Bendicht Wermuth

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.

Carbonyl reductase from human testis: purification and comparison with carbonyl reductase from human brain and rat testis

Carbonyl reductase (EC 1.1.1.184) is a cytosolic, monomeric, NADPH-dependent oxidoreductase with broad specificity for carbonyl compounds and a general distribution in human tissues. A carbonyl reductase closely resembling the human enzyme is exclusively expressed in rat reproductive tissues and adrenals (Iwata, N., Inazu, N. and Satoh, T. (1989) J. Biochem. 105, 556-564). In order to investigate the relationship between the human and rat enzyme, carbonyl reductase from human testis was purified to homogeneity. The enzyme was indistinguishable from carbonyl reductase from other human tissues on the basis of physicochemical properties, substrate specificity, inhibitor sensitivity and immunological reactivity. Likewise, the human and rat testis enzymes exhibited greatly overlapping substrate specificities for prostaglandins, steroids as well as many xenobiotic carbonyl compounds, and showed the same susceptibility to inhibition by flavonoids and sulfhydryl-blocking agents. Structural homology between the two enzymes was indicated by the mutual cross-reactivity of antibodies against carbonyl reductase from one species and the enzyme protein from the other species. Unlike the rat enzyme, which is confined to Leydig cells, the human enzyme was detectable in Leydig cells as well as Sertoli and spermatogenic cells.

[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.

Analysis of mRNA transcripts improves the success rate of molecular genetic testing in OTC deficiency

BACKGROUND Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of urea metabolism that can lead to hyperammonemic crises and orotic aciduria. To date, a total of 341 causative mutations within the OTC gene have been described. However, in about 20% of the patients with enzymatically confirmed OTC deficiency no mutation can be detected when sequencing of genomic DNA analyzing exons and adjacent intronic segments of the OTC gene is performed. METHODS Standard genomic DNA analysis of the OTC gene in five consecutive patients from five families revealed no mutation. Hence, liver tissue was obtained by needle sampling or open biopsy and RNA extracted from liver was analyzed. RESULTS Complex rearrangements of the OTC transcript (three insertions and two deletions) were found in all five patients. CONCLUSION In patients with a strong suspicion of OTC deficiency despite normal results of sequencing exonic regions of the OTC gene, characterization of liver OTC mRNA is highly effective in resolving the genotype. Liver tissue sampling by needle aspiration allows for both enzymatic analysis and RNA based diagnostics of OTC deficiency.

Immunochemical characterization of aldo‐keto reductases from human tissues

Aldose reductase, aldehyde reductase and carbonyl reductase constitute a family of monomeric NADPH‐dependent oxidoreductases with similar physical and chemical properties. Characterization of the enzymes from human tissues by immunotitration and an enzyme immunoassay indicated that, despite their apparent likeness, the three reductases do not cross‐react immunochemically.

Human liver 6-pyruvoyl tetrahydropterin reductase is biochemically and immunologically indistinguishable from aldose reductase

6-Pyruvoyl tetrahydropterin reductase has been implicated in the biosynthesis of tetrahydrobiopterin. Using immunochemical and biochemical techniques the purified human liver enzyme was shown to be identical to aldose reductase. This suggests that 6-pyruvoyl tetrahydropterin reductase may play an additional role in the reduction of aldehydes derived from the biogenic amine neuro-transmitters and corticosteroid hormones as well as in the pathogenesis of diabetic complications, as has been postulated for aldose reductase.

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

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.

[85] Aldehyde reductase from human tissues

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.

Autocatalytic modification of human carbonyl reductase by 2-oxocarboxylic acids

Carbonyl reductase occurs in multiple molecular forms. Sequence analysis has yielded a carboxyethyllysine residue in one of the enzyme forms, suggesting that pyruvate has been incorporated in a posttranslational enzymatic reaction [Krook, M., Ghosh, D., Strömberg, R., Carlquist, M. and Jörnvall, H. (1993) Proc. Natl. Acad. Sci. USA 90,502‐506]. Using highly purified carbonyl reductase from human brain we show that pyruvate and other 2‐oxocarboxylic acids are bound to the enzyme in an autocatalytic reaction. The resulting enzyme forms were indistinguishable from the native enzyme forms by electrophoresis and isoelectric focusing.

Stereospecificity of hydrogen transfer of aldehyde reductase

Aldehyde reductase from human liver catalyzes the hydrogen transfer from the pro-4R position on the dihydronicotinamide ring of the coenzyme to there face of the carbonyl carbon atom of the substrate.