Itistoehemistry 40, 89--95 (1974) 9 by Springer-Verlag 1974 The Uhrastructural Demonstration of Mitochondrial ATPase Activity by the ATP-dependent Accumulation of Ca++ Ferene Hajds, P6ter Sdtonyi, Shndor Kerpel-Fronius, Endre Somogyi and GySrgyi Bujdosd 1st Department of Anatomy and Department of Forensic Medicine Semmelweis University Medical School, Budapest Received February 15, 1974 Summary. A new method was developed for the ultrastructural demonstration of mito- chondrial ATPase activity based on the ATP dependent mitoehonch%l accumulation of Ca++. Reaction product labels the inner mitoehondrial compartment in the form of electron dense precipitate granules. Reaction could be blocked with 10 mM oligomyein. Introduetion The value of the lead coupling reaction (Wachstein and Meisel, 1957; Waeh- stein and Fernandez, 1964) in the light and electron microscopic demonstration of ATPase is increasingly argued (Deans, 1963; Gillis and Page, 1967; Hulett, 1970; Moses and Rosenthal, 1967; Moses and Rosenthal, 1968; Tiee, 1968). According to this procedure the phosphate ions liberated during the breakdown of ATP are captured by lead to form a lead-phosphate precipitate. However, the aspecific hydrolysis of ATP by lead ions proved to be disturbing factor difficult to control. Further problems emerged if the Mg ++ dependent mitochondrial ATP- ase had to be demonstrated with this technique (Grossman and tIeitkamp, 1968; Lazarus and Barden, 1964; Somogyi et al. 1970; Somogyi et al. 1971 b). It seemed that in ease of this enzyme the localization was highly dependent on the way of fixation. This indicated the active barrier function of mitoehondrial membranes. To offer an alternative at least for the mitoehondrial ATPase activity, a princi- pally new technique has now been developed based on the mitoehondrial uptake of divalent cations (c[. Lehninger, 1965). Accordingly, isolated mitoehondria accumulate Sr ++ or Ca++ on the expense of the energy either generated by coupled respiration or released by the enzymatic hydrolysis of ATP. Accumulated cations --as shown by Brierley and Slautterbaek (1964) and Greenawalt et al. (1964)- precipitate inside the mitoehondrion in the form of an electron-dense deposit. The respiration-linked accumulation of Sr ++ was previously adopted by Kerpel- Fronius and Hajds (1970) for the demonstration of energy production of in situ mitoehondria. The uptake of Ca++, on the other hand appeared to be dependent on ATP breakdown (Greenawalt et al., 1964; Vasington and Greenawalt, 1968). This was exploited for the purpose of a histochemieal reaction following the rea- soning that the energy required for mitochondrial Ca++ uptake is supplied by the enzymatic hydrolysis of ATP intramitochondrial precipitate being thus indicative of mitochondrial ATPase activity.