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Ultrastructural localization of adenosinetriphosphatase activity in skeletal muscle by calcium precipitation at high pH

Submikroskopischer Nachweis der Aktivität von Adenosintriphosphatase in Skeletmuskeln durch Calcium-Niederschlag bei hohem pH

  • Published:
Virchows Archiv B

Zusammenfassung

Der ultrastrukturelle Nachweis von ATPase-Aktivität in Skeletmuskeln der Ratte wurde durch Calcium-Niederschlag bei hohem pH untersucht. Das Gewebe wurde für elektronmikroskopische Untersuchungen in gepuffertem ATP-enthaltenden Paraformaldehyd fixiert, um die Hemmung von Fermentaktivität zu vermindern. Die Fermentreaktion wurde nach einer Modifikation nach Padykula und Herman durchgeführt, wobei Calcium als Fermentaktivator und „Fang-Ion“ für das erzeugte Phosphat diente.

In den Muskelfasern verlagerte sich das Reaktionsprodukt im sarkoplasmatischen Reticulum und in den Z-Bänden, dagegen reagierten die A-Bände allgemein nicht. Die Reaktion lief nur mit ATP als Substrat. Die Kontrollversuche ohne ATP oder Calcium und andere mit ADP, AMP, CMP und Na-β-Glycerophosphat ergaben regelmäßig negative Ergebnisse. Durch Zugabe von di-Natriumphosphat, bis ein unvollkommenes Reaktionsmedium erreicht wurde, mit Calcium aber ohne ATP, konnte Calciumphosphat nicht innerhalb der Muskelfasern niedergeschlagen werden.

Die Intensität der Reaktion variiert in verschiedenen Muskelfasern, obwohl innerhalb jeder gegebenen Faser sie verhältnismäßig konstant erschien.

Summary

Ultrastructural localization of ATPase activity in rat skeletal muscle was studied by calcium precipitation at high pH. Tissue was fixed for electron microscopy in buffered paraformaldehyde that contained ATP in order to minimize inhibition of enzyme activity. The enzyme reaction was carried out by a modification of the method of Padykula and Herman, in which calcium serves as both enzyme activator and capture ion for the generated phosphate.

Within the muscle fibers the reaction product was deposited in the sarcoplasmic reticulum and the Z-bands, whereas the A-bands were generally nonreactive. The reaction occurred only with ATP as substrate; controls omitting ATP or calcium and others substituting ADP, AMP, CMP, and sodium-β-glycerophosphate gave consistently negative results. Calcium phosphate could not be precipitated within muscle fibers by addition of dibasic sodium phosphate to an incomplete reaction medium containing calcium but lacking ATP.

The intensity of the reaction varied greatly in different muscle fibers, though within any given fiber it appeared relatively constant.

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Authors and Affiliations

  1. Laboratory of Skeletal Muscle Research, Armed Forces Institute of Pathology, 20305, Washington, D.C., USA

    M. V. Vye, D. A. Fischman & J. L. Hansen

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  1. M. V. Vye
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  2. D. A. Fischman
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  3. J. L. Hansen
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This investigation was supported in part by a research contract, Project No. 3A025601A822, from the Medical Research and Development Command, Washington, D. C.

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Vye, M.V., Fischman, D.A. & Hansen, J.L. Ultrastructural localization of adenosinetriphosphatase activity in skeletal muscle by calcium precipitation at high pH. Virchows Arch. Abt. B Zellpath. 3, 307–323 (1969). https://doi.org/10.1007/BF02901943

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