3-phospho-ethanolamine or -choline (intact plasmalogen), thus indicating enzyme specificity for a free hydroxyl group at the sn-2 position. The specific activities were 70 nmol/min/mg protein and 57 nmol/min/mg protein, respectively, for ethanolamine- and choline-lysoplasmalogen. The pH optimum was between 6.8 and 7.4. The enzyme required no known cofactors and was not affected by low mM levels of Ca2+, Mg2+, EDTA, or EGTA. The detergents, Triton X-100, deoxycholate, and octyl glucoside inhibited the enzyme. The chemical and physical properties of the lysoplasmalogenase were very similar to those of the enzyme in liver and brain microsomes. In developmental studies the specific activities of the small intestinal and liver enzymes increased markedly, 11.1- and 3.4-fold, respectively, in the first approximately 40 days of postnatal life. A plasmalogen-active phospholipase A2 activity was identified in the cytosol of the small intestines (3.3 nmol/min/mg protein) and liver (0.3 nmol/min/mg protein) using a novel coupled enzyme assay with microsomal lysoplasmalogenase as the coupling enzyme.     

Identification and characterization of alkenyl hydrolase (lysoplasmalogenase) in microsomes and identification of a plasmalogen-active phospholipase A2 in cytosol of small intestinal epithelium

A lysoplasmalogenase (EC 3.3.2.2; EC 3.3.2.5) that liberates free aldehyde from 1-alk-1′-enyl-sn-glycero-3-phospho-ethanolamine or -choline (lysoplasmalogen) was identified and characterized in rat gastrointestinal tract epithelial cells. Glycerophosphoethanolamine was produced in the reaction in equimolar amounts with the free aldehyde. The microsomal membrane associated enzyme was present throughout the length of the small intestines, with the highest activity in the jejunum and proximal ileum. The rate of alkenyl ether bond hydrolysis was dependent on the concentrations of microsomal protein and substrate, and was linear with respect to time. The enzyme hydrolyzed both ethanolamine- and choline-lysoplasmalogens with similar affinities; the K_m values were 40 and 66 μM, respectively. The enzyme had no activity with 1-alk-1′-enyl-2-acyl-sn-glycero-3-phospho-ethanolamine or -choline (intact plasmalogen), thus indicating enzyme specificity for a free hydroxyl group at the sn-2 position. The specific activities were 70 nmol/min/mg protein and 57 nmol/min/mg protein, respectively, for ethanolamine- and choline-lysoplasmalogen. The pH optimum was between 6.8 and 7.4. The enzyme required no known cofactors and was not affected by low mM levels of Ca²⁺, Mg²⁺, EDTA, or EGTA. The detergents, Triton X-100, deoxycholate, and octyl glucoside inhibited the enzyme. The chemical and physical properties of the lysoplasmalogenase were very similar to those of the enzyme in liver and brain microsomes. In developmental studies the specific activities of the small intestinal and liver enzymes increased markedly, 11.1- and 3.4-fold, respectively, in the first ～40 days of postnatal life. A plasmalogen-active phospholipase A₂ activity was identified in the cytosol of the small intestines (3.3 nmol/min/mg protein) and liver (0.3 nmol/min/mg protein) using a novel coupled enzyme assay with microsomal lysoplasmalogenase as the coupling enzyme.     

A spectrophotometric method for the assay of phospholipase D activity

Phosphatidylcholine phosphatidohydrolase (EC 3.1.4.4, phospholipase D) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. We have developed a spectrophotometric assay for phospholipase D using choline kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of choline with the oxidation of NADH. The assay was linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzyme activity, determination of initial rates of reaction, and detailed kinetic studies of phospholipase D. The method is limited to analysis of purified preparations of phospholipase D lacking competing activities to the coupled system.     

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.     

Metabolism of the lipid peroxidation product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic fractions.

The metabolism of the lipid peroxidation product 4-hydroxynonenal and of several other related aldehydes by isolated hepatocytes and rat liver subcellular fractions has been investigated. Hepatocytes rapidly metabolize 4-hydroxynonenal in an oxygen-independent process with a maximum rate (depending on cell preparation) ranging from 130 to 230 nmol/min per 10(6) cells (average 193 +/- 50). The aldehyde is also rapidly utilized by whole rat liver homogenate and the cytosolic fraction (140 000 g supernatant) supplemented with NADH, whereas purified nuclei, mitochondria and microsomes supplemented with NADH show no noteworthy consumption of the aldehyde. In cytosol, the NADH-mediated metabolism of the aldehyde exhibits a 1:1 stoichiometry, i.e. 1 mol of NADH oxidized/mol of hydroxynonenal consumed, and the apparent Km value for the aldehyde is 0.1 mM. Addition of pyrazole (10 mM) or heat inactivation of the cytosol completely abolishes aldehyde metabolism. The various findings strongly suggest that hepatocytes and rat liver cytosol respectively convert 4-hydroxynonenal enzymically is the corresponding alcohol, non-2-ene-1,4-diol, according to the equation: CH3-[CH2]4-CH(OH)-CH = CH-CHO + NADH + H+----CH3-[CH2]4-CH(OH)-CH = CH-CH2OH + NAD+. The alcohol non-2-ene-1,4-diol has not yet been isolated from incubations with hepatocytes and liver cytosolic fractions, but was isolated in pure form from an incubation mixture containing 4-hydroxynonenal, isolated liver alcohol dehydrogenase and NADH and its chemical structure was confirmed by mass spectroscopy. Compared with liver, all other tissues possess only little ability to metabolize 4-hydroxynonenal, ranging from 0% (fat pads) to a maximal 10% (kidney) of the activity present in liver. The structure of the aldehyde has a strong influence on the rate and extent of its enzymic NADH-dependent reduction to the alcohol. The saturated analogue nonanal is a poor substrate and only a small proportion of it is converted to the alcohol. Similarly, nonenal is much less readily utilized as compared with 4-hydroxynonenal. The effective conversion of the cytotoxic 4-hydroxynonenal and other reactive aldehydes to alcohols, which are probably less toxic, could play a role in the general defence system of the liver against toxic products arising from radical-induced lipid peroxidation.     

Multifunctionality of liver alcohol dehydrogenase. Studies of aldehyde dehydrogenase activity

Kinetic studies of the liver alcohol dehydrogenase catalyzed dehydrogenation of aldehydes were carried out over a wide range of octanal concentrations. The effect of specific inhibitors of liver alcohol dehydrogenase on aldehyde dehydrogenase activity was examined. The results were consistent with a steady-state random mechanism with the formation of the ternary E · NADH octanal complex at low temperatures. This ternary complex becomes inconspicuous at high temperatures. The aldehyde dehydrogenase activity was found to associate with all ethanol-active isozymes. The dual dehydrogenase reactions are catalyzed by the same molecule, presumably in the region of the same domain. However, the two activities respond differently to structural changes.     

Ternary complexes of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent system for probing the role of binding interactions with NAD(+) and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes the transfer of hydride ion from an alcohol substrate to the NAD(+) cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The enzyme is also an excellent catalyst for the reverse reaction. X-ray crystallography has shown that the NAD(+) binds in an extended conformation with a distance of 15 A between the buried reacting carbon of the nicotinamide ring and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-averaged models. Significantly, entries in the protein data bank contain both coordinates as well as temperature factors. However, enzyme function involves both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the vibrations of certain atoms in the active site where reactions take place. Ternary complexes produced during the reaction of the enzyme binary entity, E-NAD(+), with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolized via the E-NAD(+)-retinal ternary complex to retinoic acid (vitamin A acid). To unravel the mechanistic aspects of these transformations, the kinetics and energetics of interconversion between various ternary complexes are characterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Secondary kinetic isotope effects with retinol are not particularly large with the wild-type form of alcohol dehydrogenase from horse liver. We analyze alcohol dehydrogenase catalysis through a re-examination of the reaction coordinates. The ground states of the binary and ternary complexes are shown to be related to the corresponding transition states through topology and free energy acting along the reaction path.     

Microquantitative determination of intra-acinar distribution profiles of low-Km and high-Km aldehyde dehydrogenase activity in rat liver

Microquantitative measurements of total and of low-Km aldehyde dehydrogenase (ALDH) activity with millimolar and micromolar concentrations of acetaldehyde and propionaldehyde were carried out on the livers of male and female rats. Lyophilized cryostat sections of liver parenchyma were microdissected along the entire sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ALDH activity was measured in a microbiochemical assay using the oil-well technique with luminometric determination of NADH. On the basis of single measurements, mean values of total, low-Km and high-Km ALDH activity could be calculated and the specific distribution patterns graphically demonstrated. The two substrates acetaldehyde and propionaldehyde yielded similar values of ALDH activity, the intraacinar distribution profiles of which showed characteristic sex differences. In the liver of the male rat high-Km ALDH activity has two flat peaks in the periportal and the perivenous area, while low-Km ALDH activity is almost evenly distributed throughout the acinus. In the livers of female rats, both high-Km and low-Km ALDH activity shows a continuous gradient which decreases from the periportal to the perivenous zone (pp/pv = 1.4:1). It was therefore possible to demonstrate that the maxima of alcohol dehydrogenase activity and of low-Km ALDH activity are localized in opposite parts of the liver acinus of the female rat. This heterotopy should have consequences with respect to hepatotoxicity after alcohol ingestion.     

Intraparticulate localization and some properties of a clofibrate-induced peroxisomal aldehyde dehydrogenase from rat liver

A study was made of the effect of chronic administration of the hypolipidemic drug clofibrate on the activity and intracellular localization of rat liver aldehyde dehydrogenase. The enzyme was assayed using several aliphatic and aromatic aldehydes. Clofibrate treatment caused a 1.5 to 2.3-fold increase in the liver specific aldehyde dehydrogenase activity. The induced enzyme has a high Km for acetaldehyde and was found to be located in peroxisomes and microsomes. Clofibrate did not alter the enzyme activity in the cytoplasmic fraction. The total peroxisomal aldehyde dehydrogenase activity increased 3 to 4-fold under the action of clofibrate. Disruption of the purified peroxisomes by the hypotonic treatment or in the alkaline conditions resulted in the release of catalase from the broken organelles, while aldehyde dehydrogenase as well as nucleoid-bound urate oxidase and the peroxisomal membrane marker NADH:cytochrome c reductase remained in the peroxisomal 'ghosts'. At the same time, treatment by Triton X-100 led to solubilization of the membrane-bound NADH:cytochrome c reductase and aldehyde dehydrogenase from intact peroxisomes and their 'ghosts'. These results indicate that aldehyde dehydrogenase is located in the peroxisomal membrane. The peroxisomal aldehyde dehydrogenase is active with different aliphatic and aromatic aldehydes, except for formaldehyde and glyceraldehyde. The enzyme Km values lie in the millimolar range for acetaldehyde, propionaldehyde, benzaldehyde and phenylacetaldehyde and in the micromolar range for nonanal. Both NAD and NADP serve as coenzymes for the enzyme. Aldehyde dehydrogenase was inhibited by disulfiram, N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic)acid. According to its basic kinetic properties peroxisomal aldehyde dehydrogenase seems to be similar to a clofibrate-induced microsomal enzyme. The functional role of both enzymes in the liver cells is discussed.     

An enzymatic model for rat liver alcohol dehydrogenase in the presence of low-Km aldehyde dehydrogenase

Four isoenzymes of aldehyde dehydrogenase were partially purified from rat liver mitochondria by hydroxylapatite chromatography and gel filtration. While three forms display low affinity for acetaldehyde, the fourth is active at extremely low aldehyde concentrations (Km less than or equal to 2 microM) and allows the oxidation of the acetaldehyde formed by catalysis of alcohol dehydrogenase at pH 7.4. Different models of alcohol dehydrogenase have been examined by analysis of progress curves of ethanol oxidation obtained in the presence of low-km aldehyde dehydrogenase. According to the only acceptable model, when the acetaldehyde concentration is kept low by the action of aldehyde dehydrogenase, NADH no longer binds to alcohol dehydrogenase, but acetaldehyde still competes with ethanol for the active site of the enzyme. The seven kinetic parameters of the two enzymes (four for alcohol dehydrogenase and three for aldehyde dehydrogenase) and the equilibrium constant of the reaction catalyzed by alcohol dehydrogenase have been determined by applying a new fitting procedure here described.     

19F nuclear magnetic resonance observations of aldehyde dismutation catalyzed by horse liver alcohol dehydrogenase

We have examined aspects of the second catalytic activity of alcohol dehydrogenase from horse liver (LADH), which involves an apparent dismutation of an aldehyde substrate into alcohol and acid in the presence of LADH and NAD. Using the substrate p-trifluoromethylbenzaldehyde, we have observed various bound complexes by ¹⁹F NMR in an effort to further characterize the mechanism of the reaction. The mechanism appears to involve the catalytic activity of LADH · NAD · aldehyde complex which reacts to form an enzyme · NADH · acid complex. The affinity of the acid product for LADH · NADH is weak and the acid product readily desorbs from the ternary complex. The resulting LADH · NADH can then react with a second molecule of aldehyde to form NAD and the corresponding alcohol. The result is the conversion of two molecules of aldehyde to one each of acid and alcohol, with LADH and NAD acting catalytically. This sequence of reactions can also explain the slow formation of acid product observed when alcohol and NAD are incubated with the enzyme.     

Ternary complexes of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent system for probing the role of binding interactions with NAD⁺ and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes the transfer of hydride ion from an alcohol substrate to the NAD⁺ cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The enzyme is also an excellent catalyst for the reverse reaction. X-ray crystallography has shown that the NAD⁺ binds in an extended conformation with a distance of 15 Å between the buried reacting carbon of the nicotinamide ring and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-averaged models. Significantly, entries in the protein data bank contain both coordinates as well as temperature factors. However, enzyme function involves both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the vibrations of certain atoms in the active site where reactions take place. Ternary complexes produced during the reaction of the enzyme binary entity, E-NAD⁺, with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolized via the E-NAD⁺-retinal ternary complex to retinoic acid (vitamin A acid). To unravel the mechanistic aspects of these transformations, the kinetics and energetics of interconversion between various ternary complexes are characterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Secondary kinetic isotope effects with retinol are not particularly large with the wild-type form of alcohol dehydrogenase from horse liver. We analyze alcohol dehydrogenase catalysis through a re-examination of the reaction coordinates. The ground states of the binary and ternary complexes are shown to be related to the corresponding transition states through topology and free energy acting along the reaction path.     

Genetic polymorphism of enzymes of alcohol metabolism and susceptibility to alcoholic liver disease

Differences in the pharmacokinetics of alcohol absorption and elimination are, in part, genetically determined. There are polymorphic variants of the two main enzymes responsible for ethanol oxidation in liver, alcohol dehydrogenase and aldehyde dehydrogenase. The frequency of occurrence of these variants, which have been shown to display strikingly different catalytic properties, differs among different racial populations. Since the activity of alcohol dehydrogenase in liver is a rate-limiting factor for ethanol metabolism in experimental animals, it is likely that the type and content of the polymorphic isoenzyme subunit encoded at ADH2, beta-subunit, and at ADH3, the gamma-subunit, are contributing factors to the genetic variability in ethanol elimination rate. The recent development of methods for genotyping individuals at these loci using white cell DNA will allow us to test this hypothesis as well as any relationship between ADH genotype and the susceptibility to alcoholism or alcohol-related pathology. A polymorphic variant of human liver mitochondrial aldehyde dehydrogenase, ADLH2, which has little or no acetaldehyde oxidizing activity has been identified. Individuals with the deficient ALDH2 phenotype do not have altered ethanol elimination rates but they do exhibit high blood acetaldehyde levels and dysphoric symptoms such as facial flushing, nausea and tachycardia, after drinking alcohol. Because acetaldehyde is so reactive, it binds to free amino groups of proteins including a 37 kilodalton hepatic protein-acetaldehyde adduct and may elicit an antibody response. We would predict that individuals who have low ALDH2 activity because of liver disease or because they have the inactive ALDH2 variant isoenzyme might form more protein-acetaldehyde adducts and elicit a greater immune response. These adducts may represent good biological markers of alcohol abuse and may also play a role in liver injury due to chronic alcohol consumption.     

Fluorometric enzyme assay for choline and acetylcholine

A sensitive and specific assay for choline and ACh which may be applied directly to brain extracts is described. The method is based upon enzymic coupling to the oxidation of fluorescent NADH. The following enzymic sequence is utilized: acetylcholinesterase, choline phosphokinase, pyruvate kinase, and lactate dehydrogenase. The method detects as little as 0.1 mμmole of choline or ACh, which is the amount of metabolite present in 1 mg or 8 mg of whole rat brain, respectively. The specificity of the method is such that only choline and ACh of tissue samples react. Extraction of whole brain samples by either heating at pH 4 or by chloroform/methanol or perchloric acid were compared in order to find a single procedure which was useful for extraction of both ACh and free choline from brain samples. Perchloric acid extraction proved to be the most efficient of the three methods for extraction of the two constituents. By this procedure the ACh content of whole rat brain was found to be 11.5 mμmoles/gm and the choline content of the same samples was 107 mμmoles/gm. Both values are in agreement with other published results.     

Preparation of an alcohol-dehydrogenase--NAD(H)--sepharose complex showing no requirement of soluble coenzyme for its activity.

1. Horse liver alcohol dehydrogenase and an NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor. 2. The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40% of that obtained in excess of free coenzyme. 3. A molar ratio of 1:1 between the amount of bound enzyme was sufficient to obtain high activities in the absence of free coenzyme. 4. The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5. Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue. 6. The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.     

β-Lapachone alleviates alcoholic fatty liver disease in rats

Alcohol-induced liver injury is the most common liver disease in which fatty acid metabolism is altered. It is thought that altered NAD⁺/NADH redox potential by alcohol in the liver causes fatty liver by inhibiting fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. β-Lapachone (βL), a naturally occurring quinone, has been shown to stimulate fatty acid oxidation in an obese mouse model by activating adenosine monophosphate-activated protein kinase (AMPK). In this report, we clearly show that βL reduced alcohol-induced hepatic steatosis and induced fatty acid oxidizing capacity in ethanol-fed rats. βL treatment markedly decreased hepatic lipids while serum levels of lipids and lipoproteins were increased in rats fed ethanol-containing liquid diets with βL administration. Furthermore, inhibition of lipolysis, enhancement of lipid mobilization to mitochondria and upregulation of mitochondrial β-oxidation activity in the soleus muscle were observed in ethanol/βL-treated animals compared to the ethanol-fed rats. In addition, the activity of alcohol dehydrogenase, but not aldehyde dehydrogenase, was significantly increased in rats fed βL diets. βL-mediated modulation of NAD⁺/NADH ratio led to the activation of AMPK signaling in these animals. Conclusion: Our results suggest that improvement of fatty liver by βL administration is mediated by the upregulation of apoB100 synthesis and lipid mobilization from the liver as well as the direct involvement of βL on NAD⁺/NADH ratio changes, resulting in the activation of AMPK signaling and PPARα-mediated β-oxidation. Therefore, βL-mediated alteration of NAD⁺/NADH redox potential may be of potential therapeutic benefit in the clinical setting.     

Piericiden A sensitivity, site 1 phosphorylation, and reduced nicotinamide adenine dinucleotide dehydrogenase during iron-limited growth of Candida utilis.

It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. These findings were reinvestigated in the present study. The parameters and properties followed during these transitions were sensitivity of NADH oxidation to piericidin, presence or absence of coupling site 1, EPR signals appearing on reduction with NADH or dithionite, the specific activities of NADH oxidase, NADH-ferricyanide reductase, and NADH-5-hydroxy-1,4-naphthoquinone (juglone) reductase, and the kinetic behavior of NADH dehydrogenase in the ferricyanide assay. Monitoring the rates of oxidation of NADH in submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations. It was found that on gradually increasing the concentration of iron during continuous culture (transition from ironlimited to iron- and substrate-limited growth), as well as on aeration of iron-limited cells, coupling site 1, piericidin sensitivity, NADH-ferricyanide activity, and iron-sulfur centers 1 and 2 increased concurrently, with concomitant decline of NADH-juglone reductase activity. Cycloheximide prevented all these changes. Iron-sulfur centers 3 plus 4 underwent relatively little increase during these transitions. It is concluded that in both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piercidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement. No evidence was found for the development of coupling site 1 without the appearance of piericidin sensir th     

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.     

An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates

A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.     

Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase.

Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD+ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyl groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyl-CoA. These findings suggest that an S-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.