Heinz Werner Goedde

Überführung von “Aktivem acetaldehyd” (α-hydroxyyäthylthiaminpyrophosphat) in acetyl-coenzym a mit pyruvatoxydase

Abstract 14C-labeled α-hydroxyethyl-thiamine pyrophosphate (“active acetaldehyde”) was prepared from an incubation mixture of [2-14C]pyruvate with pyruvic oxidase from yeast mitochondria. After incubation of this substance with pyruvic oxidase from yeast mitochondria, arylamineacetylase from liver, and p-nitroaniline in the presence of diphosphopyridine nucleotide, CoA, and Mg++, radioactive N-acetyl-p-nitroaniline could be isolated by paperchromatography. This demonstrates α-hydroxyethylthiamine pyrophosphate to be an intermediate in the oxidation of pyruvate to acetylcoenzyme A.

THERAPY OF PROLONGED APNEA AFTER SUXAMTHONIUM WITH PURIFIED PSEUDOCHOLINESTERASE: NEW DATA ON KINETICS OF THE HYDROLYSIS OF SUCCINYLDICHOLINE AND SUCCINYLMONOCHOLINE AND FURTHER DATA ON N‐ACETYLTRANSFERASE‐POLYMORPHISM

In recent years many authors have tried to characterize different cholinesterases in human serum. If one remembers the many esterase zones of a starch gel electrophoretic pattern of human serum, one really likes to assume the existence of more than one enzyme. However, looking at the esterase patterns of the serum with the ineffective “silent” pyeudocholinesterase (phenotype S ) , there is nothing to be seen, but a faint coloration in the albumin region. But, what is the situation with the enzymatic activities on various substrates such as acetylcholine, benzoylcholine, succinyldicholine, succinylmonocholine and others? Are they metabolized by the same enzyme or not? And what is the situation with the different enzymes Berry’ postulated from saturation curves with benzoylcholine and acetylcholine, and the different inhibition of these activities by dibucaine? If, for example, the hydrolvsis of benzoylcholine at high substrate concentration with the V,:,,, at lo-’ M is due to an enzvme different from pseudocholinesterase, one would expect that it is not controlled by the alleles at the Ellocus. Tests with sera from homozygotes for the so-called “silent gene” are very appropriate to study wch questions. As I demonstrated last year2 analyzing five of these cases, and as Dietz3 found in another case of a “silent gene,” neither benzoylcholine nor acetylcholine are hydrolyzed at high as well as low concentration for more than 3% of normal rate. The same results are to be found with wccinyldicholine.

Untersuchungen zum Polymorphismus der sauren Erythrocytenphosphatasen (E C 3.1.3.2.)

The method for determination of red cell acid phosphatases is described in detail.Investigations on formal genetics (80 families with 118 children), population genetics and phylogenetics are communicated. The results agree with the assumption: “3 alleles PhA, PhB, and PhC at one autosomal locus”.ZusammenfassungDie Bestimmungstechnik der sauren Erythrocytenphosphatasen wird eingehend beschrieben. Untersuchungen zur Formalgenetik bei 80 Familien mit 118 Kindern sowie zur Populationsgenetik und Phylogenetik werden mitgeteilt. Die Ergebnisse widersprechen nicht dem formalen Modell “3 Allele PhA, PhB, PhC an einem autosomalen Locus”.

Quantitative bestimmung sowie chemisches und enzymatisches verhalten von synthetischem DL-α-hydroxyäthyl-2-thiaminpyrophosphat

Abstract Quantitative determination of synthetic DL-α -Hydroxyethyl- 2 -thiamine pyrophosphate and its chemical and enzymic behaviour DL-α -Hydroxyethyl- 2 -thiamine pyrophosphate (“active acetaldehyde” synthesized from acetaldehyde and thiamine pyrophosphate shows a lower absorption minimum at 248 mμ than thiamine pyrophosphate and thus can be differentiated from it. The isosbestic point (pH 5–8) is 270.5 mμ. Equimolar amounts of thiamine pyrophosphate and DL-α -hydroxyethyl- 2 -thiamine pyrophosphate react equally in the thiochrome assay. Enzymic dephosphorylation yields a substance the picrate of which has the same melting point as the picrate of DL -hydroxyethyl thiamine, synthesized by Krampitz et al . The thiamine pyrophosphate content of α-hydroxyethyl- 2 -thiamine pyrophosphate preparations can be determined after enzymic dephosphorylation, since thiamine and hydroxyethyl thiamine can be separated chromatographically and after elution determined quantitatively by the thiochrome assay. Synthetic DL-α -hydroxyethyl- 2 -thiamine pyrophosphate yields acetoin with pyruvic decarboxylase from brewers yeast and acetyl-coenzyme A with pyruvic oxidase from bakers yeast mitochondria as described earlier for α -hydroxyethyl- 2 -thiamine pyrophosphate isolated from enzymic incubations.

Pseudocholinesterase-variants in Thailand and Japan

Amoung 723 sera from Thailand and 371 sera from Japan the frequency of atypical genes at the E1 locus of pseudocholinesterase is estimated and compared with the results from other populations.

Studies on isoniazid conversion in Thailand

Isoniazid inactivation was studied in a sample of 100 subjects from Central Thailand. The frequency of the allele AcS (resulting in “slow” inactivation in the homozygous state) was calculated as 0.755. There is evidence for a simply additive dosage effect of the two genes AcR and AcS. The results are discussed with regard to other population studies and to recent findings concerning isoniazid inactivation and the activity of the involved enzymes in nonhuman primates.

Hydrolysis of succinyldicholine by Pseudocholinesterase at low concentrations.

I t is reported on the degradation of succinyldicholine by human pscudocholinesterase in vitro below concentrations of 5 × 10-sM expected during relaxation by this drug. The hydrolysis in serum is rapidly catalyzed by the usual enzyme (phenotype ChiUU), almost no catalytic action is found using the genetically determined enzyme variant (phenotype ChiDD ). The experiments on this pharmacogenetie problem were enabled using labelled substrate and antiserum against human pscudocholinesterase. Since 1952 succinyldicholine is used in anesthesia as short acting muscle relaxant (BourNE et M., 1952; T~ESLEFF, 1952). After application of succinyldicholine an apnoea of 4 to 5 minutes is usually observed. Some patients howewer show an apnoea prolonged up to 9 hours. In most of these cases a very low activity of the enzyme pseudocholinesterase was measured. Therefore the enzymic degradation of this drug was investigated by several laboratories. FORBAT et al. (1953) postulated that this reaction should be genetically determined; KALOW and GENEST (1957) first demonstrated an enzyme variant of human pseudocholinesterase. Other genetieM]y determined variants of pseudocholinesterase were identified. For this protein polymorphism a formal genetic model of four alleles (ChiU , Chip , ChiF , ChiS ) at one autosomal locus has been discussed (LEmgANN et M., 1963; 1V~OTULSKY, 1964; GOEDDE and BAITSC]t, 1964). No clinical difference in health has been observed between individuals with the various phenotypes of the normal allele ChiU and those of the alleles ChiD , ChiF and ChlS. I f either homozygotes or heterozygotes of the latter three alleles are subjected to the application of the muscle relaxant succinyldicholine an abnormally prolonged apnoea is observed. Heterozygotes with the usual allele do not show this sensitivity.

Biochemische Untersuchungen zur Frage der Existenz eines “silent Gene” im Polymorphismus der Pseudocholinesterasen

Two sera “without” pseudocholinesterase activity corresponding to the homozygous phenotype Ch1SS are examined by electrophoretical, manometric, and immunological methods.These “silent gene” sera show no activity under the common conditions (spectrophotometric assay).After electrophoretical separation of “silent gene” serum an esterase activity is found which can be identified as pseudocholinesterase activity, although it is weak in comparison with the activity of usual sera. The pseudocholinesterase activity of “silent gene” serum can be demonstrated only in the zone “c4” where 90% of the total activity is present if usual serum is inserted. The identification has been achieved by staining procedures applying several substrates and inhibitors.Quantitative estimation of this pseudocholinesterase activity was carried out by micromanometric assays with benzoylcholine as substrate. The activity of “silent gene” sera was 2–3% of normal serum.Antisera against human pseudocholinesterase-protein have been obtained by immunization of rabbits with a highly purified enzyme protein. Between these antisera and the homozygous “silent gene” sera precipitates were found in immuno double-diffusion tests and immunoelectrophoresis. They could be identified as pseudocholinesterase protein by esterase staining under various conditions.Quantitative estimations have been carried out by immuno-adsorption assays comparing the amount of antibody fixed by usual serum and by “silent gene” serum.The results presented in this paper suggest that the “silent gene” in pseudocholinesterase polymorphism induces in these two cases the synthesis of an enzyme protein which is similar to the enzyme protein of usual pseudocholinesterase. The weak activity is due to a qualitative difference between “silent gene” enzyme protein and the normal pseudocholinesterase protein. A structural alteration of the enzyme protein is assumed to be more likely than a quantitative difference in protein synthesis.ZusammenfassungZwei Seren des Phänotypus Ch1SS, die unter Standardtestbedingungen keine Pseudocholinesteraseaktivität aufwiesen, wurden mit Hilfe der Stärkegel-Elektrophorese, der Mikromanometrie und mit immunologischen Methoden näher untersucht.Nach elektrophoretischer Auftrennung läßt sich in diesen “silent gene”-Seren eine Pseudocholinesteraseaktivität nachweisen; im Vergleich zu der Aktivität von Normalserum erscheint diese sehr gering und läßt sich nur in der c4-Zone erkennen. Die Identifizierung gelingt mit Hilfe spezifischer Färbeverfahren unter Verwendung verschiedener Substrate und Inhibitoren.Die quantitative Bestimmung von Pseudocholinesteraseaktivität im “silent gene”-Serum mit mikromanometrischen Methoden ergab eine Aktivität von 2–3% gegenüber den Kontrollen (Benzoylcholin als Substrat).Durch Immunisierung von Kaninchen mit gereinigtem Pseudocholinesteraseprotein wurden Antiseren erhalten; zwischen “silent gene”-Serum und diesen Antiseren konnten Präzipitationsreaktionen im Immuno-Diffusionstest, in der Immuno-Elektrophorese und mit einer Immuno-Adsorptionsmethode nachgewiesen werden. Diese Ergebnisse lassen annehmen, daß bei den von uns untersuchten Fällen das “silent gene” im Pseudocholinesterasepolymorphismus eine Enzymproteinsynthese steuert; das nachgewiesene Pseudocholinesteraseprotein scheint sich qualitativ von dem Enzymprotein des Normalserums zu unterscheiden.

Untersuchungen in der Liponsäurereihe, IV. Versuche zur Synthese von Liponsäure-Antagonisten, II†

α-Liponsauren mit Methylgruppenin 4- und 7- (III bzw. VI), Oxogruppen in 5- und 7- (XIII bzw. XVI) sowie einer Hydroxylgruppe in 7-Stellung (XIX) wurden auf ubersichtlichen Wegen dargestellt. – Das UV-Spektrum der 7-Oxo-α-liponsaure zeigt Schwingungsstruktur, was auf Einebnung und vergroserte Spannung des Ringes hinweist. Hiermit steht die Geschwindigkeit der Umsetzung durch Dihydro-α-liponsaure-dehydrogenase in Einklang. – 7-Methyl-α-liponsaure erweist sich als nur schwacher Antagonist der α-Liponsaure.

Studien ber Thiamin-pyrophosphokinase

Zusammenfassung1.Ein optischer Test nach Warburg zur Bestimmung von Thiaminpyrophosphokinase wird beschrieben.2.Bei der Untersuchung von Rohextrakten aus Backhefe, Bierhefe, Escherichia coli, Spinat und Schweineleber zeigte ein alkalischer Extrakt aus getrockneter Backhefe in unserem Test die größte Aktivität.3.Dieser Backhefe-Extrakt pyrophosphoryliert 168 · 10-9 Mol Thiamin je Gramm Trockensubstanz und je Stunde. Diese Aktivität ist mehr als 10 mal größer als für die Synthese von TPP aus Thiamin beim Wachstum unter normalen, in der Fabrik üblichen Bedingungen, die den optimalen nahekommen, erforderlich ist (etwa 13 · 10-9 Mol je Gramm Trockensubstanz und je Stunde).4.Aus synthetischemD,l-2-(α-Hydroxyäthyl)-thiaminpyrophosphat (D,l-HETPP = “aktiver Acetaldehyd”) durch Phosphatase-Behandlung gewonnenes D,l-2-(α-Hydroxyäthyl)-thiamin (D,l-HET) wird von gereinigter Thiamin-pyrophosphokinase aus Backhefe mit ATP zu D,l-HETPP pyrophosphoryliert. Es verhält sich in dieser Hinsicht wie HET, das mit Phosphatase aus Enzymatisch gewonnenem HETPP bereitet wurde.Summary01.An optical test according to Warburg for the determination of thiamine pyrophosphokinase is described.2.Comparing extracts of bakers yeast, brewers yeast, Escherichia coli, spinach and pig liver in our tests, an alkaline extract of dried bakers yeast produced maximal activity.3.This extract of bakers yeast phosphorylates 168 · 10-9 moles of thiamine per gram of dry weight bakers yeast per hour. This activity is more than ten fold that required for the synthesis of TPP from thiamine under standard growth conditions in the factory.4.D,l-2-(α-hydroxyethyl)-thiamine (D,l-HET), obtained by treating synthetical D,l-2-(α-hydroxyethyl)-thiamine pyrophosphate (D,l-HETPP “active acetaldehyde”) with phosphatase is phosphorylated by purified thiamine phosphokinase and ATP to D,l-HETPP.