Jean-Pierre von Wartburg

Human Brain Aldehyde Reductases: Relationship to Succinic Semialdehyde Reductase and Aldose Reductase

Human brain contains multiple forms of aldehyde‐reducing enzymes. One major form (AR3), as previously shown, has properties that indicate its identity with NADPH‐dependent aldehyde reductase isolated from brain and other organs of various species; i.e., low molecular weight, use of NADPH as the preferred cofactor, and sensitivity to inhibition by barbiturates. A second form of aldehyde reductase („SSA reductase”) specifically reduces succinic semialdehyde (SSA) to produce γ‐hydroxybutyrate. This enzyme form has a higher molecular weight than AR3, and uses NADH as well as NADPH as cofactor. SSA reductase was not inhibited by pyrazole, oxalate, or barbiturates, and the only effective inhibitor found was the flavonoid quercetine. Although AR3 can also reduce SSA, the relative specificity of SSA reductase may enhance its in vivo role. A third form of human brain aldehyde reductase, AR2, appears to be comparable to aldose reductases characterized in several species, on the basis of its activity pattern with various sugar aldehydes and its response to characteristic inhibitors and activators, as well as kinetic parameters. This enzyme is also the most active in reducing the aldehyde derivatives of biogenic amines. These studies suggest that the various forms of human brain aldehyde reductases may have specific physiological functions.

Inhibition of aldehyde reductase isoenzymes in human and rat brain

Abstract Anticonvulsant drugs, such as barbiturates, open-chained analogues of barbiturates, glutethimide, succinimides and hydantoins have been tested in vitro as inhibitors of the isoenzymes of aldehyde reductase from human and rat brain. One major isoenzyme of both species is highly sensitive to the ionizable forms of these drugs containing the CONHCO grouping and a minimal lipophilic substitution. Differences between the isoenzymes were observed with respect to the absolute configuration of various succinimides as inhibitors. One isoenzyme of both species exerts activity with NADH as well as NADPH. The NADH-dependent activity of the human enzyme is inhibited in a noncompetitive way by NADP with a Ki-intercept of 2.2 × 10−7 M. A mixed inhibition is obtained with the biogenic acid, 4-hydroxyphenylacetic acid, as inhibitor of the main human isoenzyme. The inhibition is uncompetitive up to [I] = 1 × 10−3 M and yields a Ki value of 4.2 × 10−4M.

Studies on the subcellular localization of monoamine oxidase types A and B and its importance for the deamination of dopamine in the rat brain.

Abstract The distribution of the MAO-forms A and B between intra- and extrasynaptosomal rat brain mitochondria was studied with the aid of their known substrate and inhibitor specificities. The activities with the selective substrates serotonin, PEA and benzylamine indicated that intrasynaptosomal mitochondria have about a 3.4-fold higher MAO A:MAO B ratio than extrasynaptosomal mitochondria. However, PEA was found to be a selective substrate for MAO B only at low concentrations (such as 5 × 10 −6 M), whereas at higher concentrations (such as 10 −3 M) it was a substrate for both forms of MAO. The different ratios of the two enzyme forms in the two mitochondrial populations were confirmed when the selective inhibitors clorgyline and deprenyl were used with dopamine or 10 −3 M PEA. With these two amines, the ratios of MAO A: MAO B activities were 3–4.5 times higher in intrasynaptosomal than in extrasynaptosomal mitochondria. In particular, when the activity with dopamine was measured in intact synaptosomes, deamination being preceded by a specific uptake into these particles, the inhibitor sensitivities clearly showed that MAO activity was almost exclusively attributable to the A-form of the enzyme. Thus, mitochondria in the terminals of dopaminergic neurones have an even more pronounced enrichment in MAO A than the mitochondria obtained by osmotic lysis of a total brain synaptosomal preparation. It was also found that clorgyline and deprenyl have an inhibitory effect on the uptake of dopamine into nerve endings with IC 50 values in the range of 10 −5 to 10 −4 M. These results are discussed in terms of possible physiological significancies of the properties and distribution of the two forms of MAO.

[30] Aldose reductase from human tissues

Publisher Summary This chapter describes an assay method for the synthesis of aldose reductase from human tissues. Aldose reductase and polyol dehydrogenase constitute the sorbitol pathway converting glucose to fructose in extrahepatic tissues. Aldose reductase catalyzes the reduction of other sugar aldehydes and of several aliphatic and aromatic aldehydes. Aldose reductase is assayed spectrophotometrically by recording the decrease in nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH) absorbance at 340 nm. With crude enzyme solutions, blank reactions occur with NADPH in the absence of any aldehyde substrate; consequently the rate of the reaction is recorded before the addition of exogenous aldehyde. Depending on the substrate and on the tissue analyzed, other dehydrogenases, notably aldehyde reductase, may interfere. Interference with aldehyde reductase is diminished by the addition of diphenylhydantoin or phenobarbitone to the assay medium. The ratio of activities measured with D-xylose, DL-glyceraldehyde, and D-glucuronate is used to distinguish between the two enzymes. The chapter discusses the procedure used to isolate and purify carbonyl reducing enzymes from human tissues, along with the preparation of brain aldose reductase. Brains, either whole or without cortex and cerebellum, are obtained from legal medical autopsies.

Purification and substrate specifities of three human liver alcohol dehydrogenase isoenzymes

Human alcohol dehydrogenase (ADH, EC 1.1.1.1) occurs in multiple forms [ 1,2]. The isoenzymes are determined by 3 different gene loci, ADH-1, ADH-2 and ADH-3 which code for the different polypeptides (Y, fil, yt, which randomly associate to the dimeric enzymes [3,4]. Alleles occur at the ADH-2 and ADH-3 locus, coding for p2 (atypical ADH) and y2 [3-61. ADH from phenotypically different tissues exhibit different substrate specificities [3,4,6,7]. However, little data is available concerning purified isoenzymes of known subunit ,composition. Pure ADH is obtained by double ternary complex affinity chromatography, but it still contains all pyrazole-sensitive isoenzymes [8]. Two preliminary reports on affinity purified isoenzymes [9,10] and a comparison of normal /?1/31 with atypical /

Genotyping of human class I alcohol dehydrogenase Analysis of enzymatically amplified DNA with allele-specific oligonucleotides

2/Q from Oriental individuals have been published [ 141. Here, we report the purification of 2 homodimerit isoenzymes plpl and ylyl and the corresponding heterodimeric form ply1 from human liver of normal ADH phenotype by double ternary complex affinity chromatography and CM-cellulose ion-exchange chromatography. These isoenzymes showed large differences in catalytic properties when ethanol, n-pentanol, cyclohexanol, and benzylalcohol were used as substrates. They exhibited marked substrate inhibition as well as non-linear Michaelis-Menten kinetics. The results support the suggestion that human liver ADH subunits do not seem to act independently of one another. 2. MATERIAL AND METHODS

[85] Aldehyde reductase from human tissues

Large inter‐individual differences are noted in the susceptibility to alcohol‐related problems. Part of this variation may be due to the different isoenzyme patterns of the alcohol‐metabolizing enzymes and, consequently, different pharmacokinetics of alcohol degradation. We have used the polymerase chain reaction and oligonucleotide hybridization to amplify and analyze class I alcohol dehydrogenase isoenzyme‐specific genomic DNA. The method unambiguously distinguishes between different allelic variants and thus provides a new means of elucidating the alcohol dehydrogenase isoenzyme pattern of humans.

Atypical human liver alcohol dehydrogenase: the β 2-Bern subunit has an amino acid exchange that is identical to the one in the β 2-Oriental chain

Publisher Summary This chapter describes the assay method, purification, and properties of aldehyde reductase isolated from human tissues. Aldehyde reductase activity is measured spectrophotometrically by monitoring the oxidation of nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH) at 340 nm as a function of time. The presence of other NADPH oxidizing enzymes leads to nonspecific blank reactions in the absence of any aldehyde substrate. Depending on the substrate used, aldose reductase and alcohol dehydrogenase may interfere. The interference with alcohol dehydrogenase is excluded by the addition of pyrazole. An estimation of nonspecific contribution from other enzymes may also be obtained if aldehyde reductase is inhibited by the addition of 1.0 m M phenobarbitone or diphenylhydantoin. Human livers that appear normal are obtained from legal autopsies and the organs are frozen for 6–20 hr after death and stored at -20°C. The steps involved in the purification of aldehyde reductase are extraction, gel filtration, cibacron blue-sepharose chromatography, treatment with hydroxyapatite, and diethylaminoethyl (DEAE)-sepharose chromatography.

Uptake and metabolism of catecholamines in rat brain synaptosomes: Studies on the contribution of monoamine oxidase

Alcohol dehydrogenase Human liver isoenzyme Primary structure Mutation

cDNA sequence of the β2‐subunit of human liver alcohol dehydrogenase

Abstract The uptake of catecholamines into rat brain synaptosomes was studied without suppressing MAO activity. Control experiments confirmed that in our purified synaptosomal preparations catecholamine metabolism was attributable to intrasynaptosomal MAO activity only. Thus, total uptake was defined as the sum of accumulated and metabolised amine. The following results were found. (1) Accumulation of dopamine (DA) as well as norepinephrine (NE) reached a saturation level after about 20 min. Deaminated products were formed in a linear fashion over time. However, NE metabolites constituted less than 20% of the total NE taken up at 20 min, while DA metabolites were an important part of the total DA taken up (44 and 77% at 2 and 20 min, respectively). (2) The distribution of deaminated metabolites between the intrasynaptosomal compartment and the incubation medium was consistent with the assumption that they were released from the synaptosomes by passive diffusion. (3) Kinetic investigations of active transport gave K m values of 1 × 10 −7 M for DA uptake and 5 × 10 −7 M for NE uptake. For DA, but not for NE, results for maximal velocities were considerably higher when calculated on the basis of the sum of accumulation and metabolism, compared to accumulation alone. (4) Blocking MAO activity resulted in a reduced total DA uptake to approximately the same level of accumulation as in controls. Total NE uptake was not significantly affected by MAO inhibition. (5) In synaptosomes from reserpinized animals, a significant decrease of accumulation of both DA and NE was found. In the case of DA this reduction was largely compensated by increased MAO activity, while with NE the decrease of storage resulted in a lower total uptake of NE. It is concluded from these results that intrasynaptosomal MAO activity allows removal of DA from the extracellular space under conditions in which equilibrium between inward and outward fluxes would otherwise abolish net uptake of DA. With NE, however, MAO activity was too low to accomplish this function, and the fate of this amine subsequent to its uptake seems to depend mainly on intracellular storage.