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Isolierung und Charakterisierung von chromaffinen Noradrenalin-Granula aus Schweine-Nebennierenmark

Isolation and characterisation of chromaffin noradrenaline-granules from pig adrenal medulla

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Summary

1. The distribution of chromaffin granules (catecholamines) and of microsomes (glucose-6-phosphatase) between the subcellular fractions of pig adrenal medulla was investigated. The sediment II (large granule fraction) in which the chromaffin granules are found contained a considerable amount of microsomes. This microsomal contamination could be reduced by an additional centrifugation step (→ sediment III).

2. Centrifugation of sediment III over 1,6 M sucrose solution yielded a sediment of granules which contained an equal amount of noradrenaline and adrenaline (= mixed granules). By centrifugation over either 2.05 or 2.15 M sucrose, granules were isolated which contained 87.5±2% (n=18) of their catecholamines as noradrenaline (noradrenaline-granules). The contamination of the mixed and noradrenaline-granules by other cell organelles was investigated.

3. During incubation of granules in isotonic sucrose at 37° 20% of the noradrenaline, but only 2% of the adrenaline content, was released into the medium within the first 15 min. From 15 min onwards noradrenaline and adrenaline were released at the same rate.

4. The proteins of noradrenaline-granules were found to be highly water soluble (70% of the total proteins). Disc-electrophoresis of the soluble proteins revealed that mixed- and noradrenaline-granules contained identical proteins. Significant differences in the amino acid composition of the soluble proteins of mixed granules and the insoluble proteins of noradrenaline-granules were found.

5. The lysolecithin content of mixed granules was 13.5% of the total phospholipids, that of noradrenaline-granules 20%. Most of the lysolecithin in the adrenal medulla is localised in the membranes of the chromaffin granules.

Zusammenfassung

1. Die quantitative Verteilung von chromaffinen Granula und von Mikrosomen (durch Bestimmung der Glucose-6-Phosphatase) in den durch Differentialzentrifugation erhaltenen Fraktionen aus Schweinenebennierenmark wurde untersucht. Das Sediment II (“large granule fraction”), in dem sich die chromaffinen Granula befinden, enthält auch einen beträchtlichen Anteil der Mikrosomen. Durch neuerliche Zentrifugation (→ Sediment III) kann der Gehalt an Mikrosomen reduziert werden.

2. Durch Zentrifugation des Sediments III über 1,6 M Saccharoselösung können Granula isoliert werden, die etwa gleichviel Noradrenalin und Adrenalin enthalten (Mischgranula). Durch Zentrifugation über 2,05 bzw. 2,15 M Saccharose werden Granula isoliert, in denen 87,5±2% (n=18) der Katecholamine als Noradrenalin vorliegen (Noradrenalin-Granula). Die Verunreinigung dieser Granulafraktionen durch Mitochondrien, Lysosomen und Mikrosomen wird durch die Bestimmung charakteristischer Enzyme untersucht.

3. Chromaffine Granula, die bei 37° in isotoner Saccharose inkubiert werden, geben in den ersten 15 min 20% des Noradrenalin-, aber nur 2% des Adrenalingehaltes an das Medium ab. Nach diesem Zeitpunkt ist die Geschwindigkeit der Noradrenalin- bzw. Adrenalinabgabe gleich.

4. Der molare Katecholamin/ATP-Quotient beträgt für Misch- und Noradrenalingranula 4,5–5,0.

5. 70% der Eiweiße in den Noradrenalingranula sind wasserlöslich. Durch Disk-Electrophorese der löslichen Proteine wird gezeigt, daß Noradrenalin- und Mischgranula ähnliche Eiweiße besitzen. Signifikante Unterschiede in der Aminosäurezusammensetzung der löslichen Eiweiße der Mischgranula und der unlöslichen Eiweiße der Noradrenalingranula werden festgestellt.

6. Der Lysolecithingehalt der Mischgranula beträgt 13,5, der der Noradrenalingranula 20% des Lipidphosphors. Der Großteil des Lysolecithins im Nebennierenmark ist in den chromaffinen Granula und zwar in der Membran dieser Organellen lokalisiert.

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  1. Pharmakologisches Institut der Universität Innsbruck, Austria

    H. Winkler

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Über einen Teil dieser Ergebnisse wurde bereits auf der 32. Tagung der Deutschen Pharmakologischen Gesellschaft in Düsseldorf (1968) berichtet [Naunyn-Schmiedebergs Arch. Pharmak. exp. Path. (im Druck)].

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Winkler, H. Isolierung und Charakterisierung von chromaffinen Noradrenalin-Granula aus Schweine-Nebennierenmark. Naunyn-Schmiedebergs Arch. Pharmak. u. Exp. Path. 263, 340–357 (1969). https://doi.org/10.1007/BF00537608

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