J. P. von Wartburg

The Metabolism of Alcohol in Normals and Alcoholics: Enzymes

The knowledge about the metabolism of ethanol and the participating enzymes offers, in several ways, a basis for a better understanding of biochemical processes involved in acute and chronic alcohol intoxication. The duration of the hepatotoxic and central nervous effects of alcohol clearly depends on the rate of its metabolic breakdown. Many effects of ethanol on intermediary metabolism are known to result from its oxidation to carbon dioxide and water with the consequent process of the handling of a large excess of hydrogen derived from alcohol in the body. The recent discovery of an inducible microsomal ethanol oxidizing system offers possibilities for rationalizing alterations of the action of some drugs in alcoholics. Finally, it is conceivable that the addicting properties of alcohol are interrelated with its metabolic degradation by alcohol and aldehyde dehydrogenase.

Determination of acetaldehyde in human blood.

A method for the determination of acetaldehyde in human plasma by headspace gas chromatography is described. Chloralhydrate, an inhibitor of aldehyde dehydrogenase, is immediately added to the blood sample to prevent a rapid disappearance of acetaldehyde in the erythrocytes.

Alternative molecular forms of erythrocyte catalase.

Katalase aus Erythrocyten vom Menschen (und Pferd) lässt sich säulenchromatographisch und elektrophoretisch in drei Fraktionen A, B und C auftrennen, wobei die Fraktionen A und B die Tendenz haben, in die Fraktion C überzugehen. Durch Chromatographie unter Ausschluss von Luftsauerstoff konnte gezeigt werden, dass die Katalase in den Erythrozyten in der Form A vorliegt. Setzt man das Hämolysat dagegen einige Zeit Luftsauerstoff aus, wird die Katalase bei der Chromatographie in Form C eluiert. SH-blockierende Reagentien verhindern die Umwandlung von A in C, während C mit Mercaptoäthanol zu A reduziert werden kann. Es wird angenommen, dass dem Übergang von Fraktion A in B und C eine Bildung von Disulfidbrücken zugrunde liegt und dass es sich bei den beobachteten alternativen Formen möglicherweise um Katalase-Konformere handelt.

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.

Heterogeneity of erythrocyte catalase: Variability of the isoelectric point

Mittels isoelektrischer Fokussierung wurde der IEP von nativer Erythrocytenkatalase bestimmt (Mensch pH 6,5; Pferd pH 7,3). Die im Verlauf der Reinigung auftretende Konformationsänderung ist von einer Verschiebung des IEP nach der sauren Seite begleitet (Mensch ΔpH —1,3; Pferd ΔlpH −1,6).

Kinetic Studies on NADPH-Linked Aldehyde Reductase from Human Liver

The mechanism of D-glucuronate reduction by human liver NADPH-dependent aldehyde reductase was investigated. At pH 7.4 the Km values for NADPH, NADP+, D-glucuronate and L-gulonate were 2.2 microM, 6 microM, 3.2 mM and 6 mM, respectively. Product inhibition studies in the forward direction (reduction of glucuronate) gave a competitive pattern for the inhibition of NADPH oxidation by NADP+ and non-competitive patterns for the other three inhibitions. In the backward direction all patterns appeared to be competitive. Deuterium isotope effects were dependent on the concentration of D-glucuronate and decreased to unity at infinite concentrations of D-glucuronate. Our findings suggest for aldehyde reductase a kinetic mechanism with sequential ordered binding of NADPH and D-glucuronate and random dissociation of NADP+ and L-gulonate.

LOCALIZATION AND PROPERTIES OF ALDEHYDE REDUCTASE

Publisher Summary This chapter discusses the localization and properties of aldehyde reductase. It presents the purification and the physico-chemical properties of human liver aldehyde reductase. Purification of human liver aldehyde reductase was achieved by passing crude liver homogenate over the following chromatography resins: DEAE-cellulose, Sephadex G-lOO, hydroxyl-apatite, and DEAE-Sephadex A-50, which all had been equilibrated against 10 mM Na-phosphate buffer pH 7.0. The Sephadex G-100 and hydroxylapatite columns were developed with the same buffer; from the ion exchange resins the enzyme was eluted by a gradient 10 to 100 mM Na-phosphate pH 7.0. Three independent methods were used to determine the molecular weight: SDS-gel electrophoresis, ultracentrifugation, and gel filtration on Sephadex G-l00. The values obtained by SDS-gel electrophoresis and gel filtration differ significantly. An unspecific adsorption to dextran could be the cause for the lower molecular weight found by gel filtration. Such a phenomenon was excluded by the use of Bio-Gel, which gave the same results as Sephadex.

ALTERNATIVE MOLECULAR FORMS OF ERYTHROCYTE CATALASE

Publisher Summary This chapter focuses on alternative molecular forms of erythrocyte catalase. Catalase of different sources is known to be heterogeneous in nature. Purified fractions A and C of human and horse erythrocyte catalase, as well as hemolysates were prepared. Fraction A of horse erythrocyte contains a total of about 16 sulfhydryl groups. The isoelectric point (IEP) of erythrocyte catalase, as determined by isoelectric focusing, varies considerably according to the species as well as the pretreatment of the enzyme sample. Experiments with human erythrocyte catalase gave analogous results. The transition of fraction A of human as well as horse erythrocyte catalase to fraction C is characterized by an irreversible oxidation of sulfhydryl groups and is paralleled by a considerable shift of the IEP. Conformational alterations of similar nature have also been observed with liver catalase and other red-cell enzymes such as glucose-6-phosphate dehydrogenase and aspartate aminotransferase, stressing the importance of a reducing environment for maintenance of the structure of intracellular enzymes.