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.     

Comparative subcellular distribution of benzaldehyde and acetaldehyde dehydrogenase activities in two hepatoma cell lines and in normal hepatocytes.

The NAD- and NADP-dependent aldehyde dehydrogenase (ALDH) activities were evaluated in two rat hepatoma cell lines, namely the well-differentiated MH1C1 line and the less differentiated HTC line. Each activity was determined in parallel in isolated rat hepatocytes, for comparison. The aliphatic aldehyde acetaldehyde (ACA) and the aromatic aldehyde benzaldehyde (BA) were used as substrates. With the first substrate the ALDH activities found in the crude cytoplasmic extracts were lower in hepatoma cells than in normal hepatocytes, especially when measured with NADP as coenzyme (ACA/NADP). Otherwise, with benzaldehyde as substrate the NAD-dependent enzyme activity (BA/NAD) was increased about 9-fold in HTC cells over hepatocytes and decreased in MH1C1 cells, while the NADP-dependent (BA/NADP) activity was increased 38- and 2.5-fold in HTC and MH1C1 cell lines, respectively. Studies on the subcellular distribution of these enzyme activities showed that the activity measured with acetaldehyde and NAD (ACA/NAD) was almost equally distributed between the cytosol and the subcellular particles in the three cell populations, but the ACA/NADP activity was shifted towards the cytosolic compartment in hepatomas, especially in HTC cells. The BA/NAD and BA/NADP ALDH activities found in the organelles of hepatoma cells were markedly reduced in comparison with hepatocytes, in favour of the cytosol. The most striking difference between the normal and the transformed cells was the 94-fold increase over hepatocytes of the BA/NADP activity, found in the cytosolic fractions of HTC cells. MH1C1 cells showed a less pronounced (7.5-fold) enhancement of this tumour-associated specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of aldehyde dehydrogenase from Proteus vulgaris.

NADP-linked aldehyde dehydrogenase (aldehyde : NADP+ oxidoreductase, EC 1.2.1.4) was purified from Proteus vulgaris to the stage of homogeneity as judged by ultracentrifugation and polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 130000 by gel filtration. The enzyme which was crystallized from ammonium sulfate solution, lost its activity. The enzyme did not require coenzyme A, and the reaction was completely dependent on ammonium ions which could be partially replaced by Rb+ or K+. The optimum pH was about 9. Broad substrate specificity was observed and Km values for propionaldehyde, acetaldehyde and isovaleraldehyde were 1.7 - 10(-5), 4 - 10(-5) and 3 - 10(-5) M, respectively. The physiological role of the enzyme in living cells is obscure, but might account for another degradative pathway of L-leucine in P. vulgaris differing from the established pathway.     

Initial characterization of aldehyde dehydrogenase from rat testis cytosol

Aldehyde dehydrogenase was purified 187-fold from cytosol of rat testis by chromatographic methods and gel filtration with a yield of about 50%. The enzyme exhibits absolute requirement for exogenous sulfhydryl compounds and strong dependence on temperature. Addition of 0.4mM Ca2 or Mg2 ions results in 50% inhibition. Optimally active at pH 8.5 and 50 degrees C, aldehyde dehydrogenase displays broad substrate specificity; saturation curves with acetaldehyde and propionaldehyde are non-hyperbolic, with Hill coefficients comprised between 0.8 and 0.7. Strong substrate inhibition can be observed with both aromatic and long-chain alyphatic aldehydes. According to mathematical models, Km decreases from 246 microM for acetaldehyde to 4 microM for capronaldehyde and Ki decreases from about 4mM for butyraldehyde to 0.2 mM for capronaldehyde.     

Bovine lens aldehyde dehydrogenase. Purification and preliminary characterization.

Cytoplasmic aldehyde dehydrogenase from bovine lens was purified to apparent homogeneity by using ion-exchange and affinity chromatography. Sedimentation-equilibrium ultracentrifugation, gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis show that the enzyme is a dimer of Mr 114000, with subunits of Mr 57000. The enzyme does not dissociate into monomers in the presence of Ca2+ or Mg2+. The enzyme has a pI of 5.0, an activation energy of 35.1kJ/mmol and a pK value of 8.6 with acetaldehyde as substrate. The enzyme is a prolate ellipsoid with a Stokes radius of 4nm. Progesterone, deoxycorticosterone and chlorpropamide inhibited enzyme activity, and this inhibition may play a role in cataract formation in patients maintained on systemic corticosteroids and in tablet-dependent diabetics.     

Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).     

Acetaldehyde oxidation in rat liver mitochondria, action of Mg2+, ATP and rotenone on acetaldehyde oxidation in intact mitochondria, and on some purified aldehyde dehydrogenase isozymes

In intact rat liver mitochondria acetaldehyde is oxidized by three functionally distinct dehydrogenase systems. Two of these reduce intramitochondrial nicotinamide adenine dinucleotide (NAD): one is operative with micromolar acetaldehyde concentrations and is stimulated by Mg2+, the other is operative with millimolar acetaldehyde concentrations and is stimulated by adenosine 5'-triphosphate (ATP). The third system reduces added NAD and is stimulated by rotenone. Connected to these systems, three aldehyde dehydrogenase isozymes (ALDH) have been purified: a low-Km ALDH activated by Mg2+, a high-Km ALDH activated by ATP and Mg2+, a high-Km ALDH activated by rotenone. The properties of some isozymes are affected by detergents. Thus, deoxycholate augments the stimulation of low-Km isozyme by Mg2+ and confers sensitivity to Mg2+ and ATP on one of the high-Km isozymes. A fourth isozyme has been purified. Its affinity for acetaldehyde is so low that it is very unlikely that acetaldehyde is the physiological substrate.     

The effect of phosphorylation on oxidative and CoA- and NAD+-independent turnover of pyruvate by pyruvate dehydrogenase complex in the brain

It was shown that in the presence of ATP and Mg2+ the phosphorylation of the partially purified pyruvate dehydrogenase complex and the enzyme in isolated brain mitochondria inhibited the oxidative activity of the pyruvate dehydrogenase complex. The phosphorylation did no affect essentially the nonoxidative decarboxylation of pyruvate to form CO2 and acetaldehyde. In native mitochondria from the bovine brain the nonoxidative activity of the pyruvate dehydrogenase complex reached about 10% as compared to the oxidative activity of enzyme.     

Kinetics of membrane-bound aldehyde dehydrogenase from Acinetobacter calcoaceticus

In recent investigations we were able to demonstrate that the NADP-dependent aldehyde dehydrogenase of Acinetobacter calcoaceticus is an inducible enzyme localized in intracytoplasmic membranes limiting alkane inclusions. Long-chain aliphatic hydrocarbons and alkanols are inducers of the enzyme. It was purified by us and now kinetically characterized using the enzyme-micelle form, which contains bacterial phospholipids and a detergent (sodium cholate), too. The pH optimum of aldehyde dehydrogenase was determined to be at pH 10. The enzyme showed substrate inhibition (by aldehyde excess). The Ks and Km values of the leading substrate NADP+ were found to be 8.6 X 10(-5) and 10.3 X 10(-5)M independent of the chain-length of the aldehydes. The Km values of the aldehydes decreased depending on increasing chain-length (butanal: 1.6 X 10(-3), decanal: 1.5 X 10(-6)M). The Ki values (for inhibition by aldehyde excess) showed a similar behaviour (butanal: 7.5 X 10(-3), decanal: 3.5 X 10(-5)M) as well as the optimal aldehyde concentrations inducing the "maximal" reaction velocity (butanal: 5mM, decanal: 6 microM). The number of inhibiting aldehyde molecules per enzyme-substrate complex was determined to be n = 1. NADPH showed product inhibition kinetics (Ki(NADPH) = 2.2 X 10(-4)M), fatty acids did not. We were unable to measure a reverse reaction. The following ions and organic compounds were non-competitive inhibitors of the enzyme: Sn2+, Fe2+, Cu2+, BO3(3-), CN-, EDTA, o-phenanthroline, p-chloromercuri-benzoate, mercaptoethanol, phenylmethylsulfonyl fluoride, and diisopropylfluorophosphate; iodoacetate did not influence enzyme activity. Chloral hydrate was a competitive inhibitor of the aldehydes. Ethyl butyrate activates the enzyme, dependent on the chain-length of the aldehyde substrates.     

Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803.

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.     

NAD and NADP-dependent glutamate dehydrogenase in Hydrogenomonas H 16

Summary Hydrogenomonas H 16 synthetized two chromatographically distinct forms of glutamate dehydrogenase which differed in their thermolability. One glutamate dehydrogenase utilized NAD, the other NADP as a coenzyme.Low specific activity of NAD-dependent glutamate dehydrogenase was found in cells grown with glutamate as sole nitrogen source or in cells grown with a high concentration of ammonium ions. In the presence of a low concentration of ammonium ions or in a nitrogen free medium, the specific activity of the NAD-dependent enzyme increased. Corresponding to the formation of the NAD-dependent glutamate dehydrogenase the enzyme glutamine synthetase was synthesized. The ratio of NAD-dependent glutamate dehydrogenase to glutamine synthetase activity differed only slightly in cells grown with different nitrogen and carbon sources.The NADP-dependent glutamate dehydrogenase was found in high specific activity in cells grown with an excess of ammonium ions. Under nitrogen starvation the formation of the NADP-dependent glutamate dehydrogenase ceased and the enzyme activity decreased.     

Physiological role of yeasts NAD(P)+ and NADP+-linked aldehyde dehydrogenases.

The activity of NAD+ and NADP+-linked aldehyde dehydrogenases has been investigated in yeast cells grown under different conditions. As occurs in other dehydrogenase reactions the NAD(P)+-linked enzyme was strongly repressed in all hypoxic conditions; nervetheless, the NADP+-linked enzyme was active. The results suggest that the NAD(P)+ aldehyde dehydrogenase is involved in the oxidation of ethanol to acetyl-CoA, and that when the pyruvate dehydrogenase complex is repressed the NADP+-linked aldehyde dehydrogenase is operative as an alternative pathway from pyruvate to acetyl-CoA: pyruvate leads to acetaldehyde leads to acetate leads to acetyl-Coa. In these conditions the supply of NADPH is advantageous to the cellular economy for biosynthetic purposes. Short term adaptation experiments suggest that the regulation of the levels of the aldehyde dehydrogenase-NAD(P)+ takes place by the de novo synthesis of the enzyme.     

Inhibition of mitochondrial aldehyde dehydrogenase and acetaldehyde oxidation by the glutathione-depleting agents diethylmaleate and phorone

Experiments were carried out to study the effect of two commonly used glutathione-depleting agents, diethylmaleate and phorone, on the oxidation of acetaldehyde and the activity of aldehyde dehydrogenase. The oxidation of acetaldehyde by intact hepatocytes was inhibited when the cells were incubated with diethylmaleate. Washing and resuspending the cells in diethylmaleate-free medium afforded protection against the inhibition of acetaldehyde oxidation. The oxidation of acetaldehyde by isolated rat liver mitochondria as well as by disrupted mitochondria in the presence of excess NAD+ was inhibited by diethylmaleate or phorone, indicating inhibition of the low-Km aldehyde dehydrogenase. In addition, diethylmaleate inhibited oxidation of acetaldehyde by the high-Km cytosolic aldehyde dehydrogenase. Significant accumulation of acetaldehyde occurred when ethanol was oxidized by hepatocytes in the presence, but not in the absence, of diethylmaleate. Thus, diethylmaleate blocks the oxidation of added or metabolically generated acetaldehyde, analogous to results with other inhibitors of the low-Km aldehyde dehydrogenase such as cyanamide. These results suggest that caution should be used in interpreting the effects of diethylmaleate or phorone on metabolic reactions, especially those involving metabolism of aldehydes such as formaldehyde, because, in addition to depleting glutathione, these agents inhibit the low-Km aldehyde dehydrogenase.     

Aldehyde Dehydrogenases in Rat Brain. Subcellular Distribution and Properties

Abstract: Kinetic studies suggested the presence of several forms of NAD-dependent aldehyde dehydrogenase (ALDH) in rat brain. A subcellular distribution study showed that low- and high- K _m activities with acetaldehyde as well as the substrate-specific enzyme succinate semialdehyde dehydrogenase were located mainly in the mitochondrial compartment. The low- K _m activity was also present in the cytosol (<20%). The low- K _m activity in the homogenate was only 10–15% of the total activity with acetaldehyde as the substrate. Two K _m values were obtained with both acetaldehyde (0.2 and 2000 μ m ) and 3,4-dihydroxyphenylacetaldehyde (DOPAL) (0.3 and 31 μ m ), and one K _m value with succinate semialdehyde (5 μ m ). The main part of the aldehyde dehydrogenase activities with acetaldehyde, DOPAL, and succinate semialdehyde, but only little activity of the marker enzyme for the outer membrane (monoamine oxidase, MAO), was released from a purified mitochondrial fraction subjected to sonication. Only small amounts of the ALDH activities were released from mitochondria subjected to swelling in a hypotonic buffer, whereas the main part of the marker enzyme for the intermembrane space (adenylate kinase) was released. These results indicate that the ALDH activities with acetaldehyde, DOPAL and succinate semialdehyde are located in the matrix compartment. The low- K _m activity with acetaldehyde and DOPAL, but not the high- K _m activities and succinate semialdehyde dehydrogenase, was markedly stimulated by Mg²⁺ and Ca²⁺ in phosphate buffer. The low- and high- K _m activities with acetaldehyde showed different pH optima in pyrophosphate buffer.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells. Purification and properties

NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed in transformed or established mammalian cell lines in vitro but only in the developmental tissues of normal adult animals (Mejia, N. R. and MacKenzie, R. E. (1985) J. Biol. Chem. 260, 14616-14620). The enzyme, which contains methenyltetrahydrofolate cyclohydrolase activity as well, has been purified 6000-fold from Ehrlich ascites tumor cells. The preparation is homogeneous by sodium dodecyl sulfate gel electrophoresis (Mr = 34,000), and results from cross-linking with bis(sulfosuccinimidyl)suberate are consistent with a dimeric structure (Mr = 68,000) for the native bifunctional enzyme. The dehydrogenase is specific for NAD and requires both a divalent cation, Mg2+ or Mn2+, for activity and as well is stimulated by inorganic phosphate. When compared to the usual NADP-dependent methylenetetrahydrofolate dehydrogenase from mouse liver, the NAD-dependent dehydrogenase activity of the murine tumor enzyme shows a greater affinity for the polyglutamate forms of folate.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

An induced aliphatic aldehyde dehydrogenase from the bioluminescent bacterium, Beneckea harveyi. Purification and properties.

A NAD+-dependent aldehyde dehydrogenase, the activity of which induces at the same time as luceriferase, has been purified from the bioluminescent bacterium Beneckea harveyi, and its chemical and physical properties have been investigated. The purification is accomplished in three steps resulting in an enzyme preparation that gives a single protein band on three different gel electrophoresis systems. The molecular weight of the purified enzyme was estimated to be 120,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 59,000 indicating that aldehyde dehydrogenase has a dimeric structure with subunits of similar molecular weight. The purified enzyme has a high specificity for long chain aliphatic aldehydes; the Michaelis constants for aldehydes decrease with increasing chain length as also observed for bacterial aldehyde dehydrogenases involved in the metabolism of hydrocarbons. The aldehyde specificity of the aldehyde dehydrogenase is similar to that of luciferase indicating that the functional role of the enzyme may be linked with the bioluminescent system.     

NAD-linked, GSH- and factor-independent aldehyde dehydrogenase of the methylotrophic bacterium, Hyphomicrobium X

Cell-free extracts of Hyphomicrobium X showed NAD-dependent aldehyde dehydrogenase activity, provided that NAD addition preceded that of aldehyde. Activity was lost rather rapidly, especially during purification attempts, but this could be partially masked by including a time-dependent restoration step with thiol compounds in the protocol. The nature of the assay buffer appeared to be critical and stimulation occurred on incorporation of K+ ions in the mixture. An even higher specific activity could be achieved by 1,4-dithiothreitol (DTT) treatment of the preparation, followed by removal of DTT, and assaying in the absence of thiol compounds under anaerobic conditions. Exposure of such a preparation to O2 led to a significant decrease in activity within a couple of hours. Immediate inactivation occurred on addition of H2O2, but this could be prevented completely by prior addition of NAD. Since GSH does not participate in the reaction and no stimulating factor was detected, the role of thiol compounds is most probably confined to restoration or prevention of damage to an O2-sensitive, necessary thiol group. Since the same features were found for cell-free extract as for the partially purified enzyme, only one enzyme type seems to be present. Although the enzyme is a general aldehyde dehydrogenase, the kinetic parameters and the specific activity of the cell-free extract for formaldehyde indicate that it may play a role in formaldehyde dissimilation by Hyphomicrobium X. The NAD-linked, GSH- and factor-independent aldehyde dehydrogenase described here appears to be different in several respects from the formaldehyde dehydrogenase of Pseudomonas putida (EC 1.2.1.46) (despite showing similar behavior toward coenzymes and factors) but resembles the aldehyde dehydrogenase from baker's yeast (EC 1.2.1.5).     

Substrate Specificity of the Purified Primary Alcohol Dehydrogenases from Methanol-Oxidizing Bacteria

Hyphomicrobium strain WC, Pseudomonas strain TP-1, and Pseudomonas strain W1 are capable of growth on methanol as the sole source of carbon and energy. Methanol-grown cells of each organism contain a primary alcohol dehydrogenase that has been purified to homogeneity. Each enzyme has a molecular weight of 120,000 and shows an in vitro requirement for phenazine methosulfate and ammonium ions for enzymatic activity. Normal aliphatic alcohols are oxidized rapidly by each enzyme. The presence of a methyl group on the carbon atom adjacent to the primary alcohol group lowers the enzymatic activity. This effect is reduced as the methyl substituent is moved further away from the hydroxyl group. The effect of other substituents on enzymatic activity is reported. Methanol, formaldehyde, and to a limited extent acetaldehyde are oxidized by the primary alcohol dehydrogenases. Higher aldehydes are not oxidized. A possible explanation for this specificity, with regard to aldehydes, is presented in terms of degree of hydration of the aldehyde.