Gerhard Michal

Neue Methode zur enzymatischen Bestimmung von Äthanol in Lebensmitteln

In the presence of the enzyme alcohol dehydrogenase (from yeast, ADH, EC 1.1.1.1), ethanol is oxidized to acetaldehyde by nicotinamide-adenine-dinucleotide (NAD). The unfavourable equilibrium of this reaction is displaced to the side of the reaction products by removing acetaldehyde in another enzymatic reaction, using aldehyde dehydrogenase (from yeast, Al-DH, EC 1.2.1.5). The assay conditions were optimized. The reaction time is shortened to a few minutes. In this way, ethanol can be determined in various foodstuffs, with particular advantage if the alcohol content is low. This is due to the high sensitivity of the reaction. The assay is largely free from disturbances and yields measurements of high precision.ZusammenfassungEs wird eine neue enzymatische Methode zur Bestimmung von Äthanol in Lebensmitteln beschrieben. Äthanol wird in Gegenwart des Enzyms Alkohol-Dehydrogenase (aus Hefe, ADH, EC 1.1.1.1) durch NAD zu Acetaldehyd oxidiert. Die für diese Reaktion ungünstige Gleichgewichtslage wird durch Abfangen des Acetaldehyds mit Aldehyd-Dehydrogenase (aus Hefe, Al-DH, EC 1.2.1.5) verschoben. Die Versuchsbedingungen wurden optimiert. Die Reaktionszeit beträgt nur mehr wenige Minuten. Die Methode ist allgemein anwendbar. Besonders vorteilhaft ist auf diese Weise die Äthanolbestimmung in alkoholarmen Lebensmitteln wegen der großen Empfindlichkeit der Reaktion auszuführen. Der Test ist weitgehend störungsunabhängig und erlaubt Messungen von hoher Präzision.

Cyclophosphates VI cyclophosphates as substrates and effectors of phosphodiesterase

Summary The synthesis and the chemical and physical properties of a major number of 3′,5′-cyclophosphates is described. These compounds are derivatives of 3′,5′-AMP, 3′,5′-IMP or 3′,5′-GMP, substituted in the 6-, 8-, 2- or 2′-position. Furthermore, the partial purification of phosphodiesterase from beef pituitary is described. The cyclophosphate analogues were tested for their splitting behavior at mM substrate concentration with several different phosphodiesterases. Due to the heterogenicity of this enzyme, the selection of the proper reference compound for calculating the relative splitting rates is of importance. The patterns of splitting rate changes caused by different substituents are discussed. Based on additional measurements of the mutual inhibition behavior, some conclusions on the relationship of splitting rates and inhibition effects are drawn.

Cyclophosphates: I. Effect of various cyclophosphates on phosphorylase b kinase activation

Abstract 1. 1. The influence of various cyclophates on phosphorylase b kinase activation was tested in a liver extract and purified muscle protein fraction. 2. 2. The compounds showed a maximum acceleration of activation within 90–113% of that caused by adenosine 3′,5′-monophosphate (3′,5′-AMP); the concentration range in which this acceleration took place different from compound to compound. 3. 3. Substitution in the 6-position of the purine moiety does not influence the activation reaction greatly. Some compounds are even active in lower concentration than 3′,5′-AMP. 8-Substituted derivatives of 3′,5′-AMP require slightly higher concentrations for activation, the corresponding derivatives of 3′,5′-APM need about a 10-fold concentration. Pyrimidine derivatives require about a 50-fold concentration, molecules with altered 2′-position about a 100-fold concentration. The shifts are multiplicative. 4. 4. Comparison of results show that activation in the liver system is observed at lower cyclophosphate concentrations than in the muscle system. 5. 5. The biochemical implications of the results are discussed.

The effect of acute ethanol ingestion on in vitro metabolism of choline and ethanolamine derivatives in rat liver

Abstract The effect of acute ethanol ingestion on hepatic phospholipid metabolism has been investigated in the rat. No significant increase in the content of either lecithin or phosphatidylethanolamine occured in the liver after 12 hr from treatment. The triglyceride content of the liver increased three-fold. An increase of lecithin and phosphatidylethanolamine synthesis by the Kennedy pathway was found in vitro in the homogenates and microsomal fractions prepared from the ethanol-treated rats. A higher rate of conversion of phosphorylcholine and phosphorylethanolamine to CDP-choline and CDP-ethanolamine was also found in the experimental animals. The breakdown of CDP-choline to phosphorylcholine was noticeably decreased in the liver fractions of the ethanol-treated rats. No changes in the sequential methylation pathway for lecithin synthesis in the liver were observed after acute ethanol ingestion.

Cyclophosphates V in vivo metabolic and cardiovascular effects of new cyclophosphates

Summary New synthetic derivatives of natural cyclic nucleotides have been tested for their metabolic (blood glucose and plasma corticosterone) and cardiovascular (heart rate and blood pressure) effects in the rat. A clear cut specificity of the effects emerges from the comparison between the different series of substituted nucleotides (N 6 , 2′O-8-and 2-substitutions). A long lasting effect on plasma steroid levels has been demonstrated with the compound 6-(3′4′-dimethoxyphenyl-)ethyl-amino-3′,5′-PuMP + , at doses and times where there is no effect on the cardiovascular parameters or blood glucose levels.

ANALOGUES OF CYCLIC AMP AND THEIR PHYSIOLOGICAL RESPONSE

ABSTRACT Sutherlands second messenger model and the intracellular cAMP†-system is presented. The key-points of its manipulation by externally applied compounds are discussed. They form the guidelines for development of chemical analogues of the cAMP molecule effective on phosphodiesterases and protein kinases. Special attention is devoted to their influence on glycogenolysis, steroidogenesis, lipolysis, hormone secretion and contractility of various muscles. Some of these analogues show relatively high specificity of physiological responses, such as separation of metabolic and contractile effects, while others show general enhancement of the multivalent responses of cAMP itself. It is shown that a good correlation between in vitro and in vivo data exist. The pharmacological significance of these findings is briefly discussed.

Empfindliche Methoden zur Coenzym-A-Analyse

For higher CoA concentrations, spectrophotometric endpoint determinations are the methods of choice. However, in biological samples the sensitivity is frequently insufficient. As an alternate, two kinetic assay methods, employing phosphotransacetylase or carnitine acetyltransferase, respectively, and the fluorometric adaptation of the test with β-hydroxyacyl-CoA-dehydrogenase are described. The tests are compared with respect to specificity and sensitivity.ZusammenfassungFür höhere CoA-Konzentrationen sind spektralphotometrische Endwertbestimmungen die Methoden der Wahl. Bei biologischem Material reicht jedoch häufig die Empfindlichkeit nicht aus. Als Alternative werden zwei kinetische Tests, welche Phosphotransacetylase bzw. Carnitin-Acetyltransferase benützen, sowie die fluorimetrische Ausführungsform des Tests mit β-Hydroxyacyl-CoA-Dehydrogenase beschrieben. Die Tests werden hinsichtlich Spezifität und Empfindlichkeit miteinander verglichen.